Roote Prokop SupplMat 1v3.3

A.
Prokop - A rough guide to Drosophila mating schemes
A rough guide to Drosophila mating schemes (version 3.2) 1
This document is one part of a Drosophila genetics training package,

the entire strategy of which is described in detail elsewhere [85].
Content
1. Why work with the fruitfly Drosophila melanogaster?
2. The importance of genetic mating schemes
3. Handling flies in the laboratory
3.1 Keeping flies
3.2. Performing crosses
4. How to design a mating scheme

4.1. Genetic rules
4.1.1. Law of segregation
10
4.1.2. Alleles
10
4.1.3. Independent assortment of chromosomes
12
4.1.4. Linkage groups and recombination
13
4.2. Marker mutations
16
4.3. Balancer chromosomes
17
5. Transgenic flies
19
5.1. Generating transgenic fly lines
19
5.2 Important classes of transgenic fly lines
21
a. Enhancer/reporter construct lines
21
b. Enhancer trap lines
22
c. Protein trap lines
22
d. Gal4/UAS lines
23
e. FRT lines
24
f. RNAi lines
24
6. Classical strategies for the mapping of mutant alleles or transgenic constructs
25
a. Determining the chromosome
25
b. Meiotic mapping
25
Updated versions of this document can be downloaded @ dx.doi.org/10.6084/m9.figshare.106631 and a lite version
for short term training @ dx.doi.org/10.6084/m9.figshare.156395
A. Prokop - A rough guide to Drosophila mating schemes
c. Deletion mapping
26
d. Complementation tests with known loss-of-function mutant alleles
27
7. Concluding remarks and next steps (Powerpoint presentation)
28
8. Acknowledgements
29
9. References
29
Boxes
Box 1. Nobel prizes for work on Drosophila
Box 2. Concepts for genetic research: forward versus reverse genetics & LOF versus GOF
Box 3. Fly stocks available for Drosophila research
Box 4. Keeping information about laboratory stocks
Box 5. How to select flies
Box 6. Awareness of potential genetic modifiers
11
Box 7. Gene descriptors and locators
14
Box 8. Examples of balancer chromosomes
18
Box 9. How to design mating schemes
28
Figures
Fig. 1. The life cycle of Drosophila melanogaster
Fig. 2 A typical flow diagram of how genetic screens in Drosophila contribute to research
Fig. 3. Maintaining and handling flies in the laboratory
Fig. 4. Criteria for gender selection
Fig. 5. Drosophila chromosomes
10
Fig. 6. Independent assortment of alleles & comparison of recessive and dominant inheritance
12
Fig. 7. Inheritance of genes on the same chromosome (linked genes)
13
Fig. 8. Anatomy of adult Drosophila
15
Fig. 9. Examples of typical marker mutations used during genetic crosses
16
Fig. 10. The use of balancers in stock maintenance
17
Fig. 11. First chromosome balancer, FM7
19
Fig. 12. Using P-elements to generate and map transgenic insertions
20
Fig. 13. Enhancer trap and enhancer/reporter lines
22
Fig. 14. The versatile Gal4/UAS system for targeted gene expression
23
Fig. 15. Clonal analysis using MARCM
24
Fig. 16. Maternal gene product and germline clones
25
Fig. 17. Determining the chromosome of insertion of a P-element
26
Fig. 18. Deletion mapping
27
Appendix
Appendix 1. A recombination scheme
33
Appendix 2. A trihybrid cross
35
1. Why work with the fruitfly Drosophila melanogaster?

More than a century ago the fruitfly Drosophila melanogaster was introduced as the invertebrate
model organism that founded the field of classical genetics. It has been argued that Drosophila, as
an omnipresent follower of human culture, was easy to obtain and maintain in laboratories, and that
it was kept in many laboratories as a cheap model for student projects suitable in times of neoDarwinism (the study of Darwinian evolution with Mendelian genetics) [60]. Several laboratories
started using the fly for their main research, but it was the serendipitous discovery of the white
mutation and recognition of its linkage to the X chromosome in 1910 by T.H. Morgan which kickstarted the systematic use of the fly for genetic research, essentially fuelled by Morgan's graduate
students Sturtevant and Bridges [2,22,60,97]1. Building on the sophisticated fly genetics gained
during the early decades, research during the second half of the 20th century gradually turned flies
into a powerful "boundary object" linking genetics to other biological disciplines [57]. Thus, fly
genetics was systematically applied to the study of development, physiology and behaviour,
generating new understanding of the principal genetic and molecular mechanisms underpinning
biology, many being conserved with higher animals and humans [2,9,12,45,57,63,67,72,105].
Notably, it has been estimated that ...about 75% of known human disease genes have a
recognisable match in the genome of fruit flies [84]. Therefore, besides remaining a powerhouse
for unravelling concepts and fundamental understanding of basic biology, Drosophila is nowadays
often used as a test tube to screen for genetic components of disease-relevant processes or
pathways, or to unravel their cellular and molecular mechanisms, covering a wide range of disease
mechanisms including neurodegeneration and even neurotoxicology [13,51,55,83]. It is therefore
not surprising that Drosophila is the insect behind six Nobel laureates (Box 1).
Box 1. Nobel prizes for work on Drosophila (www.nobelprize.org/nobel_prizes/medicine/laureates/)
1933
1946
1995
2011
Thomas Hunt Morgan - the role played by chromosomes in heredity

Hermann Joseph Muller - the production of mutations by means of X-ray irradiation
Edward B. Lewis, Christiane Nsslein-Volhard, Eric F. Wieschaus - the genetic control of early
embryonic development
Jules A. Hoffmann - the activation of innate immunity
Drosophila's enormous success originates from the numerous practical advantages this tiny insect
and the community of fly researchers have to offer to the experimenter. The most important
2 3
advantages are briefly listed below (and tongue-in-cheek here) & :
Fruit flies are easy and cheap to keep. High numbers of different fly stocks can be kept in a
handful of laboratory trays, thus facilitating high-throughput experiments and stock
management (section 3).
A fruit fly generation takes about 10 days (Fig.1), thus fly research progresses rapidly and
pedigrees over several generations can be easily planned and monitored.
The fly genome is of low redundancy, i.e. only one or very few genes code for members of
one protein class. In contrast, higher organisms tend to have more paralogous genes
encoding closely related proteins that often display functional redundancy and complicate
loss-of-function analyses.
A particular strength of Drosophila is the possibility to perform unbiased screens for genes
that regulate or mediate biological processes of interest, often referred to as forward genetics
(Fig. 2; Box 2). Highly efficient and versatile strategies have been developed that can be
adapted to the experimenter's needs [17,42,53,91,94].
Virtually every gene of Drosophila is amenable to targeted manipulations through a wide
range of available genetic strategies and tools, ideal to perform reverse genetics (Box 2).
1
2
Dan Lindsley (2008) Drosophila genetics - The first 25 years @ hstalks.com/?t=BL0341788
for an excellent overview of Drosophila genetics see the appendix of the book by Hartwell [49] (http://highered.mcgrawhill.com/sites/007352526x/student_view0/genetic_portrait_chapters_a-e.html)
3
Informative lay descriptions of fly research can be found on the Wellcome Trust Blog:
The portrait of a fly (Part 1) - wellcometrust.wordpress.com/2012/11/20/feature-the-portrait-of-a-fly-part-1/
The portrait of a fly (Part 2) - wellcometrust.wordpress.com/2012/11/23/the-portrait-of-a-fly-part-2-fly-on-the-wall/
Figure 1. The life cycle of Drosophila melanogaster

Fertilised females store sperm in their receptaculum
seminis for the fertilisation of hundreds of eggs to be laid
over several days. At 25C embryonic development lasts
st
for ~21hr. The hatched larvae (1 instar) take 2 days to
nd
rd
rd
molt into 2 then 3 instar larvae. 3 instar larvae
continue feeding for one more day (foraging stage) before
they leave their food source and migrate away
(wandering stage) and eventually pupariate (prepupa
then pupa). During the pupal stages, all organs
degenerate (histolysis) and restructure into their adult
shapes (metamorphosis). 10d after egg-lay, adult flies
emerge from the pupal case. After eclosure, males
require up to 8 hr to mature sexually, which can be
capitalised on for virgin female collection (section 3). The
times mentioned here need to be doubled when flies are
raised at 18C [3]. Image modified from FlyMove [104].
Figure 2 A typical flow diagram of how genetic
screens in Drosophila contribute to research
A) To induce random mutations, large numbers of
flies are treated chemically (e.g. using EMS, ethyl
methanesulfonate - highly carcinogenic!),
manipulated genetically (e.g. through P-element
mutagenesis; section 5.1) or exposed to irradiation
(e.g. applying X-ray). Other unbiased approaches
are screens with large collections of transgenic
RNAi lines to systematically knock down genes
one by one (section 5.2f) or with EP-line
collections to systematically over-express genes
(section 5.2d). B) The essential task is to select
those mutant or genetically manipulated animals
that display phenotypes representing defects in the
biological processes to be investigated. C) The
responsible gene is either indicated by the specific
RNAi- or EP-line inducing the phenotype, or can
be identified using classical genetic or molecular
strategies to map newly induced mutations to
defined genes within the fly genome (Fig. 12B and
section 6). D) Once the gene is identified, its
nature and normal function can be studied. E)
Vertebrate or human homologues of Drosophila
genes are usually known (listed under "Orthologs"
in FlyBase) and their principal mechanisms are often well conserved. Therefore, capitalising on
knowledge from fly research, they can be studied with informed, focussed experiments in vertebrate/
mammalian model organisms, or human patients can be screened for mutations in these genes.
Experimental manipulations and observations of cells and tissues in vivo are relatively easy.
Thus, organs are of relatively low complexity and size, and can usually be studied live or via
straightforward fixation and staining protocols in the whole organism. Only in exceptional
cases are these experiments subject to legal requirements or procedures, thus enormously
facilitating the fast implementation of experimental ideas. Furthermore, there is a "parallel
universe" of complementary Drosophila research in cell culture. Firstly, an impressive number
of Drosophila cell lines is readily available (dgrc.cgb.indiana.edu/cells/Catalog), of which
especially S2 cells have achieved considerable recognition beyond the community of fly
researchers [29]. Secondly, primary cell cultures (cells directly harvested from the organism)
are well established, especially for neurons and haemocytes [81,88], and offer important
complementary readouts amenable to the full range of versatile Drosophila genetics.
Finally, more than a century of fly work has produced a huge body of knowledge and a rich
resource of genetic tools. From early days of Drosophila genetics up to this day, the fly
community has maintained a highly collaborative spirit which facilitates research enormously
through generous exchange of materials and information [59,60]1. Well organised databases
and stock centres provide easy access to knowledge, fly strains and materials, all of which are
well integrated and curated in FlyBase (flybase.org) the central point of reference for fly
researchers worldwide [73,93] 2.
Box 2. Concepts for genetic research: forward versus reverse genetics & LOF versus GOF
Gene manipulations are generally employed to serve two principal strategies: forward and reverse
genetics [92]. FORWARD GENETICS is the approach to identify the genes that are responsible for a
particular biological process or function. In Drosophila, this is usually performed through using unbiased
large-scale screens for genetic aberrations that disturb the process/function in question, and the
subsequent identification of the genes affected through these aberrations (Fig. 2). REVERSE
GENETICS is the approach to unravel the functions behind specific genes of interest, for example when
trying to understand molecular mechanisms or functions of genes known to cause human disease (using
the fly as a "test tube"). For this, loss- or gain-of-function (LOF, GOF) approaches are employed, using
existing mutant alleles and a wide range of transgenic fly lines that are often readily available (Box 3).
GOF approaches attempt to obtain functional information by creating conditions where the gene is
excessively or ectopically expressed or its function exaggerated. This can be achieved through classical
GOF mutant alleles (section 4.1.2) or through targeted expression of genes, either of their wild type
alleles or of constitutive active versions (section 5).
LOF approaches attempt to eliminate a genes function partially or completely. This can be achieved
by employing classical LOF mutant alleles (section 4.1.2), transposable element insertions (existing for
virtually all gene loci; section 5.2b-d), knock-down of genes using RNA interference strategies (readily
available as transgenic lines for virtually every gene; section 5.2f), the targeted expression of dominantnegative constructs (e.g. catalytically dead versions of enzymes titrating out the function of the
endogenous healthy enzyme), or the use of targeted expression of single domain antibodies (Box 3).
Furthermore, there are constantly improving strategies for the manipulation of genes in situ, i.e. in their
chromosomal location, including...
classical mutagenesis strategies in which mutations are first generated at random and then selected
over chromosomal deficiencies uncovering the targeted gene, thus enriching for candidate mutant
alleles of this gene (section 6c)
generation of targeted deletions at the gene locus through recombinase-mediated mobilisation or
homologous recombination of local transposable elements (section 5.1)
targeted manipulations of the gene locus through genomic engineering, using recombinase-based
strategies [52], TALEN strategies (transcription activator-like effector nuclease) [62,68] or CRISPR
technology (clustered regularly interspaced palindromic repeats) strategies [7,43].
2. The importance of genetic mating schemes

Daily life in a fly laboratory requires performing classical genetic crosses. In these crosses, mutant
or genetically modified flies are used (Box 3). These different fly variants are the bread-and-butter
of fly research, providing the tools by which genes are manipulated or visualised in action in order
to investigate their function. The art of Drosophila genetics is to use these tools, not only in isolation
but often combined in the same flies. This combinatorial genetic approach significantly enhances
the information that can be extracted.
For example, you investigate a certain gene called Mef2. You have isolated a candidate
mutation in this gene which, when present in two copies in embryos, correlates with aberrant
muscle development. You hypothesise that this phenotype is caused by loss of Mef2 function. A
standard approach to prove this hypothesis is to carry out "rescue experiments" by adding back a
wild type copy of the gene into the mutant background, analogous to gene therapy. For this, you will
need to clone the Mef2 gene and generate transgenic fly lines for the targeted expression of Mef2
(section 5.1). To perform the actual experiment, you now need to bring the Mef2 transgenic
construct into Mef2 mutant individuals. This last step requires classical genetic crosses and the
1
All issues of the legendary Drosophila Information Service can be browsed here: www.ou.edu/journals/dis/
For an easy guide to FlyBase see: http://flybase.org/static_pages/docs/pubs/FlyBase_workshop_2009.pdf
careful design of genetic mating schemes.

These mating schemes are a key prerequisite for successful Drosophila research. The rules
underpinning these schemes are simple. However, they often require thinking ahead for several
generations, comparable to planning your moves during a game of chess. To enable you to design
such mating schemes, this manual will provide you with the key rules of the game and explain the
main parameters that need to be considered.
Box 3. Fly stocks available for Drosophila research
1. Flies carrying classical loss- and gain-of-function mutations (including marker mutations) or
deficiencies (section 4.1b)
2. Flies with chromosomal rearrangements (duplications, inversions, translocations etc.) [3,46]
3. Flies with balancer chromosomes (section 4.3; Box 8)
4. Flies with transgenic constructs encoding a range of products (section 5.2) including..
o ..wildtype or mutant versions of genes (including dominant negative constructs) from Drosophila or
other organisms
o ..whole chromosomal fragments for rescue, gain-of-function or targeted mutagenesis experiments
[101,103]
o ..reporter genes (encoding -Gal, fluorescent proteins, calcium indicators, pH indicators etc.) fused
to gene-specific or inducible promoters, or under the control of position-specific activating
elements at their chromosomal insertion site (section 5.2a-c)
o ..exogenous transcription factors (e.g. Gal4, tTA, LexA) with known expression patterns to induce
targeted expression of a gene of choice (section 5.2d)
o ..small interfering RNAs to knock down gene expression (section 5.2f)
o ..single-domain antibodies against endogenous proteins [64] or designed into anti-GFP nanobodies
for the targeted degradation of GFP-tagged proteins [25]
o ..recombinases (e.g. flippase, C31) or their recombination target sites (e.g. FRT, attP) at specific
chromosomal locations; they are jointly used for site-directed insertion of transgenes (section 5.1)
or to generate mosaics of mutant cells in the germline or in somatic tissues (section 5.2e)
o ..genetically encoded toxins (e.g. ricin, tetanus toxin), cell death inducers (e.g. hid, DTS; Box 8),
optogenetic tools (e.g. channel rhodopsin) [54] or other physiological tools (e.g. Kir channels,
ts
Shibire ) for the analysis and/or experimental manipulation of cells
3. Handling flies in the laboratory

3.1. Keeping flies
Before starting the theoretical part, it is necessary to give a brief insight into the practical aspects of
fly husbandry and how the genetic crosses are performed. This should allow you to imagine the
actual "fly pushing" work required to execute the mating schemes designed on the drawing board.
As indicated in Box 3, many different fly stocks are available for fly work. Drosophila
research laboratories usually maintain considerable numbers of stocks relevant to their projects
(Fig. 3A). But always be aware that stock keeping is work intense since you deal with live animals
which need to be cared for like pets! Therefore, you should have a good reason for keeping stocks.
For example, they may be unique (in this case also consider to send them on to stock centres or
interested colleagues for back-up), or you may want to have them readily available to be able to
kick-start practical work on experimental ideas that arise through daily discussion and thought.
Always consider that most stocks can be ordered from public or commercial stock centres (FlyBase
/ Resources / Stock Collections) or by sending requests to colleagues all over the world, most of
whom are willing to freely share fly stocks, especially when they are already published in scientific
journals. Note that new flies coming into the laboratory should be properly filed (Box 4) and kept in
quarantine under observation for a couple of generations in order to exclude diseases or parasites
they may carry. Fly stocks are kept in small vials (Fig. 3B) containing food (the main ingredients of
which are corn flour, glucose, yeast and agar)1 and they can easily be transferred to fresh vials for
maintenance. These vials are usually stored on trays in rooms or incubators (Fig. 3A) which are
temperature-controlled since temperature influences the developmental time of flies (Fig. 1).
1
fly media recipies: Bloomington / Fly Work / Media recipies and for efficient storage see here
Figure 3. Maintaining and handling flies in the laboratory

1
A) Fly stocks are stored in large numbers on trays in temperature controlled rooms/incubators (the trays
shown here each hold two copies of 50 stocks). B) Each fly stock is kept in glass or plastic vials which
contain food at the bottom and are closed with foam, cellulose acetate, paper plugs or cotton wool. Larvae
live in the food and, at the wandering stage, climb up the walls (white arrow) where they subsequently
pupariate (white arrow head). C-E) To score for genetic markers and select virgins and males of the
desired phenotypes, flies are immobilised on CO 2-dispensing porous pads (E), visualised under a
dissecting scope (C, D) and then discarded into a morgue or transferred to fresh vials via a paint brush,
2
forceps or pooter / aspirator (C, E). For further information on how a typical fly laboratory is organised see
3
other sources [3,4,5,95] .
Box 4. Keeping information about laboratory stocks
Work in a fly laboratory involves constant influx of new fly stocks, although only a small percentage of
these will eventually be kept in your stock collection. Follow good practice by making it a rule to instantly
document the essential information for each incoming stock in a dedicated folder or data sheet/base
before it gets lost and forgotten in daily routine:
1. Keep the full original genetic description and any other information you may find on vials or
accompanying notes (e.g. stock centre references or other seemingly meaningless numbers). Note
that genetic descriptions you are given by the donor may be incomplete, and your accessory notes
may provide unique identifiers for this fly stock when communicating with the donor laboratory.
2. Note down the donor laboratory/person and contact. You will need this information for further
enquiries and acknowledgements in future publications.
3. When introducing stocks into your collection, transfer the above information into the accompanying
data base/sheet. Make sure there is a proper genetic description, a clearly assigned short hand for
daily use, info on the donor and the key reference publication. This information will be most useful
when writing up your project and for people succeeding you in the laboratory.
Stock keeping is usually done at 18C (generation time of about 1 month). It is good practice to
keep one young and one two week older vial of each stock. Every fortnight, freshly hatched flies
from the month-old vial are flipped into a fresh vial, whilst the two-week-old vial should have
produced larvae and serves as back-up. Such a routine allows you to spot any problems on
time, such as infections (mites, mould, bacteria, viral infections) [3], the need to add water (if the
food is too dry and coming away from the wall) or to reduce humidity (if vials are too moist so
that fungus accumulates and/or flies get stuck in the food and at walls).
Experiments with flies tend to take place at room temperature or at certain conventional
temperatures, such as 25C for well-timed experiments or 29C to speed up development or
enhance targeted gene expression with the Gal4/UAS system (section 5.2).
1
Incubators need to be fly-proof: copper is aggressively corroded in the presence of flies and should be replaced by
stainless steel or needs at least to be well protected (e.g. thoroughly coated with resin).
2
for use & construction of pooters see: files.figshare.com/1417904/PootersForDrosophilaGenetics_v2_1.pdf
3
detailed stock-keeping instructions: flystocks.bio.indiana.edu/Fly_Work/culturing.htm
Figure 4. Criteria for gender selection

st
Images show females (top) and males (bottom): lateral whole body view (1 column), a magnified view of
nd
rd
th
the front legs (2 column), dorsal view (3 column) and ventral view (4 column) of the abdomen. Only
males display sex combs on the first pair of legs (black arrow heads). Females are slightly larger and
display dark separated stripes at the posterior tip of their abdomen, which are merged in males (curved
arrows). Anal plates (white arrows) are darker and more complex in males and display a pin-like extension
in females. The abdomen and anal plate are still pale in freshly eclosed males and can be mistaken as
female indicators at first sight. Photos are modified from [1] and [30]. During a very short period after
eclosion, flies display a visible dark greenish spot in their abdomen (meconium; not shown) which can be
taken as a secure indicator of female virginity even if fertile males are present.
3.2. Performing crosses

To perform crosses, females and males that carry the appropriate genotypes are carefully selected
and transferred into one vial for mating (Box 5). Some aspects need consideration:
Males and females need to be distinguished using the criteria explained in Figure 4.
Selected females have to be virgin, i.e. selected before they are randomly fertilised by sibling
males in their vial of origin. To select virgins, choose vials containing many dark mature pupae
from which adult flies are expected to eclose. To start the selection procedure, discard all flies
from the vial and thoroughly check that all eclosed flies (including those that transiently stick to
the food or walls) have been removed or otherwise eliminated. The key rationale of this
procedure is that freshly eclosed males remain sterile for a period of several hours and will not
court females. Hence, after clearing vials, all females eclosed within this period will be virgin.
This period lasts for 5-8 hrs at 25C, about double the time at 18C, and considerably longer at
even lower temperatures (we use 11C to maintain crosses up to two days for subsequent virgin
collection). Therefore, a typical routine for virgin collection is to keep vials overnight at low
temperatures (ideally below 18C) and harvest virgins first thing in the morning. During the day,
they are kept at higher temperatures (to enhance yield) and harvested again around lunchtime
and early evening, before moving them back to lower temperature for the night.
Flies have to be selected for the right phenotypic markers. When designing a mating
scheme, combinations of markers need to be wisely chosen so that the correct genotypes of
both sexes can be unequivocally recognised at each step of the scheme (often from parallel
crosses). Phenotypic markers will be explained in section 4.2, and the rules how to choose them
will become clear from later sections.
In general, more female flies are used in a cross than male flies (unless males are expected to be
of low fitness), with two thirds being female as a reasonable approximation. In general, consider
that di- and trihybrid crosses (see example in Fig. 6) will have a low yield of the required offspring
and that the numbers of flies available for crosses in a complex mating scheme may gradually
reduce from generation to generation. Complex mating schemes should therefore be initiated with
large volume crosses (also see Box 5).
A frequent problem is that genetic combinations required for your mating scheme may
render flies morbid, so that the numbers in which they hatch are far lower than statistically expected
(referred to as semi- or sub-lethality). If you need to perform crosses with such animals and
gender choice is an option, choose males from the morbid stock/genotype and females from the
more viable stock/genotype to enhance your chances of establishing the next generation.
Furthermore, make sure that you improve the yield of these morbid flies by taking a number of
measures:
Avoid over-population of vials, which tends to negatively select against morbid individuals. For
this, transfer parents to new vials when sufficient eggs have been laid (within a time frame of 1
day to 1 week, depending on the fertility of stocks used and numbers of parental flies).
Morbid flies tend to get stuck and lost in the food. Therefore monitor crosses daily from start of
eclosion, even if you want to collect only males. Alternatively you may resort to gender scoring
at the larval or pupal stage (Box 5).
Morbid animals tend to hatch late. For example, males carrying the balancer chromosome FM7
(Fig. 11) tend to eclose days after their heterozygous female siblings. Therefore, continue
scoring for as long as possible, but stop and discard the tube before potential individuals of the
grandchild generation start emerging (after ~19 d in a modestly populated tube at 25C).
Make sure that strains are free of bacterial or viral diseases as well as fungal or mite infection
[3]. These conditions can pose a threat to the feasibility of mating schemes. The best
prophylaxis is careful and regular husbandry of your fly stocks.
Note that these same measures should also be taken when you need to quantitatively assess the
relative abundance of different phenotypes emerging from a cross. This is required, for example,
when carrying out meiotic mapping experiments (section 6b), or when you want to perform geno/phenotypic counts of homozygous mutant versus heterozygous/balanced animals, in order to
determine the degree of lethality as a measure of allelic strength. In these cases you need to take
care that morbid animals are not disadvantaged by the stock keeping and harvesting prodecures.
Box 5. How to select flies
Early drosophilists commonly used ether to select flies for gender and phenotypic markers. Nowadays
flies are tipped from their vials onto porous pads dispensing CO2 which acts as a narcotic and is not
harmful if exposure is kept to a few minutes (www.youtube.com/watch?v=S7FkmBjrnAs). Using a
dissection microscope, flies can be easily inspected and selected on this pad (Fig. 3C-E):
Small numbers of flies are efficiently selected using a pooter/aspirator, which is a simple rubber tube
with a mouth piece at one end and glass pipette at the other (Fig. 3C, E and footnote 2 on page 7). A
pooter is far more efficient than a paint brush, since flies can be simultaneously selected and collected
and then directly be blown into vials, even if these already contain flies.
When scoring large numbers of flies, arrange them into a line across the pad and pull out one
phenotypic class at a time. In this way you only have 2 piles of flies on the pad at any one time. Note
that a simple iPhone App can be used to count flies efficiently. Use a funnel to tip the selected flies
from the pad into vials. Note that large-scale virgin collection can be automated using stocks carrying
inducible lethal factors on the Y chromosome [see l(2)DTS in Box 8].
Morbid fly stocks/crosses do not tolerate overnight storage at lower temperatures (used to guarantee
virginity of eclosed individuals in the morning) very well. To maximise yields, individuals can be sexed
as larvae or pupae [32], removed from their tubes with a wet paint brush, and transferred into vials
separated by gender. After eclosion, flies can be screened for phenotypic markers.
Especially in complex mating schemes involving multiple markers, a safe way of phenotype and
gender selection is to merely separate males from females into distinct vials during your daily routine.
Only when enough animals have been collected, perform the marker selection in one single session.
This mode is safer and less time-consuming, especially for the inexperienced fly pusher or when
various crosses are running in parallel so that keeping an overview becomes a challenge.
Selected flies are added to fresh standard vials properly labelled with gender and genotype (Fig. 3B) and
kept at your temperature of choice. Non-selected flies are disposed of in a fly morgue (usually a bottle
containing 70% alcohol) and never returned to their vials of origin.
4. How to design a mating scheme

4.1. Genetic rules
In order to design mating schemes for Drosophila, the typical rules of classical genetics can be
applied. These rules are briefly summarised here and described in greater depth elsewhere [3,46].
10
4.1.1. Law of segregation

Drosophila is diploid, i.e. has two homologous sets of chromosomes, and all genes exist in two
copies (except X-chromosomal genes in males; Fig. 5). By convention, homologous alleles are
separated by a slash or horizontal line(s) (Fig. 6, Box 9). According to the first law of Mendel (law of
segregation), one gene copy is inherited from each parent or, vice versa, the two copies of a gene
are separated during meiosis and only one copy is passed on to each offspring (Fig. 6). Nondisjunction events are rare exceptions in which both copies pass to one gamete.
Figure 5. Drosophila chromosomes

Cytological images of mitotic Drosophila chromosomes. Left: Female and male cells contain pairs of
heterosomes (X, Y) and three autosomal chromosomes. Right: Schematic illustration of Drosophila
salivary gland polytene chromosomes which display a reproducible banding pattern which can be used
for the cytogenetic mapping of gene loci (black numbers; see FlyBase / Tools / Genomic/Map Tools /
nd
rd
Chromosome Maps for detailed microscopic images); 2 and 3 chromosomes are subdivided into a left
(L) and right (R) arm, divided by the centrosome (red dot). Detailed descriptions of Drosophila
chromosomes can be found elsewhere [50].
4.1.2. Alleles1
Genes exist in different alleles. Most loss-of-function mutant alleles (hypo- or amorphic/null) are
recessive. Their phenotypes are not expressed in heterozygous (-/+) but only in homozygous
animals (-/-), i.e. the wildtype allele mostly compensates for the functional loss of one gene copy
(see w, vg or e in Fig. 6). Loss-of-function mutant alleles can also be dominant. For example,
phenotypes are observed in animals heterozygous for Ultrabithorax (Ubx/+), Polycomb (Pc/+), or
Notch (N/+) loss-of-function alleles, i.e. the wildtype allele is insufficient to compensate for loss of
one functional gene copy (haplo-insufficiency). Dominant alleles can also be gain-of-function,
usually caused by over-expression of a gene product (hypermorph or "dominant negative"
antimorph) or by ectopic expression or activation of a gene product, potentially conveying novel
gene functions (neomorph). For example, BarH1 over-expression in the eye causes kidney-shaped
eyes in Bar1/+ individuals (Fig. 6) [61], ectopic Antp expression in antennae the antenna-to-leg
transformations in Antp73b/+ (Fig. 9) [41], and Krppel mis-expression the reduced eyes in If1/+
animals (Fig. 9) [23]. Dominant alleles may display intermediate inheritance showing a stepwise
increase in phenotype strength from heterozygous to homozygous animals. Thus, the eyes of
heterozygous flies (B1/+) are kidney-shaped, whereas they display a stronger slit-shaped phenotype
in homo- (B/B) or hemizygous (B/Y) flies (Fig. 6). Animals carrying the loss-of-function mutant allele
abd-AMX1 in heterozygosis are viable and show a weak dominant cell proliferation phenotype,
whereas homozygous animals are lethal and show a strong cell proliferation phenotype [80]. Note,
that the phenotype distribution in pedigrees involving dominant mutant alleles differs from those
with recessive mutant alleles (Fig. 6). Also note that the existence of dominant and recessive alleles
has impacted on gene names (capitalisation of the first letter), which can be confusing or even
misleading (Box 7). As a further matter of complication, a phenotype you observe may not always
be caused by the gene or mutant allele you believe to study, but a whole range of potential
independent factors in the background of your fly stock/cross might modify the strength or quality of
the observed phenotype, or be causing the whole phenotype all together. Be aware of this and
carry out appropriate control experiments before drawing hasty conclusions (Box 6).
1
see also http://en.wikipedia.org/wiki/Muller's_morphs
11
Box 6: Awareness of potential genetic modifiers

Apart from alleles or constructs that you want to study, fly stocks often carry additional features such as
marker mutations which have remained as left-overs from gene mapping exercises (section 4.2). These
are often not indicated in the genotypic descriptions, but be aware of them since they may have unwanted
side effects. For example, the ebony marker mutation not only causes dark body colour, but it encodes a
multi substrate enzyme that influences circadian rhythm, vision and courtship behaviour [98] (references
therein). Furthermore, hidden genetic modifiers throughout the genome can have a significant impact on
phenotypes you study [26,27,28,35,100]. A number of factors, singly or in combination, can cause genetic
modifications:
UNKNOWN SECOND-SITE MUTATIONS: Unknown second-site mutations (for example on the
balanced chromosome that carries a mutation you want to study) may cause false phenotypes [44].
Note that mutations (especially homozygous-viable or haploinsufficient ones) kept in a stock over a
long period, may cause positive selection for second-site mutations which ameliorate the original
phenotype. Whilst this effect is unwanted in stock keeping, it is actively capitalised on in enhancersuppressor screens [91].
SYMBIONTS AND VIRAL INFECTIONS: For example, the intracellular bacterial symbiont Wolbachia
is highly abundant in many Drosophila colonies and can impact on fly development, behaviour and
longevity [74]. It can be detected by PCR or DAPI staining and eliminated using tetracyclin treatment
[3]. Far less is known about viral infections, but the finding that about 130 genes including many
signalling pathway components were activated upon infection with Drosophila C virus (DCV) [38],
suggests that such a condition can impact on seemingly unrelated gene functions.
B-CHROMOSOMES: They are supernumerary chromosomes inherited in a non-Mendelian fashion
(like plasmids in bacteria), and they can impact on gene regulation and meiotic processes [8].
SATELLITE DNA: heterochromatic repetitive elements serve important epigenetic functions; they
account for ~30% of the D. melanogaster genome, but values may vary in different strains and
therefore impose modifying potential [18].
TRANSPOSABLE ELEMENTS: Transposable elements can insert into the genome in a random
manner, impacting on gene function (section 5.1). There are close to 100 different species of
transposable elements of euchromatin in Drosophila and ~1500 insertions were determined in one
single genome [56], indicating their enormous potential as modifiers [107].
EPIGENETIC INHERITANCE: PIWI-interacting RNAs (piRNAs) act as a kind of immune system
against transposable elements, but only if they are maternally deposited in the oocyte. Inbred
laboratory fly strains are poor in piRNAs and natural strains are rich, and crosses between them lead
to the phenomenon of hybrid dysgenesis where laboratory females crossed to natural males cause
infertility but not vice versa [87,89]. Another form of epigenetic inheritance called genetic imprinting
seems absent from Drosophila (likely due to lack of gene methylation in fly) [34].
MITOCHONDRIAL DNA: Mitochondria are producers of energy and reactive oxygen species
important for cell physiology. Mitochondria are inherited from the mother, yet 90% of their functional
genome is encoded by the cell nucleus and, accordingly, the combination of mitochondrial and
somatic genomes can generate unpredictable biological variations, for example of longevity [82].
Y-CHROMOSOME: The Y-chromosome of Drosophila is primarily heterochromatic, yet has
regulatory power over autosomal genes which can vary significantly between Y-chromosomes from
stocks of different origin [110].
To control for any unknown genetic backgrounds, you must seek independent ways of confirming
observed phenotypes, ideally through heteroallelic combinations using mutant stocks from different
sources, performing gene knock-down (section 5.2f) and/or rescue experiments (section 2). For highly
complex phenotypes (e.g. behaviour, longevity), these strategies will not suffice. In this case, stocks need
to be cleaned from infection and isogenised (i.e. the genome around mutant loci has to be replaced by
that of a reference stock). Already the classical geneticists, when generating detailed linkage maps of
Drosophila chromosomes, used standardised inbred stocks, the most famous of which are Canton-S,
Berlin-K and Oregon-R [60]. More recently, a powerful community resource of 192 fully sequenced inbred
lines derived from natural stocks was generated by the Drosophila melanogaster Genetic Reference Panel
(DGRP) [69]. Inbred fly lines are available at the Bloomington Stock Centre (All Browsing Options/WildType Stocks) and can now be used for analysis of population genomics and quantitative traits in genomewide association studies (GWAS) (e.g. [71]). Finally, always consider that genetic traits are sensitive to
environmental conditions. For example, several marker mutations change penetrance of their phenotypes
when flies are reared at different temperatures [32]. Furthermore, the choice of food can essentially
influence development, behaviour, longevity and reproduction rates, also dependent on the gut flora
[47,108]. One way to standardise the influence of food, is to use chemically defined (holidic) diet [65,78].
12
Figure 6. Independent assortment of alleles & comparison of recessive and dominant inheritance
Two examples of crosses between heterozygous parents (P) involving recessive alleles (top left) and a
dominant allele (green box top right) are shown. Homologous alleles are separated by a horizontal line;
maternal alleles are shown in black, paternal ones in blue. Mutant alleles are w (white; white eyes), vg
(vestigial; reduced wings), B (Bar; reduced eyes); phenotypes are indicated by fly diagrams (compare Fig.
9). When comparing inheritance of the eye marker mutations w (left) and B (right), it becomes apparent
that the allele assortments are identical, yet only the heterozygous B mutant females show an intermediate
eye phenotype.
nd
The left example is a dihybrid cross involving mutant alleles on X and 2 chromosomes (separated
by semicolons). In the first offspring/filial generation (F1) each chromosome has undergone independent
assortment of alleles (demarcated by curly brackets) and each of the four possible outcomes per
chromosome can be combined with any of the outcomes of the other two chromosomes resulting in 4 x 4 =
16 combinations. In case of two autosomal genes, the phenotypic distribution would be 9:3:3:1 (9 white : 3
blue : 3 yellow : 1 pink coloured fields in the Punnett square), as compared to 3:1 in a monohybrid cross
(vg/+ X vg/+ only one of 4 animals displays vg phenotype). In the above cross, w is X-chromosomal
which changes the phenotypic distribution to 6:6:2:2 (6 white : 6 plain/hatched blue : 2 yellow : 2
plain/hatched pink in the Punnett square). The Punnett square lists all possible combinations (symbols
explained on the right); red and blue stippled boxes in the curly bracket scheme and Punnett square show
the same examples of two possible offspring. Note that the Punnett square reflects the numerical outcome
of this cross in its full complexity, whereas the curly bracket strategy only qualitatively reflects potential
combinations and is easier to interpret for the purpose of mating scheme design (Box 9). The complexity of
Punnett squares becomes even more obvious when dealing with trihybrid crosses (Appendix 2).
4.1.3. Independent assortment of chromosomes

Drosophila has one pair of sex chromosomes (heterosomes: X/X or X/Y) and three pairs of
autosomes (Fig. 5). Usually, non-homologous chromosomes behave as individual entities during
meiosis and are written separated by semicolon in crossing schemes (Fig. 6, Box 9). According to
the second law of Mendel (law of independent assortment), they assort independently of one
another during gamete formation, leading to a high number of possible genotypes (Fig. 6). A good
strategy to deal with this complexity during mating scheme design is to define selection criteria for
each chromosome independently (curly brackets in Fig. 6; see Box 9). The 4th chromosome
13
harbours very few genes and its genetics slightly differs from other chromosomes [46]. It plays a
negligible role in routine fly work and will therefore not be considered here.
4.1.4. Linkage groups and recombination
Genes located on the same chromosome are considered a linkage group that tends to segregate
jointly during meiosis. However, when homologous chromosomes are physically paired during
meiotic prophase (synapsis), the process of intra-chromosomal recombination (crossing-over)
can lead to exchange of genetic material between homologous chromosomes (Fig. 7; note, that
recombination does not occur on the 4th chromosome). As a rule of thumb, the recombination
frequency increases with distance between gene loci, but non-uniformly across the chromosome
arms (map expansion/compression). Frequencies are usually high in the middle of chromosome
arms and low in regions adjacent to heterochromatin-rich telomeres and centromeres.
Recombination frequencies have been used to generate spatial chromosomal maps of gene loci
(recombination maps), defining 1% chance of crossing-over between two loci as 1 map unit (or
centimorgan, cM) [46]1. 50% is the maximum detectable crossing-over frequency because crossingover is happening at the 4-strand stage; only 2 strands are involved in any one event and exchange
between sister chromatids produces no observable changes. If two genes are 50 cMs apart then
they are equivalent to being unlinked (due to the increase in multiple crossing-over events occurring
between them). If the location of two loci is known relative to the cytogenetic map, their position on
the recombination map can be roughly estimated and the recombination frequency between them
deduced (Fig. 7B and bottom of Box 7).
Figure 7. Inheritance of genes on the same chromosome (linked genes)

rd
(P) A cross between flies heterozygous for viable recessive mutations of the 3 chromosomal loci rosy
(ry; brownish eyes, 87D9-87D9) and ebony (e; black body colour, 93C7-93D1); female chromosomes are
shown in beige, male in blue. According to the law of segregation, homologous chromosomes are
distributed to different gametes (egg and sperm) during gametogenesis. Male chromosomes do not
undergo crossing-over. In females, crossing-overs are possible (red zigzag lines), and recombination
between any pair of genes may (yes) or may not (no) occur (at a frequency dependent on their location
and distance apart), thus increasing the number of different genotypes. In the first filial generation (F1),
three potential genotypes and two potential phenotypes would have been expected in the absence of
recombination (strict gene linkage); this number is increased to 7 genotypes and 4 phenotypes when
including crossing-over.
For mating schemes, recombination can be a threat as well as an intended outcome:

There are two key remedies to prevent unwanted recombination during mating schemes. The
first strategy is to use balancer chromosomes (section 4.3). The second strategy is to take
advantage of the recombination rule. The recombination rule states that there is no
1
The first ever linkage map [96]: www.esp.org/foundations/genetics/classical/holdings/s/ahs-13.pdf
14
crossing-over in Drosophila males (Fig. 7). The reason for this is not clear but might relate
to the observation that, the type of genes expressed in male meiosis "is much more similar to
mitosis than to female meiosis" [106], albeit reductional divisions occur and haploid gametes
are produced.
Box 7. Gene descriptors and locators
Drosophila genes have different descriptors: name, symbol, synonyms, the annotation symbol and
the FlyBase ID. As an example, go to the FlyBase home page flybase.org. In the "Quick Search" box
click on the "Data Type" tab, select "Data Class / genes" and type "shot" into the text field. This will
direct you to the gene page where you will find a full description of the gene short stop including
various identifiers and locators in the top section and further synonyms in the second last bottom
section [e.g. kakapo/kak, groovin/grv, kopupu/kop, l(2)CA4]. The naming of genes and chromosomal
aberrations follows agreed rules (FlyBase / Documents / Nomenclature), as summarised here:
o The names of Drosophila genes (and their associated short forms or symbols) reflect the
classical (and certainly most human) way to describe a gene or marker mutation. They are
most commonly used in daily life and publications and tend to reflect the mutant phenotypes of
genes - often in very creative ways (e.g. faint sausage, ether-a-gogo, couch potato). For
example, white loss-of-function mutations cause white eyes, indicating that white gene function
is normally required for red eye colour. However, not everybody has followed this tradition when
naming genes. Furthermore, mutations of genes which were identified as homologues to known
mouse or human genes tend to be named after their mammalian relatives. Note that genes
encoding products of similar molecular function may be given names/symbol with identical
prefixes (usually indicating the protein class) and unique suffixes (usually referring to a gene's
cytogenetic location; e.g. Actin-5C, Actin-42A, Actin-57B). For an entertaining radio feature
about fly names listen to www.bbc.co.uk/programmes/b00lyfy1.
o As illustrated by the shot example, genes have often been called differently by independent
researchers (Synonyms & Secondary IDs), and these names come with their independent
symbols. FlyBase usually follows the rule that the first published name for a mutation of a gene
(usually not the wildtype locus or protein) becomes official, but a searchable list of all
synonymous names is maintained. In any case, FlyBase is your key point of reference and you
are advised to use their official naming.
o The annotation symbol (CG number, the Computed Gene identifier) originates from the
genome sequencing projects and has only been assigned to genetic loci that have been
identified as genes. For example, Cy/Curly is a mutation of unknown molecular nature and has
therefore no CG number. CG numbers are primarily used if no other name has been given yet.
o The FlyBase ID (FBgn = FlyBase gene) is the only unique identifier available for both
annotated genes and non-annotated marker mutations. It is often the prime reference during
database searches.
As a general convention, genes/symbols that were FIRST named after recessive mutant alleles
(section 4.1.2) start with lower case, those FIRST identified by dominant alleles are capitalised. For
example, abd-A is lower case due to its original classification as a recessive gene, although
subsequent analyses have revealed dominant loss-of-function mutant phenotypes [80]. Capitalisation
can be confusing, since identical symbols starting with either upper or lower case represent different
gene or marker names (e.g. syn/syndrome versus Syn/Synapsin). Furthermore, genes named after
vertebrate homologues are capitalised, regardless of whether their mutant alleles are dominant or
recessive (e.g. Nrx-IV /Neurexin IV or Syn/Synapsin).
-
Be aware that short hand for mutant alleles in daily use can differ. For example, "w;+;+" or "w" or "w "
or "w /w " all mean the same thing, i.e. a white mutant fly. Whereas the first two versions do not
discriminate gender, the fourth option clearly indicates a female.
Note that genes and their mutant alleles are usually italicised, whereas proteins are written in plain
sf20
and often capitalised (the shot gene, the shot
mutant allele, the Shot protein)
The genomic location of a gene is given in up to 4 ways: the chromosome (arm), cytogenetic map
position (both Fig. 5), the sequence location within the fully sequenced Drosophila genome and, for
marker mutations, also the recombination map position (e.g. the shot gene is on chromosome arm
2R, in cytogenetic map position 50C6-50C9, in sequence location 2R:9,751,742..9,829,615
corresponding to the recombination map position 2-[68]). Use the "Map Conversion Table"
(importable in Excel) for determining recombination map positions of other genes (section 4.1.4).
15
Figure 8. Anatomy of adult Drosophila

Lateral (A) and dorsal (A') view of the head and thorax region of an imago; body parts and bristles are
indicated. B) Ventral views of a female (left) and male (right) abdomen; note differences of the anal plate
in B which provide easy markers to determine gender (Fig. 4). C) Morphology of the wing and its
characteristic veins. Image modified from [19].
In other occasions it can be the intended outcome of a mating scheme to recombine

mutations onto the same chromosome. For example, in reverse to what is shown in Fig. 7,
you may want to combine the rosy (ry) and ebony (e) mutations from different fly stocks onto
one chromosome in order to perform studies of ry,e double-mutant flies. A typical mating
scheme for this task is explained in Appendix 1.
16
Figure 9. Examples of typical marker mutations used during genetic crosses

nd
rd
Mutations are grouped by body colour (top), eye markers (2 row), wing markers (3 row), bristle markers
(bottom row), and "other" markers (top right). Explanations in alphabetic order:
73b
rd
o Antennapedia (dominant; 3 ; antenna-to-leg transformation)
1
st
o Bar (dominant; 1 ; kidney shaped eyes in heterozygosis, slit-shaped eyes in homo-/hemizygosis)
nd
o Curly (dominant; 2 ; curled-up wings; phenotype can be weak at lower temperatures, such as 18C)
rd
o Dichaete (dominant; 3 ; lack of alula, wings spread out)
rd
o Drop (dominant; 3 ; small drop-shaped eyes)
rd
o ebony (recessive; 3 chromosome; dark body colour)
rd
o Humeral (dominant; 3 ; Antennapedia allele, increased numbers of humeral bristles)
nd
o Irregular Facets (dominant; 2 ; slit-shaped eyes)
o mini-white (dominant in white mutant background, recessive in wildtype background; any chromosome;
hypomorphic allele commonly used as marker on transposable elements)
nd
o Pin (dominant; 2 ; short pointed bristles)
rd
o Serrate (dominant; 3 ; serrated wing tips)
st
o singed (recessive; 1 ; curled bristles)
rd
o Stubble (dominant; 3 ; short, blunt bristles)
nd
o vestigial (recessive; 2 ; reduced wings)
st
o white (recessive: 1 ; white eye colour)
st
o yellow (recessive; 1 ; yellowish body colour)
1
Photos of flies carrying marker mutations have been published elsewhere [30,32] .
4.2. Marker mutations

The anatomy of the fly is highly reproducible with regard to features such as the sizes and positions
of bristles, the sizes and shapes of eyes, wings and halteres, or the patterns of wing veins (Fig. 8).
Many mutations have been isolated affecting these anatomical landmarks in characteristic ways
and described in great detail [66]2. On the one hand these mutations can be used to study
biological processes underlying body patterning and development (by addressing what the mutant
1
2
see also images available on FlyBase or as an App, or download the poster "Learning to Fly".
available on FlyBase at the bottom of "Summary Information" for genes that were listed in the red book
17
phenotypes reveal about the normal gene function). On the other hand these mutations provide
important markers to be used during genetic crosses and, hence, for mating scheme design. A few
marker mutations commonly used for fly work are illustrated in Fig. 9.
Figure 10. The use of balancers in stock maintenance

A) Chromosome 3 of wildtype (WT; left arm, green gradient; CM, centromere; right arm, turquoise
gradient) compared to a TM3 balancer chromosome. Chromosomal rearrangements of TM3 are shown as
boxes which correspond to analogous boxes on the wildtype chromosome and display the original colourcode and cytogenetic location in their new orientation (data taken from the Bloomington site: Balancers /
Inversion breakpoints present on balancers). The asterisk indicates a non-rearranged region prone to
recombination. B) A cross of two parents (P) heterozygous for LamininA (LanA; a homozygous embryonic
lethal mutation on 3L, 65A8-65A9) and the recessive and viable marker mutation e (ebony, dark body
rd
colour; 3R, 93C7-93D1). Both mutations are on the 3 chromosome and kept over a balancer. The
mutant chromosome is shown in light green, the balancer chromosome in red, parental alleles in blue,
maternal in black. The first filial generation (F1) is shown on the right. It is compared to a parallel cross
(left) where the balancer was replaced by a wildtype chromosome (white). In the parallel cross, only the
two combinations containing LanA in homozygosis are lethal (black strikethrough). Out of 6 viable
combinations, only two are identical to the parents. In the cross with balancers, also the homozygous
balancer constellation is eliminated (blue strikethrough) as well as all combinations involving
recombination (red strikethrough). Only the combinations identical to the parental genotype are viable,
ideal for stock maintenance.
4.3. Balancer chromosomes

Balancer chromosomes are essential for the maintenance of mutant fly stocks as well as for mating
scheme design. Balancer chromosomes carry multiple inversions through which the relative
positions of genes have been significantly rearranged (Fig. 10A) [3]. Balancer chromosomes
segregate normally during meiosis, but they suppress recombination with a normal sequence
18
chromosome and the products of any recombination that does occur are lethal due to duplications
and deletions of chromosome fragments (aneuploidy of chromosome fragments). The cytological
order of breakpoints for each balancer is listed on the Bloomington site (Balancers / Inversion
breakpoints present on balancers) and shown as pictograms in THE ATLAS [32], nicely illustrating
the weak spots where balancers are prone to recombination (asterisk in Fig. 10A). In addition, most
balancer chromosomes are lethal in homozygosis. Together these properties are essential for
stock maintenance, since they eliminate all genotypes that differ from the parental combination (Fig.
10B). First chromosomal balancers (FM7, first multiply-inverted 7) are usually viable in homo- or
hemizygosis, but carry recessive mutations such as snX2 and lzs that cause female sterility in
homozygosis. The principal outcome for stock maintenance is the same (Fig. 11). The third key
feature of balancer chromosomes is the presence of dominant and recessive marker mutations.
Through their dominant marker mutations, balancer chromosomes are easy to follow in mating
schemes. For example, by making sure that a recessive mutant allele of interest is always kept over
dominantly marked balancers, the presence of this allele can be "negatively traced" over the various
generations of a mating scheme - especially since recombination with the balancer chromosomes
can be excluded. Examples of balancer chromosomes are listed in Box 8.
Box 8. Examples of balancer chromosomes
Numerous balancer stocks are available from Drosophila stock centres (e.g. Bloomington / Balancers):
Typical standard balancers (most marker mutations explained in Fig. 9):
a
1
o FM7a (1st multiply-inverted 7a) - X chromosome - typical markers: y, w , sn, B
st
1
o FM7c (1 multiply-marked 7c) - X chromosome - typical markers: y, sc, w, oc, ptg, B
nd
o CyO (Curly derivative of Oster) - 2 chromosome - typical markers: Cy (Curly), dp (dumpy;
2
bumpy notum), pr (purple; eye colour), cn (cinnabar; eye colour)
nd
o SM6a (2nd multiply-inverted 6a) - 2 chromosome - typical markers: al, Cy, dp, cn, sp
rd
bx-34e
o TM3 (3rd multiply-inverted 3) - 3 chromosome - typical markers: Sb, Ubx
, (bithorax; larger
halteres) e, Ser
o TM6B (3rd multiply-inverted 6B) - 3rd chromosome - typical markers: AntpHu, e, Tb (Tubby;
rd
physically shortened 3 instar larvae and pupae)
Balancers with extra features which can make your life easier:
st
nd
o most 1 and 2 chromosomal balancers carry the same dominant markers (B and Cy,
4
respectively); additional dominant markers, such as Star/S* on CyO or Lobe/L on SM1, can be
helpful to distinguish paternal and maternal balancers, e.g. in back-crosses.
o balancers may carry l(2)DTS or transgenic insertions of hs-hid (Wrinkled) constructs, which cause
cellular lethality when elevating the temperature to ~29C the individuals carrying these balancers
are automatically eliminated, thus enriching for animals homozygous for the non-balancer
chromosome. Note, that having these features on the Y chromosome can be used to collect
virgins at large scale by simply elevating the temperature during development.
o green/blue balancers carry constructs expressing GFP or -Gal, ideal to select against balanced
animals also in embryos, larvae or pupae - live or in fixed/stained preparations. However, note
that some of these balancers were generated through double-insertion of a Gal4 construct (e.g.
Kr-Gal4, twi-Gal4) and a UAS-GFP construct [24,48]; the Gal4 constructs on these balancers will
activate any other UAS-constructs kept in the same stock, thus causing potential phenotypes or
accumulation of unwanted suppressor mutations over time.
Multiple-balancer stocks carry balancers on more than one chromosome, ideal to cross together
and keep mutations / markers on different chromosomes (see also Fig. 15).
Translocation balancer stocks also carry two balancers, but these act as one balancer across
different chromosomes; large fragments have been exchanged between these balancers [e.g.
T(2;3)CyO-TM3] causing lethality in animals that do not inherit both of them.
Compound-X / attached-X chromosomes [e.g. C(1)DX] are not true balancers but can be used in
similar ways; they consist of two X chromosomes fused together so that they do not segregate during
meiosis and are jointly passed on to one gamete. Stocks are maintained by C(1)DX/Y females which
inherit the attached-X from their mothers and the Y from their fathers, whereas C(1)DX/X females
carry three X chromosomes and are lethal or sterile. The X/Y males are the only individuals passing
on the non-attached-X chromosome - ideal for maintaining dominant female sterile mutations.
19
Figure 11. First chromosome balancer, FM7

A stable stock carrying a recessive, homozygous lethal allele of myospheroid (mys) balanced over the
a
FM7 chromosome carrying the following marker mutations: recessive y (yellow body colour), recessive w
1
(bright orange eyes), dominant Bar (reduced eyes; Fig. 6). In the F1 generation, hemizygous mys mutant
males die as embryos, females homozygous for FM7 are viable but sterile. Therefore, only the parental
genotypes contribute to subsequent generations, thus maintaining the mys mutant stock.
Note that the 4th chromosome does not require balancers since it does not display
recombination. Instead the ciD mutant allele is used to maintain stocks with lethal/sterile mutations
of genes on the 4th chromosome; ciD is a recessive lethal, dominant marker mutation caused by a
chromosome rearrangement that led to a fusion protein encoded by the cubitus interruptus and pan
genes.
5. Transgenic flies
5.1. Generating transgenic fly lines
Transgenic flies have become a key resource for Drosophila genetics with many important
applications (see below). Accordingly, transgenic animals are omnipresent in mating schemes, and
it is important to understand their principal nature and some of their applications. The generation of
transgenic fly lines is based on the use of transposable elements/transposons. Transposable
elements are virus-like DNA fragments that insert into the genome fairly randomly, where they can
be maintained in position over many generations, replicate like endogenous genes and follow
Mendelian rules of inheritance. There are ~100 types of natural transposons in Drosophila
melanogaster and thousands of insertions per individual genome [56]. Transposons encode
specialised enzymes called transposases which catalyse mobilisation of the transposons into other
genomic locations, either through excision/re-integration or through replication (Fig.12A). In
Drosophila, the most frequently used class of transposon is the P-element which will be dealt with
primarily in this manual. For the purpose of transgenesis, transposons are modified genetically.
The transposase gene is removed and replaced by the genes the experimenter wants to introduce
into the fly genome, in addition to marker genes and genes/motifs for the selective cloning of the
transposable element in bacteria (Fig. 12B), as well as further potential features enhancing the use
of these constructs (section 5.2).
To introduce purpose-tailored transposons into the fly genome, they are injected into early
embryos at the syncytial blastoderm stage. Injection has to take place at the posterior pole where
the pole cells will form, which are the precursors of sperm and egg cells (Fig. 12) [6]. If successfully
integrated into the genome of some pole cells, the injected transposons will give rise to transgenic
offspring. To catalyse genomic insertion of these P-elements, injections are performed in the
presence of transposases, through using transgenic fly lines expressing transposase in the
germline, or co-injecting helper elements (Fig. 12C, D). Transgenic transposases are crossed out
in the next generation, helper elements don't integrate into the genome and get lost subsequently
(Fig. 12D). Through this disappearance of the enzymatic transposase activity, successful P-element
insertions are stabilised and can be maintained as stocks. Generating transgenic fly lines through
transposon/helper element injection requires technical expertise and specialised equipment, such
as micromanipulators and glass needle pullers. It is often more economical to outsource this task to
specialised companies (of which there are a number existing worldwide), instead of establishing
and maintaining this capacity in individual laboratories.
20
Figure 12. Using P-elements to generate and map transgenic insertions

A) The insertion of natural P-elements into the genome (grey line) requires two key features: firstly,
flanking IS motifs (insertion sequences) mediating stem-loop conformation important for the insertion
process (blue arrow); secondly, catalytic transposase activity (scissors and dashed blue arrow), and this
+
enzyme is encoded by the P-element itself. B) P{lacZ,w } is a classic example of an engineered Pelement used for transgenesis where the transposase gene is replaced by: the lacZ from E. coli (dark
blue box) as reporter gene, a mini-white gene (red box) as selection marker in F1 (see F), an antibiotic
resistance gene (e.g. ampicillin; white box) and an origin of replication (ori; grey box). Once a fly strain
with a stable genomic P-element insertion is established, the exact insertion site can be mapped:
genomic DNA from these flies is extracted and then digested using defined restriction sites in the Pelement (e.g. EcoR1; red asterisk) and random sites for the same enzyme (light blue box) in the nearby
genome (grey letters); the obtained restriction fragment contains the C-terminal part of the P-element
permitting selective cloning along with the adjacent genomic sequence; the obtained gene sequence can
be blasted against the fly genome to map its precise position (box with blue letters) and deduce the
cytogenetic map position (box with green letters; see Box 3). C-F) Making transgenic flies: P-element
solution (red) is injected into the posterior pole of early embryos (C); transposase is either (i) expressed in
the embryo or (ii) co-injected with the P-element solution in form of helper elements which lack IS motifs
(D) and will therefore not insert but disappear during subsequent cell divisions. P-elements become
randomly inserted into the genome of posterior pole cells (D) which will differentiate into egg/sperm cells
when the injected individuals mature into w adults (E). When these adults are crossed to other w
animals, the transgenic individuals amongst the F1 offspring can be selected by their red eye colour (F),
encoded by the mini-white gene marker on the inserted P-element (B, D). Note that P-element insertions
are still heterozygous in these F1 animals.
21
Existing P-element insertions can be mobilised to produce excisions and transpositions

into new chromosomal locations. For this, the P{2-3} strain (carrying a non-excisable transposaseencoding insertion) is crossed with P-element-carrying flies to induce transposition. In the next
generation, P{2-3} is crossed out again to stabilise any newly generated P-element insertions [53].
P-element mobilisation is used for a number of reasons. For example, random P-element insertions
into genes can disrupt their functions and provide new mutant alleles for these genes (P-element
mutagenesis) [53]. In other approaches, reporter genes on P-elements (e.g. lacZ, Gal4 or GFP)
are used to interrogate the genome for gene expression patterns (enhancer/gene/protein trap
screens; details in section 5.2.). Mobilisation of mapped P-element insertions can also be used to
induce deletions at their insertion sites. This can occur through a process called imprecise
excision where the P-element may remove genetic material either side of the insertion site.
Deletions can also be generated through homologous recombination, a strategy that removes the
genomic sequence between two adjacent P-element insertions [70]. For these latter approaches,
countless mapped transposable element insertions are readily available for most gene loci, which
are carefully listed in FlyBase and the Berkeley Drosophila Genome Project (BDGP) [10].
A number of problems with P-elements have been identified and led to improved strategies.
For example, P-elements have size limitations for the DNA inserts they can successfully insert into
the fly genome. Fragment sizes can be significantly increased through the use of BAC (Bacterial
Artificial Chromosome) technology, which allows whole genomic loci of greater than 100 kb to be
used for transgenesis [101,102,103]1. Another problem is the so called position effect, referring to
the fact that identical P-element constructs can have different levels of expression, as a function of
their individual genomic insertion sites. This is due to the fact that each genomic locus displays a
reproducible base-level of transcriptional activity, caused through factors such as site-specific
degrees of chromosomal condensation. One way to deal with this problem is to use specific
features that increase the expression strength of the transposable elements [77], so that they
generate stronger signal even if inserted in less favourite sites. A second strategy is to avoid
position effect by using reproducible site-directed integration of transposons into specific genomic
positions. For example, C31 integrase (as an alternative to the P-transposase) promotes
recombination between attP and attB motifs. Consequently, when attB-bearing transposons are
injected into C31-expressing fly strains carrying attP sites at defined genomic locations, a high
percentage of transposons will insert only at the defined attP site [15]. Note that C31-mediated
recombination can also be used to engineer genes or genomic regions within their natural
chromosomal location (genomic engineering; see Box 2) [52]. Finally, P-elements display a
pronounced non-random insertion spectrum (insertion hot & cold spots), meaning that certain
classes of transposons are biased to insert in certain regions of the genome and avoid others, or
that they show preferential insertion in 5' regulatory rather than coding regions of genes. This can
be advantageous in some cases, but primarily poses a problem, in particular for unbiased genomewide genetic screens (Fig. 2). To circumvent this problem, a number of alternative vectors with
different or less pronounced preferences are available, such as the lepidopteran piggyBac or the
Minos transposon [10,53].
5.2 Important classes of transgenic fly lines
There is a great variety of transgenic fly lines (Box 3) and their nomenclature is complex (see
FlyBase / Documents / Nomenclature). This nomenclature takes into consideration the respective
class of transposon, the molecular components it contains including dominant markers, the
insertion site and other unique identifiers. Here we use a "light" version of this nomenclature (Figs.
12 and 13), with P indicating P-element as the vector, information between curly brackets naming
the key transgenic components including w+ as the dominant marker, and further information
behind brackets may indicate the gene locus of insertion. Usually further identifiers in superscript
are required to unequivocally describe each individual insertion line but will not be considered here.
In the following some important classes of transgenic lines will be explained.
a. Enhancer/reporter construct lines (Fig. 13 A): Enhancers are gene regulatory elements which
induce/facilitate the transcriptional activation at gene promoters, in some cases acting over
distances of several kilo bases. Usually enhancers act on the promoters of endogenous
1
P[acman] clones of genes or genomic regions can be found at pacmanfly.org and are distributed at bacpac.chori.org
22
genes in their region, but they can also activate the promoters on transgenic constructs.
Therefore, to identify and characterise enhancers in (non-coding) regulatory regions of genes,
genomic fragments containing these regions can be cloned in front of a P-element promoter
(which alone is too weak to initiate gene expression) fused to a reporter gene (e.g. GFP or
lacZ/-Gal from E. coli). Transgenic animals carrying these constructs can then be analysed
for the spatiotemporal expression pattern of the reporter gene as a readout for enhancer
activity, and also be used as genetic tools to label certain tissues for experimental purposes.
Figure 13. Enhancer trap and enhancer/reporter lines

+
A) P{Ubx-lacZ,w } illustrating an enhancer/reporter line. An enhancer element that usually activates the
promoter of the Ubx gene at cytogenetic map position 89D (light green box with right pointing arrow) is
cloned (stippled black line) into a P-element, next to a lacZ reporter gene with a basal promoter (dark box
with right pointing arrow) that alone is insufficient to drive lacZ expression. After genomic insertion
(scissors; here at cytogenetic map position 36C), Ubx-E activates (black arrow) transcription of the basal
promoter in a Ubx-like pattern translating into a Ubx-like Gal expression pattern in the transgenic flies
+
(blue). B) P{lacZ,w }Ubx illustrating an enhancer trap line. A P-element (curly bracket; colour code as in
Fig. 12) carrying lacZ with a basal promoter is inserted in the Ubx gene locus at 89D. The endogenous
Ubx-E activates expression of the lacZ gene on the P-element (blue in fly). Note that the inserted Pelement may disrupt (red stippled T) expression or function of the endogenous gene (red stippled X), thus
generating a mutant allele (red stippled arrow).
b. Enhancer trap lines (Fig. 13 B): The P-element promoter alone is too weak to initiate gene
expression of fused reporter genes. Therefore, transposable elements carrying such a Pelement promoter fusion construct will display reporter gene expression only if inserted in a
genomic site which lies within the activity range of endogenous enhancers. By generating
many random insertions of such P-elements, the genome can therefore be screened for
enhancers which are active in specific tissues at certain stages. Such activity often indicates
the presence of genes which are expressed and therefore potentially relevant in these tissues.
This procedure is referred to as an enhancer trap screen [11]. Since P-element insertions
frequently affect the function of genes at their insertion site (stippled red T in Fig. 13 B), they
can be used for systematic P-element mutagenesis screens [53] (see also Fig. 2). Once Pinduced insertions have been generated, reporter gene patterns may reveal when and where
the gene is active (Fig. 13 B), and efficient cloning strategies can be used to map the insertion
and identify the targeted gene (Fig. 12 B).
c. Protein trap lines: A protein trap screen is an advanced version of an enhancer trap screen.
It uses transposons which carry protein tag-encoding sequences (e.g. GFP) flanked by splice
acceptor and donor sites. If such a transposon inserts into an intron (within or flanking a
gene's coding region), the tag gets spliced into the host gene's natural product. This produces
tagged versions of endogenous proteins which are otherwise under their normal regulation (in
contrast to GFP-tagged proteins expressed via Gal4/UAS), so that GFP reflects their natural
expression and localisation patterns [20,58,90].
23
d. Gal4/UAS lines: Gal4 is a transcription factor from yeast that activates genes downstream of
UAS (upstream activating sequence) enhancer elements. Gal4 does not exist endogenously
in flies and does not act on any endogenous loci in the fly genome. Very many transgenic
Gal4 fly lines have been and are still being generated. To illustrate this point, the simple
search term "Gal4" produces almost 6000 hits representing individual fly stocks at the
Bloomington Stock Centre. Of these, numerous Gal4 lines are readily available that display
Gal4 expression in different tissues or cells at specific developmental stages (Fig. 14 a, b). By
simply crossing Gal4-expressing flies to UAS construct lines (Fig. 14 c, d) or enhancerpromoter (EP) lines [86] (Fig. 14 e), the genes downstream of UAS enhancers are being
activated. UAS-linked genes can be of very different nature including reporters, different
isoforms of fly genes (or of other species), optogenetic or physiological tools, small interfering
RNAs or cytotoxins (Box 3). Once crossed to a Gal4 line, the offspring will display expression
of these UAS-coupled genes in the chosen Gal4 pattern. This provides an impressively
versatile and powerful system for experimentation, the spatiotemporal pattern of which can be
further refined through technical improvements such as the use of Gal80 (a Gal4 repressor),
Split Gal4 or the use of alternative strategies (alone or in combination), such as the LexAbased binary expression system [36,40,76]. A further important feature of the Gal4/UAS
system is that its expression strength can be decreased/increased by keeping Gal4/UAS
individuals at lower (e.g. 18C)/higher (e.g. 29C) temperatures.
Figure 14. The versatile Gal4/UAS system for targeted gene expression
The Gal4/UAS system is a two component system where flies carrying Gal4-expressing constructs are
crossed to flies carrying UAS-constructs (inset). Gal4 (black knotted line) binds and activates UAS
enhancers (dotted-stippled lines), so that the pattern in which Gal4 is expressed (here ubiquitously in the
fly) will determine the expression pattern of any genes downstream of the UAS enhancer (here -Gal or
Ubx). The two components can be freely combined providing a versatile system of targeted gene
expression. For example, Gal4-expressing constructs can be enhancer construct lines (a) or enhancer
trap lines (b). The shown Gal4 lines are analogous to those in Fig. 12 with some modifications: Pelements carry Gal4 instead of lacZ, the enhancer trap line is inserted into the ubiquitously expressed
Act42A actin gene at cytogenetic map position 42A, and the enhancer element is the Act42A enhancer
(actin-E) which activates expression of Gal4 ubiquitously in the fly (black). Three examples of UAS lines
+
are shown: c) P{UAS-lacZ,w } carries a UAS enhancer in front of the lacZ reporter gene; d) P{UAS+
+
Ubx,w } carries the UAS enhancer in front of the Ubx gene; e) P{EP,w }Ubx is an enhancer-promoter
(EP) line with a random insertion into the Ubx locus at 89D (analogous to enhancer trap line in Fig. 12 A);
P-elements of EP lines carry an UAS enhancer plus basal promoter which, on Gal4 binding, jointly
activate genes that lie downstream of their random insertion sites (here the Ubx gene).
24
e. FRT lines: FRT (FLP recognition target) sites are specifically targeted by the yeast FLP
recombinase ("flippase"). The FLP/FRT system is widely used in Drosophila as an inducible
recombination system that has mostly replaced former X-ray based strategies [14,99]. It is
used to excise genetic material (to activate/inactivate genes or markers) or to cause
somatic recombination between homologous chromosomes, an event that would normally
only occur during meiosis (Fig. 7). Somatic recombination requires specific insertions of FRTbearing P-elements close to the centromere of both homologous chromosomes. At these FRT
sites, FLP will catalyse breakage and exchange of the homologous chromosome arms which
can distribute into different cells in subsequent cell divisions. When starting from heterozygous
individuals, this method can produce mosaic tissues with homozygous clones of cells
surrounded by heterozygous cells [14]. Somatic recombination is used for MARCM (Mosaic
Analysis with a Repressible Cell Marker) analysis studying the behaviour of single mutant
cells or cell groups in normal or mutant tissue [109] (Fig. 15). Another important application is
the generation of germline clones using Flippase/FRT-mediated recombination at the larval
stage. After such animals have developed into female adults, their ovaries contain
homozygous mutant germline stem cells which will give rise to oocytes/embryos without
maternal gene product (maternal mutant; Fig. 16) [31].
Figure 15. Clonal analysis using
MARCM:
A) MARCM scheme: Activation of
UAS-GFP through a tissue-specific
Gal4 driver (blue arrow) is suppressed
(grey T) by the Gal4 inhibitor Gal80
present in heterozygosis on a
chromosome with an FRT site; the
homologous chromosome carries a
mutation of interest and an equivalent
FRT site; activation of Flippase (Flp)
causes somatic recombination at these
FRT sites (red arrows); during
subsequent somatic cell divisions the
mutant allele may assort into the same
daughter cell and become homozygous
(m/m) creating a parallel twin clone
(+/+) that carries both the wildtype
allele and Gal80 in homozygosis.
Subsequent divisions multiply cells with
these
genotypes
embedded
in
heterozygous tissue. B-D) Illustration
for the use of MARCM in research: B)
Image of a normal wing imaginal disc
at the late larval stage displaying blue
marker gene expression along the
antero-posterior
compartment
boundary. C) In larvae carrying a
hypothetical mutation in homozygosis (m/m), wing discs express the marker gene throughout and are
under-grown and aberrant to a degree that no sensible conclusions can be made about the gene's
function. D) Small MARCM clones do not disturb the overall morphology of the wing disc and allow the
study of mutant cells unequivocally identifiable by their GFP-expression (green outline). In this example,
m/m mutant cells away from the compartment boundary display ectopic expression of the blue marker
gene, suggesting that the wildtype M protein negatively regulates expression of the marker gene.
f.
RNAi lines: Application of RNA interference strategies in flies has become a powerful
alternative to the use of mutant alleles. As one key advantage, fly lines carrying UAS-RNAi
constructs (available for virtually every gene) [37,75] allow the targeted knock-down of specific
genes in a reproducible tissue or set of cells, often at distinct stages of development. Like
analyses using mutant FRT-clones (section 5.2e), this approach can therefore overcome
problems caused by systemic loss of gene function, such as early lethality (often impeding
analyses at postembryonic stages) or complex aberrations of whole tissues that can be
25
difficult to interpret. However, the use of RNAi lines needs to be well controlled.
Demonstration of reduced protein or RNA levels of the targeted gene is not sufficient, since
phenotypes can still be due to additional off-target effects (i.e. knock-down of independent
gene functions). Therefore, it is advised to use more than one independent RNAi line targeting
different regions of the gene. Other proof of specificity can come from enhancement of the
knock-down phenotype in the presence of one mutant copy of the targeted gene or, vice
versa, suppression of the knock-down phenotype through co-expression of a rescue construct
for the targeted gene (using the degenerate code to protect rescue RNA from knock-down).
Figure 16. Maternal gene product and germline clones

A) Many genes are expressed in the female germline and their gene products (RNA or protein; blue) are
deposited in oocytes (but not in sperm; white), often perduring into embryonic or larval stages or even
beyond (depending on the half life of the particular RNA and/or protein); the red + indicates the haploid
gamete genotypes, the black +/+ below, describes the somatic phenotype of ovaries. B) If mutant alleles
of such genes are homozygous lethal, heterozygous parents (m/+) have to be used in crosses to
generate homozygous mutant F1 individuals (m/m); these mutant individuals display maternal gene
product (derived from their mother's wildtype copy of the gene) that may mask mutant phenotypes
especially at earlier stages of development. C) By using Flippase/FRT-mediated recombination at the
larval stage (analogously to Fig. 15A), females with homozygous mutant germline stem cells can be
generated that will give rise to oocytes without maternal product [31]. If the homologous chromosome
D
D1
D2
carries a dominant, female sterile ovo mutant allele (ovo or ovo ) oogenesis is blocked, and only stem
D
cells that lost ovo and have become homozygous mutant (m/m) can give rise to oocytes. Homozygous
mutant embryos developing from these eggs display neither maternal nor zygotic gene function, whereas
heterozygous embryos have zygotic expression of the wildtype allele starting at/after embryonic stage 8
(~3.5 hr after fertilisation at 25C) [21].
6. Classical strategies for the mapping of mutant alleles or transgenic constructs

You may encounter situations in which the location of a mutant allele or P-element insertion is not
known, for example after having conducted a chemical or X-ray mutagenesis (Fig. 2) or when using
a P-element line of unknown origin (unfortunately not a rare experience). To map such mutant
alleles, a step-wise strategy can be applied to determine the chromosome, the region on the
chromosome and, eventually, the actual gene locus. Nowadays, mapping can often be achieved
through molecular strategies, such as plasmid rescue (Fig. 12 B), inverse or splinkerette PCR
[79] or high-throughput genome sequencing [16]. However, classical genetic strategies remain
important and are briefly summarised here.
a. Determining the chromosome: You hold a viable P{lacZ,w+} line in the laboratory that serves
as an excellent reporter for your tissue of interest, but it is not known on which chromosome
the P-element is inserted. To determine the chromosome of insertion you can use a simple
two-generation cross using a w- mutant double-balancer stock (Fig. 17).
b. Meiotic mapping: During meiosis, recombination occurs between homologous chromosomes
and the frequency of recombination between two loci on the same chromosome provides a
measure of their distance apart (section 4.1.4). To make efficient use of this strategy, multi
marker chromosomes have been generated that carry four or more marker mutations on the
same chromosome (Bloomington / Mapping stocks / Meiotic mapping). Each marker provides
26
an independent reference point, and they can be assessed jointly in the same set of crosses,
thus informing you about the approximate location of your mutation [17,46]. Note that multimarker chromosomes can also be used to generate recombinant chromosomes where other
strategies might fail. For example, recombining a mutation onto a chromosome that already
carries two or more mutations, or making recombinant chromosomes with homozygous viable
mutations is made far easier with multi-marker chromosomes.
Figure 17. Determining the chromosome of insertion of a P-element

+
st
nd
rd
A homozygous viable transgenic fly line carries a P{lacZ,w } insertion on either 1 , 2 or 3 chromosome
+
(Pw ?). P) To determine the chromosome of insertion, males of this line (paternal chromosomes in blue)
nd
rd
are crossed to a white mutant double-balancer line carrying balancers on both 2 and 3 chromosome
(note, that the same can be done in two parallel crosses to single balancer stocks carrying balancers on
nd
rd
only 2 and only 3 ; try it out!). F1) In the first filial generation potential X chromosome insertions can be
determined; if X is excluded, complementary chromosome combinations are selected for a second cross;
males can be used for the dominant marker combination (If and Ser) since recombination is excluded by
default in males (section 4.1.4.), whereas recombination in the females is suppressed through using the
nd
rd
balancers (CyO and TM3). F2) In the second filial generation, potential 2 or 3 chromosomal insertions
can be determined (note that helpful stocks for follow-up crosses can be selected at this stage, e.g.
+
+
nd
If/CyO;Pw /Pw would facilitate future combinations of the P-element insertion with a mutation on the 2
chromosome); if w/w;If/CyO;Ser/TM3,Sb flies in F2 are still orange, you have a rare event in which your
th
insertion is on the 4 or the Y chromosome.
c. Deletion mapping: Deficiencies are chromosomal aberrations in which genomic regions

containing one, few or many genetic loci are deleted. Large collections of balanced
deficiencies are available through stock centres (e.g. Bloomington / Deficiencies) and listed in
FlyBase. Using improved technology the Bloomington Deficiency Kit now covers 98.4% of the
euchromatic genome [33]. These deficiencies provide a rich resource to map genes through
classical complementation testing. For this, you cross your mutant to deficiencies of the region
determined by meiotic mapping. If your mutation crossed to the deficiency displays its known
27
phenotype (e.g. lethality) you can infer that the gene of interest is uncovered by this deficiency
(hemizygous constellation). Note that, when dealing with lethal mutations, only 25% of your
offspring are expected to carry the phenotype, so you look for presence/absence of balancerfree animals in F1 (Fig. 6). Absence of the phenotype excludes the group of genes uncovered
by the deficiency. By using various deficiencies in the area, the mapping of the gene can be
further refined (Fig. 18).
Figure 18. Deletion mapping

A mutation (red triangle) in the yellow highlighted gene locus has been mapped (e.g. through meiotic
mapping) to a region of the right arm of chromosome 2 (2R). To refine its mapping, the mutant allele is
crossed to deficiencies (Df) that have their breakpoints in this region (red bars indicate the deleted
chromosomal region for each deficiency). Closest breakpoints of deficiencies that complement the
mutation (+) indicate the region in which the gene is located (blue double-arrow). Closest breakpoints of
non-complementing deficiencies (-) may lie within the gene in question and, in this example, clearly
identify the mutated gene (red double-arrow).
d. Complementation tests with known loss-of-function mutant alleles: Once the location of your
gene has been narrowed down by deletion mapping, you can cross your mutation to available
loss-of-function mutations for the genes in this area, basically following the same strategy as
for deletion mapping. Presence of the phenotype indicates that the mutations are alleles of the
same gene (hetero-allelic constellation). Absence of the phenotype suggests that these
alleles belong to different genes (trans-heterozygous constellation). However, be aware
that the nature of a gene may be complex and lead to false interpretations of your
complementation analysis:
Genes may display transvection, a phenomenon where different homozygous mutant
alleles affecting different areas of the same gene may complement each other [39].
Genes can be nested, i.e. complete genes can be lying within introns of another gene, or
they may map to the complementary strand of DNA at the same locus.
Adjacent genes with separate coding regions might still share common gene regulatory
regions, and therefore display unusual complementation behaviour.
Finally, non-coding RNAs are encoded by independent loci that may often be considered
to represent genes themselves. These loci have important gene regulatory functions and
can complicate the analyses of other genes in their vicinity1.
To circumvent some of these problems, other strategies are available. For example,
collections of UAS-RNAi fly lines (section 5.2f) can be used to systematically knock down
the functions of genes in the area of interest. This strategy only works if your mutation has
phenotypes characteristic enough to be unequivocally identifiable upon gene knock-down.
Furthermore, important clarification can often be obtained from the detailed transcriptional
nice example: http://biobabel.wordpress.com/2012/05/30/a-dual-purpose-rna-and-hox-regulation/
28
profiles displayed for every gene on FlyBase (at the bottom of the "Expression/Regulation"
view in GBrowse).
Box 9. How to design mating schemes (illustrated in Figs. 6 and 17)

write 'X' between maternal and paternal genotypes to indicate the crossing step
genes on the same chromosome may be separated by comma, and also the names of balancer
chromosomes may be separated by comma from the list of their markers (e.g. TM3,Sb,e)
genes on homologous/sister chromosomes are separated by a slash or horizontal lines (usually one,
sometimes two)
genes on different chromosomes are separated by a semicolon
st
nd
rd
always write chromosomes in their order (1 ; 2 ; 3 ); to avoid confusion indicate wildtype
th
chromosomes as "+" (e.g. y/Y ; + ; Sb/+); note, that the 4 chromosome is mentioned only in the
th
relatively rare occasions that 4 chromosomal loci are involved in the cross
the first chromosome represents the sex chromosome; always assign a Y chromosome to the male of
a cross (see Fig. 6); note that the Y chromosome is sometimes indicated by a horizontal line with a
check on its right side (
)
especially as a beginner, stick to a routine order, such as...
o ...the female genotype is always shown on the left side, male on right
o ...the maternal chromosomes (inherited from mother) are shown above, paternal chromosomes
(grey) below the separating line
especially as a beginner, always write down all possible combinations resulting from a cross; carefully
assign phenotypes to each genotype, define selection criteria and check whether these criteria
unequivocally identify the genotype you are after
to keep this task manageable, use curly brackets for chromosome separation and assess each
chromosome individually (Fig. 6). At the end, cross-check whether criteria might clash (for example, a
mini-white marker on the second chromosome only works as a selection criterion if the first
chromosome is homo- or hemizygous for white)
always make sure that you avoid unwanted recombination events by using balancer chromosomes
and/or the recombination rules (no crossing-over in males or on the 4th chromosome). If
recombination is the task of your cross, make sure you use females during the crossing-over step
(usually in F1).
be aware of fly nomenclature which can be confusing, especially with respect to capitalisation and the
indication of whether an allele is recessive, dominant, loss- or gain-of-function (Box 3). Be aware that
you understand the nature of the involved alleles, since dominant alleles behave differently to
recessive ones in a cross (Fig. 6)
The nomenclature of transposable elements or chromosomal aberrations can be tedious. To work
more efficiently, feel free to use your own unequivocal short hand during the task. For example,
+
+
+
+
"P{UAS-lacZ,w }" and "P{eve-Gal4,w }" could be shortened to "PUw " and "PGw ".
7. Concluding remarks and next steps (Powerpoint presentation)

You should now have gained the key knowledge and terminology required to design mating
schemes for Drosophila and to function in a fly laboratory. However, the information given is still
basic and requires that you further explore the details behind the various aspects mentioned here.
For this, some literature has been provided throughout the text. Should there be mistakes,
passages that are hard to understand or information that is missing or wrong, please, be so kind to
let me know ([email protected]).
To apply and consolidate your knowledge you can now download and study the PowerPoint
presentation "Roote+Prokop-SupplMat-3.ppt" (shar.es/YcX2f). The presentation briefly reiterates
the principal features of meiosis versus mitosis and the key rules of fly genetics. You will then be
confronted with a standard crossing task, and the presentation will lead you step-by-step through
the solution of this task, illustrating how the rules of Drosophila genetics are applied and explaining
the various strategic considerations and decisions that have to be taken. Make use of this
opportunity to test your knowledge by making your own suggestions first, before being presented
with a possible solution. To answer queries, revisit this manual, thus consolidating your knowledge.
29
8. Acknowledgements
I am most grateful to Casey Bergman, Ian Dworkin, Sanjai Patel and John Roote for comments and
feedback on this manual. I would like to thank figshare.com for providing the facilities to upload and
manage this document so easily.
9. References
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2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
Ahuja A, Singh RS (2008) Variation and evolution of male sex combs in Drosophila: nature of selection
response and theories of genetic variation for sexual traits. Genetics 179: 503-509.
Ashburner M (1993) Epilogue. In: Bate M, Martnez Arias A, editors. The development of Drosophila
melanogaster. Cold Spring Harbor: CSH Laboratory Press. pp. 1493-1506.
Ashburner M, Golic KG, Hawley RS (2005) Drosophila: a laboratory handbook. Cold Spring Harbor,
New York: Cold Spring Harbor Laboratory Press. 1409 p.
Ashburner M, Roote J (2007) Culture of Drosophila: the laboratory setup. CSH Protoc 2007: pdb ip34.
Ashburner M, Roote J (2007) Maintenance of a Drosophila laboratory: general procedures. CSH Protoc
2007: pdb ip35.
Bachmann A, Knust E (2008) The use of P-element transposons to generate transgenic flies. In:
Dahmann C, editor. Drosophila Methods and Protocols. 2008/07/22 ed: Humana Press. pp. 61-77.
Bassett AR, Liu J-L (2014) CRISPR/Cas9 and genome editing in Drosophila. Journal of Genetics and
Genomics 41: 7-19.
Bauerly E, Hughes SE, Vietti DR, Miller DE, McDowell W, et al. (2014) Discovery of supernumerary B
chromosomes in Drosophila melanogaster. Genetics.
Beckingham KM, Armstrong JD, Texada MJ, Munjaal R, Baker DA (2005) Drosophila melanogaster the model organism of choice for the complex biology of multi-cellular organisms. Gravit Space Biol Bull
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Appendix 1. A recombination scheme

You want to recombine mutant alleles of the viable, recessive, 3rd chromosomal loci rosy (ry; dark
brown eyes) and ebony (e; black body colour) onto one chromosome. According to FlyBase, ry
localises to recombination map position 3-52, and e to 3-70.7. Hence, they lie 18.7cM apart,
indicating that slightly less than 1 in 5 oocytes will carry the desired recombination event.
For this, you start by crossing ry females with e males or vice versa (P, parental cross). In the first
filial generation (F1), all flies are trans-heterozygous (ry,+/+,e). Note that the different fly stocks
used in this cross will be colour-coded to allow you to easily trace the origin of each chromosome.
According to the recombination rule, you need to take females so that recombination can occur.
Note that crossing-over during oogenesis in these females occurs at random, i.e. their eggs which
give rise to the second filial generation (F2) represent a cocktail of recombination events with a
statistical likelihood of 18.7% as calculated above. Note that only half of the tested animals carry
the first marker ry, out of which only 18.7% display the wanted recombination. Therefore, 9.35% of
the single F2 individuals carry a recombinant chromosome with both markers, and 9.35% a
recombinant chromosome with wildtype alleles of both markers. The key task is to identify and
isolate these recombination events through a step-wise process.
In the first step, recombination events need to be "stabilised" to prevent further recombination. For
this, F1 females are crossed to a balancer stock carrying a balancer chromosome (Bal1) over a
dominantly marked chromosome (M1; sections 4.2. and 4.3). In the third filial generation (F3), you
determine whether one of the markers (here ry) is present (remember that, according to the law of
segregation, only 50% of balanced F2 individuals carry ry). To determine the presence of ry, you
cross F2 animals back to a ry mutant stock. Two important issues need to be considered here.
Firstly, each individual in F2 is the result of an individual recombination event in its mother's
germline. Therefore, single animals need to be tested for the presence of ry. For practical
reasons, use single males since they can fertilise several females and therefore have a higher
likelihood to generate enough offspring.
34
Secondly, you have to cross back to ry mutant flies, but need to be able to distinguish your
recombinant chromosome from the ry chromosome of the back-cross. For this, cross the ry
stock to a balancer stock (Bal2) that can be distinguished from Bal1.
In F3, use simple selection to separate out two groups of flies: non-balanced flies allow you to
determine whether flies have brownish eyes (i.e. carry ry on their potentially recombinant
chromosome). If this is the case, flies carrying Bal2 over the potentially recombinant paternal
chromosome (rather than the ry chromosome of their mothers) can be used to establish a stable fly
stock. The fourth filial generation (F4) emerging from these newly established fly stocks will contain
non-balanced animals (ry and e are viable mutations). Stocks in which non-balanced flies have
brownish eyes and dark body colour bear the desired recombinant chromosome and will be kept,
the rest discarded.
For consideration:
To have a statistical chance of isolating recombination events, more than 10 single crosses in F2
should be used to match the 9.35% chance of obtaining a recombinant.
The example of ry and e represents an unusual case, since they are common marker mutations that
are found on several balancer chromosomes (section 4.3.). Using balancers with these markers would
allow you to immediately identify the presence of the desired mutations on the potentially recombinant
chromosomes. Try it yourself.
35
Appendix 2. A trihybrid cross
nd
Example of a trihybrid cross between heterozygous parents (P, top) involving recessive alleles on X, 2 and
rd
3 chromosomes (separated by semicolons). Homologous alleles are separated by a horizontal line; maternal
alleles are shown in black, paternal ones in blue. Mutant alleles are w (white; white eyes), vg (vestigial;
reduced wings), e (ebony; dark body colour); phenotypes are indicated by fly diagrams (compare Fig. 9). In
the first offspring/filial generation (F1) each chromosome has undergone independent assortment of alleles
(demarcated by curly brackets) and each of the four possible outcomes per chromosome can be combined
with any of the outcomes of the other two chromosomes resulting in 4 x 4 x 4 = 64 combinations. The Punnett
square at the bottom systematically lists all possible combinations (different phenotype classes are colourcoded and display a 18:18:6:6:6:6:2:2 distribution; symbols are explained at the bottom). Red and blue
stippled boxes show the same examples of two possible offspring in both the curly bracket scheme and the
Punnett square. Note that the Punnett square reflects the numerical outcome of this cross in its full
complexity, whereas the curly bracket strategy only qualitatively reflects potential combinations and is easier
to interpret for the purpose of mating scheme design (Box 9).

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A.

Prokop - A rough guide to Drosophila mating schemes

A rough guide to Drosophila mating schemes (version 3.2) 1

This document is one part of a Drosophila genetics training package,

2. The importance of genetic mating schemes

3. Handling flies in the laboratory

3.1 Keeping flies

3.2. Performing crosses

4. How to design a mating scheme

4.1.1. Law of segregation

4.1.3. Independent assortment of chromosomes

4.1.4. Linkage groups and recombination

4.2. Marker mutations

4.3. Balancer chromosomes

5.1. Generating transgenic fly lines

5.2 Important classes of transgenic fly lines

a. Enhancer/reporter construct lines

b. Enhancer trap lines

c. Protein trap lines

6. Classical strategies for the mapping of mutant alleles or transgenic constructs

a. Determining the chromosome

A. Prokop - A rough guide to Drosophila mating schemes

d. Complementation tests with known loss-of-function mutant alleles

7. Concluding remarks and next steps (Powerpoint presentation)

Box 1. Nobel prizes for work on Drosophila

Box 3. Fly stocks available for Drosophila research

Box 4. Keeping information about laboratory stocks

Box 5. How to select flies

Box 6. Awareness of potential genetic modifiers

Box 7. Gene descriptors and locators

Box 8. Examples of balancer chromosomes

Box 9. How to design mating schemes

Fig. 1. The life cycle of Drosophila melanogaster

Fig. 3. Maintaining and handling flies in the laboratory

Fig. 4. Criteria for gender selection

Fig. 5. Drosophila chromosomes

Fig. 7. Inheritance of genes on the same chromosome (linked genes)

Fig. 8. Anatomy of adult Drosophila

Fig. 9. Examples of typical marker mutations used during genetic crosses

Fig. 10. The use of balancers in stock maintenance

Fig. 11. First chromosome balancer, FM7

Fig. 12. Using P-elements to generate and map transgenic insertions

Fig. 13. Enhancer trap and enhancer/reporter lines

Fig. 15. Clonal analysis using MARCM

Fig. 16. Maternal gene product and germline clones

Fig. 17. Determining the chromosome of insertion of a P-element

Fig. 18. Deletion mapping

Appendix 1. A recombination scheme

Appendix 2. A trihybrid cross

A. Prokop - A rough guide to Drosophila mating schemes

1. Why work with the fruitfly Drosophila melanogaster?

Thomas Hunt Morgan - the role played by chromosomes in heredity

Dan Lindsley (2008) Drosophila genetics - The first 25 years @ hstalks.com/?t=BL0341788

A. Prokop - A rough guide to Drosophila mating schemes

Figure 1. The life cycle of Drosophila melanogaster

A. Prokop - A rough guide to Drosophila mating schemes

2. The importance of genetic mating schemes

For an easy guide to FlyBase see: http://flybase.org/static_pages/docs/pubs/FlyBase_workshop_2009.pdf

A. Prokop - A rough guide to Drosophila mating schemes