Seminar on

Alternative To Animal Screening Procedure,

In Vitro Models, Molecular Biology Technique
Alternatives to animal testing

Most scientists and governments say they agree that animal testing
should cause as little suffering as possible, and that alternatives to
animal testing need to be developed.
 The "three Rs" first described by Russell and Burch in 1959, are
guiding principles for the use of animals in research in many countries:
Replacement refers to the preferred use of non-animal methods over
animal methods whenever it is possible to achieve the same scientific
aim.
Reduction refers to methods that enable researchers to obtain
comparable levels of information from fewer animals, or to obtain
more information from the same number of animals.
Refinement refers to methods that alleviate or minimize potential
pain, suffering or distress, and enhance animal welfare for the animals
still used.
Two major alternatives to in vivo animal testing are in vitro
cell culture techniques and in silico computer simulation.
However, some claim they are not true alternatives since
simulations use data from prior animal experiments and
cultured cells often require animal derived products, such as
serum.
Others say that they cannot replace animals completely as
they are unlikely to ever provide enough information about
the complex interactions of living systems.
 Other alternatives, not subject to this criticism, involve the
use of humans for skin irritancy tests and donated human
blood for pyrogenicity studies.
 Another alternative is so-called microdosing, in which the
basic behaviour of drugs is assessed using human volunteers
receiving doses well below those expected to produce whole-
body effects.
 Origin
In 1954, Charles Hume, founder of the Universities Federation for
Animal Welfare (UFAW) made an original proposal for the Three Rs
to the UFAW to take in consideration alternatives for animal testing
and change scientific study in laboratory animal experiments.
Committee under the chairmanship of Sir Peter Medawar, the
Nobel prize-winning immunologist, along with Christine Stevens,
founder of the Animal Welfare Institute (AWI) in the U.S, and
William Lane-Petter, the Secretary of the Research Defense Society
of Great Britain provided financial support and managed the project
to publish the concept of animal testing alternatives. The
microbiologist R.L. Burch and the zoologist W.M.S. Russell were
chosen to publish the work. "The Principles of Humane
Experimental Technique" was published in London in 1959, and the
book defined animal testing alternatives as “The Three R's:
Refinement, Reduction, and Replacement.”
 Types

Cell culture
Cell culture is currently the most
successful, and promising, alternative to
animal use. For example, cultured cells
have been developed to create
monoclonal antibodies, prior to this
production required animals to undergo a
procedure likely to cause pain and
distress.
MONOCLONAL ANTIBODY PRODUCTION
 Skin corrosion

Human skin equivalent tests can be used to replace

animal-based corrosive studies. Two products,
EpiDerm and EpiSkin are derived from human skin
cells which have been cultured to produce a model
of human skin.
These methods are currently accepted replacements
in Canada and the European Union.
Another synthetic replacement uses a protein
membrane to simulate a skin barrier and is
approved as a partial replacement by the US
Department of Transportation and European Union.
 Human-based

Skin irritation
A skin patch test has been designed and is used in Canada
to measure development of rashes, inflammation, swelling
or abnormal tissue growth on human volunteers. Unlike
corrosives, irritants cause only reversible skin damage.
Pyrogenicity
Pyrogens are most often pharmaceutical products or
intravenous drugs that may cause inflammation or fever
when they interact with immune system cells. This
interaction can be quickly and accurately tested in vitro
using donated human blood.
Computer simulation

Examples of computer simulations available include models of

diabetes, asthma and drug absorption, though potential new
medicines identified using these techniques are currently still
required to be verified in animal and human tests before
licensing.
Computer operated mannequins, also known as
crash test dummies, complete with internal sensors and video,
have replaced live animal trauma testing for automobile crash
testing.
The first of these was “Sierra Sam” built in 1949 by Alderson
Research Labs (ARL) Sierra Engineering. These dummies
continue to be refined.
 Prior to this, live pigs were used as test subjects for crash testing.
 Molecular biology

 Molecular biology is the study of biology at a molecular level.

 The field overlaps with other areas of biology and chemistry, particularly
genetics and biochemistry.
 Molecular biology chiefly concerns itself with understanding the
interactions between the various systems of a cell, including the
interactions between DNA, RNA and protein biosynthesis as well as
learning how these interactions are regulated.
 Molecular biology also introduced new methods to pharmacology, such
as the polymerase chain reaction (PCR), Northern, Western and Southern
blotting.
 Very recently, microarray technology , Allele Specific Oligonucleotide
and mass spectroscopy were added as novel in vitro methods.
In Vitro Techniques of molecular biology
Expression cloning
Polymerase chain reaction (PCR)
Gel electrophoresis
Macromolecule blotting and probing
Southern blotting
Northern blotting
Western blotting
Eastern blotting
Arrays
Antiquated technologies
 Expression cloning

 One of the most basic techniques of molecular biology to study protein

function is expression cloning.
 In this technique, DNA coding for a protein of interest is cloned (using PCR
and/or restriction enzymes) into a plasmid (known as an expression vector).
 This plasmid may have special promoter elements to drive production of the
protein of interest, and may also have antibiotic resistance markers to help
follow the plasmid.
 This plasmid can be inserted into either bacterial or animal cells.
 Introducing DNA into bacterial cells can be done by transformation (via
uptake of naked DNA), conjugation (via cell-cell contact) or by transduction
(via viral vector). Introducing DNA into eukaryotic cells, such as animal cells,
by physical or chemical means is called transfection.
 Several different transfection techniques are available, such as calcium
phosphate transfection,electroporation, microinjection and liposome
transfection.
 The plasmid may be integrated into the genome, resulting in a stable
transfection, or may remain independent of the genome, called transient
transfection.
 Polymerase chain reaction (PCR)

The polymerase chain reaction is an extremely versatile

technique for copying DNA.
In brief, PCR allows a single DNA sequence to be copied
(millions of times), or altered in predetermined ways. For
example, PCR can be used to introduce restriction enzyme
sites, or to mutate (change) particular bases of DNA, the
latter is a method referred to as "Quick change".
 PCR can also be used to determine whether a particular
DNA fragment is found in a cDNA library. PCR has many
variations, like reverse transcription PCR (RT-PCR) for
amplification of RNA, and, more recently, real-time PCR (
QPCR) which allow for quantitative measurement of DNA
or RNA molecules.
 Gel electrophoresis
Gel electrophoresis is one of the principal tools of
molecular biology.
The basic principle is that DNA, RNA, and proteins
can all be separated by means of an electric field.
 In agarose gel electrophoresis, DNA and RNA can
be separated on the basis of size by running the
DNA through an agarose gel.
 Proteins can be separated on the basis of size by
using an SDS-PAGE gel, or on the basis of size and
their electric charge by using what is known as a
2D gel electrophoresis.
 Macromolecule blotting and probing
The terms northern, western and eastern blotting are
derived from what initially was a molecular biology joke
that played on the term Southern blotting, after the
technique described by Edwin Southern for the
hybridization of blotted DNA.
Patricia Thomas, developer of the RNA blot which then
became known as the northern blot actually didn't use the
term. Further combinations of these techniques produced
such terms as southwesterns (protein-DNA hybridizations),
northwesterns (to detect protein-RNA interactions) and
farwesterns (protein-protein interactions), all of which are
presently found in the literature.
 Southern blotting
Named after its inventor, biologist Edwin Southern, the Southern blot is a method
for probing for the presence of a specific DNA sequence within a DNA sample.
 DNA samples before or after restriction enzyme digestion are separated by gel
electrophoresis and then transferred to a membrane by blotting via capillary action.
The membrane is then exposed to a labeled DNA probe that has a complement
base sequence to the sequence on the DNA of interest.
 Most original protocols used radioactive labels, however non-radioactive
alternatives are now available.
Southern blotting is less commonly used in laboratory science due to the capacity
of other techniques, such as PCR, to detect specific DNA sequences from DNA
samples.
These blots are still used for some applications, however, such as measuring
transgene copy number in transgenic mice, or in the engineering of gene knockout
embryonic stem cell lines.
 Northern blotting

The northern blot is used to study the expression patterns of a specific type
of RNA molecule as relative comparison among a set of different samples
of RNA.
It is essentially a combination of denaturing RNA gel electrophoresis, and a
blot.
In this process RNA is separated based on size and is then transferred to a
membrane that is then probed with a labeled complement of a sequence of
interest.
The results may be visualized through a variety of ways depending on the
label used; however, most result in the revelation of bands representing the
sizes of the RNA detected in sample.
The intensity of these bands is related to the amount of the target RNA in
the samples analyzed.
The procedure is commonly used to study when and how much gene
expression is occurring by measuring how much of that RNA is present in
different samples.
It is one of the most basic tools for determining at what time, and under
what conditions, certain genes are expressed in living tissues.
 Western blotting

 Antibodies to most proteins can be created by injecting small amounts of

the protein into an animal such as a mouse, rabbit, sheep, or donkey (
polyclonal antibodies)or produced in cell culture (monoclonal antibodies).
 These antibodies can be used for a variety of analytical and preparative
techniques.
 In western blotting, proteins are first separated by size, in a thin gel
sandwiched between two glass plates in a technique known as SDS-PAGE (
sodium dodecyl sulfate polyacrylamide gel electrophoresis).
 The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon
or other support membrane.
 This membrane can then be probed with solutions of antibodies.
 Antibodies that specifically bind to the protein of interest can then be
visualized by a variety of techniques, including colored products,
chemiluminescence, or autoradiography.
 Often, the antibodies are labeled with an enzymes. When a
chemiluminescent substrate is exposed to the enzyme it allows detection.
Using western blotting techniques allows not only detection but also
quantitative analysis.
 Eastern blotting

Eastern blotting technique is to detect

post-translational modification of
proteins. Proteins blotted on to the PVDF
or nitrocellulose membrane are probed for
modifications using specific substrates.
 Arrays
A DNA array is a collection of spots attached to a solid support such as a
microscope slide where each spot contains one or more single-stranded DNA
oligonucleotide fragment.
Arrays make it possible to put down a large quantity of very small (100 micrometre
diameter) spots on a single slide.
Each spot has a DNA fragment molecule that is complementary to a single DNA
sequence (similar to Southern blotting).
In this technique the RNA in a tissue is isolated and converted to labeled cDNA.
 This cDNA is then hybridized to the fragments on the array and visualization of the
hybridization can be done.
Since multiple arrays can be made with the exact same position of fragments they are
particularly useful for comparing the gene expression of two different tissues, such as
a healthy and cancerous tissue.
There are many different ways to fabricate microarrays; the most common are silicon
chips, microscope slides with spots of ~ 100 micrometre diameter, custom arrays, and
arrays with larger spots on porous membranes (macroarrays).
There can be anywhere from 100 spots to more than 10,000 on a given array.
Arrays can also be made with molecules other than DNA.
For example, an antibody array can be used to determine what proteins or bacteria are
present in a blood sample.
 Antiquated technologies
In molecular biology, procedures and
technologies are continually being developed
and older technologies abandoned.
For example, before the advent of DNA gel
electrophoresis (agarose or polyacrylamide),
the size of DNA molecules was typically
determined by rate sedimentation in sucrose
gradients, a slow and labor-intensive technique
requiring expensive instrumentation; prior to
sucrose gradients, viscometry was used.
 REFERENCES:

Drug Discovery and Evaluation by H. Gerhard

Vogel , pg 4.
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