Cloning Vectors

Cloning vector A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. Types There are many types of cloning vectors. Genetically engineered plasmids and bacteriophages (like phage ) are most common. Other types include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).
The pGEX-3x plasmid vector.

Expression vectors (or constructs) are used for the expression of the transgene in the target cell, and generally have a promoter sequence that drives expression of the transgene. The main purpose of expression vectors is controlled expression of a particular gene inside a convenient host (e.g. E. coli). Transcription vectors are simpler only capable of being replicated and transcribed but not translated, and so, are used to amplify their insert. Insertion of a vector into the target cell is generally called transfection, although insertion of a viral vector is often called transduction. Common features For convenient and favorable insertions, expression vectors have had nearly all their normal restriction sites removed and a synthetic multiple cloning site (MCS) inserted that contains many restriction sites. MCS allows for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientation. A selectable marker for an antibiotic resistance (e.g. -lactamase) is often carried by the vector to allow the selection of positively transformed cells. All plasmids must carry a functional origin of replication (ORI; not shown in figure). Some other possible features present in cloning vectors are: vir genes for plant transformation, integrase sites for chromosomal insertion, lac Z fragment for complementation and blue-white selection, and/or reporter genes in frame with and flanking the MCS to facilitate the production of recombinant proteins [e.g. fused to the Green fluorescent protein (GFP) or to the glutathione S-transferase. Modern vectors may encompass additional features besides the transgene insert and a backbone: Promoter: Necessary component for all vectors: used to drive transcription of the vector's transgene. Some commonly used promoters are T7 promoters, lac promoters (bla promoter) and cauliflower mosaic virus 35s promoter (for plant vectors). Genetic markers: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA. Antibiotic resistance: Vectors with antibiotic-resistance open reading frames allow for identification of the cells that have taken up the vector through antibiotic selection.
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Cloning Vectors
Epitope: Vector contains a sequence for a specific epitope that is incorporated into the expressed protein. Allows for antibody identification of cells expressing the vector.
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-galactosidase: Vector's MCS contains sequence for -galactosidase, an enzyme that digests galactose, to either side of the region intended for an insert. If the insert has not successfully ligated into the vector, cells expressing the empty vector will generate -galactosidase and digest galactose. However, cells that express a vector with a transgene will NOT have the coding sequence for galactosidase and be unable to digest galactose, and a subsequent color dye for galactose (X-gal) subsequently identifies cells expressing a vector with an insert, although it is unknown whether the insert is the intended one.
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Targeting sequence: Expression vectors may include encoding for a targeting sequence in the finished protein that directs the expressed protein to a specific organelle in the cell. Screening : Blue/White selection General purpose vectors like pUC19 usually include a system for detecting the presence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used is the lac Z gene coding for E. coli -galactosidase, whose integrity can easily be detected by the ability of the enzyme it encodes to hydrolyze the soluble, colourless substrate X-gal (5bromo-4-chloro-3-indolyl--D-galactoside) into an insoluble, blue product (5,5'-dibromo-4,4'dichloro indigo). Cloning a fragment of DNA within the vector-based gene encoding the galactosidase prevents the production of an active enzyme. If X-gal is included in the selective agar plates, transformant colonies are generally blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA. Plasmids Plasmid vectors minimally consist of an origin of replication that allows for semi-independent replication of the plasmid and also the transgene insert in the host. Modern plasmids generally have many more features, notably including a "multiple cloning site" which includes nucleotide overhangs for insertion of an insert, and multiple restriction enzyme consensus sites to either side of the insert. In the case of plasmids utilized as transcription vectors, incubating bacteria with plasmids generates hundreds or thousands of copies of the vector within the bacteria in hours, and the vectors can be extracted from the bacteria, and the multiple cloning site can be cut by restriction enzymes to excise the hundredfold or thousandfold amplified insert. These plasmid transcription vectors characteristically lack crucial sequences that code for polyadenylation sequences and translation termination sequences in translated mRNAs, making protein expression from transcription vectors impossible. Viral vectors Viral vectors are generally genetically-engineered viruses carrying modified viral DNA or RNA that has been rendered noninfectious, but still contain viral promoters and also the transgene, thus allowing for translation of the transgene through a viral promoter. However, because viral vectors frequently are lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Viral vectors are often designed for permanent incorporation of the insert into the host genome, and thus leave distinct genetic markers in the host genome after incorporating the transgene (e.g. retroviruses leave a

Cloning Vectors
characteristic retroviral integration pattern after insertion that is detectable and indicates that the viral vector has incorporated into the host genome). Transcription Expression vectors have a variety of expression patterns: constitutive (consistent) expression or inducible expression under specific conditions. This is based on different promoter activities, not post-transcriptional activities. Viral promoters are often used for constitutive expression in plasmids and in viral vectors because they normally reliably force constant transcription in many cell lines and types. Inducible expression depends on promoters that respond to the induction conditions: for example, the murine mammary tumor virus promoter only initiates transcription after dexamethasone application and the Drosophilia heat shock promoter only initiates after high temperatures. Expression vectors require not only transcription but translation of the vector's insert, thus requiring more components than simpler transcription-only vectors. Sequences Necessary for Expression vectors: Polyadenylation tail: Creates a polyA tail at the end of the transcribed premRNA that protects the mRNA from exonucleases and ensures transcriptional and translational termination: stabilizes mRNA production. Minimal UTR length: UTRs contain specific characteristics that may impede transcription or translation, and thus the shortest UTRs or none at all are encoded for in optimal expression vectors. Kozak sequence: Vectors should encode for a Kozak sequence in the mRNA, which assembles the ribosome for translation of the mRNA. Strong termination codon. Expression vectors must also have expression signals such as a strong promoter, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence). These conditions are necessary only for Expression vectors in eukaryotes, and not prokaryotes. The goal of a well-designed expression vector is the production of large amounts of stable messenger RNA. After expression of the gene product, purification of the protein is required; but since the vector is introduced to a host cell, the protein of interest should be purified from the host proteins. Therefore, to make the purification process easy, the cloned gene should have a tag. This tag could be histidine (His) tag or any other marker peptide. Expression vectors are used for molecular biology techniques such as site-directed mutagenesis. In general, DNA vectors that are used in gene-cloning experiments need not result in the expression of a protein. Expression vectors are basic tools for biotechnology and the production of proteins such as insulin that are important for medical treatments of specific diseases like diabetes. Viral vectors Viral vectors are commonly used to deliver genetic material into cells in vivo or in vitro. Delivery of genes by a virus is termed transduction and the infected cells are described as transduced.

Cloning Vectors
Key properties of a viral vector Viral vectors are tailored for specific applications but generally share a few key properties. are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells but, once the infection has taken place, requires a helper virus to provide the missing proteins for production of new virions. cell it infects.

Safety: Although viral vectors are occasionally created from pathogenic viruses, they

Low toxicity: The viral vector should have a minimal effect on the physiology of the Stability: Some viruses are genetically unstable and can rapidly rearrange their

genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design. of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell. certain marker genes which aid for the easy identification of which cells have up-taken the viral genes. A common marker is antibiotic resistance to a certain antibiotic. The cells can then be isolated easily as those which have not taken up the viral vector genes will not have antibiotic resistance and so will not be able to grow in a culture with antibiotics present.
MLV based vector system. pgk = murine internal promoter driving the expression of a selectable marker; neo = neomycin; pac = puromycin; hph = hygromycin.

Cell type specificity: Most viral vectors are engineered to infect as wide a range

Identification: Viral vectors are often given

Types of viral vectors Retrovirus Retroviruses are mostly used in current gene therapy approaches. Recombinant retroviruses like the Moloney murine leukemia virus (MLV) contain a reverse transcriptase which allows integration into the host genome stably. They have been used in a number of FDA-approved clinical trials such as the SCID-X1 trial. Retroviral vectors can either be replicationcompetent or replication-defective. Replicationdefective vectors are the most common choice because the viruses have had the coding regions for the genes necessary for additional rounds of virion replication and packaging deleted. These can infect their target cells and deliver their viral payload, but then fail to continue the typical lytic pathway, which would typically result in cell

Cloning Vectors
death. The typical maximum length of an allowable DNA insert in a replication-defective viral vector is usually about 8-10 kB, and most cDNA sequences can still be accommodated. Conversely, replication-competent viral vectors contain all the necessary genes for virion synthesis, and will continue to propagate themselves once infection occurs. Because the viral genome for these vectors is much lengthier, the length of the actual inserted gene of interest is limited compared to the replication-defective vectors. The primary drawback to use of retroviruses like MLV is that they are effective on actively dividing cells. So, non-dividing cells like neurons are very resistant to transduction by them. There is a concern for insertional mutagenesis due to the integration into the host genome which can lead to cancer or leukemia. In a retroviral gene therapy trial for severe combined immunodeficiency (SCID) conducted in 2002, four patients developed leukemia as a consequence of the treatment. Lentivirus
Lentiviral vector system. SD = splicing donor site; RRE = rev response element; [ga] = initial fragment of gag. The dashed line reported in the first packaging construct indicates the deletions been made in the HIV-1 genome.

Lentiviruses are a subclass of Retroviruses, and have recently been adapted as gene delivery vectors. Their unique feature is their ability to integrate into the genome of non-dividing cells. Upon entry into the host, the viral RNA is reverse-transcribed to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme. The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. Site of integration is unpredictable, which may disturb the function of cellular genes and lead to cancer. The provirus can disturb the function of cellular genes and lead to activation of oncogenes promoting the development of cancer, which raises concerns for their possible applications in gene therapy. However, lentiviral vectors show a lower tendency than the -retroviral vectors to integrate in places that potentially cause cancer. Lentiviral vectors never carry the genes required for their replication. To produce a lentivirus, several plasmids are transfected into a so-called packaging cell line, commonly HEK 293. One or more plasmids, generally called packaging plasmids, encode the virion proteins like capsid and reverse transcriptase. Another plasmid contains the transgene to be delivered. It is transcribed to produce the ssRNA viral genome and is marked by the presence of the (psi) sequence. This sequence is used to package the genome into the virion.

Cloning Vectors
Adenovirus As opposed to lentiviruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division. This limits their use in basic research, although adenoviral vectors are occasionally used in in vitro experiments. Their primary applications are in gene therapy and vaccination. Since humans commonly come in contact with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences. To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity. Adeno-associated virus Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy. Nanoengineered substance Nonviral substances such as Ormosil have been used as DNA vectors and can deliver DNA loads to specifically targeted cells in living animals. (Ormosil stands for organically modified silica or silicate.) Bacterial artificial chromosome A Bacterial Artificial Chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids contain partition genes that promote the even distribution of plasmids after bacterial cell division. The usual insert size is 150-350 kbp, but can be >700 kbp. A similar cloning vector called PAC has also been produced from the bacterial P1-plasmid. BACs are often used to genome sequencing. A short piece of the organism's DNA is amplified as an insert in BACs, sequenced, and finally, the sequenced parts are rearranged in silico. Common gene components

oriS, repE F for plasmid replication and regulation of copy number.

stable maintenance of the BAC.

parA and parB for partitioning F plasmid DNA to daughter cells during division and ensures

Selectable markers for antibiotic resistance, some BACs also have lacZ at the cloning site for blue/white selection.

T7 & Sp6 phage promoters for transcription of inserted genes.

Inherited disease BACs are now being utilized to a greater extent in modeling genetic diseases, often alongside transgenic mice. BACs have been useful in this field as complex genes may have several regulatory sequences upstream of the encoding sequence, including various promoter sequences that will govern a gene's expression level. BACs have been used to some degree of success with mice when studying neurological diseases such as Alzheimer's disease or as in the case of aneuploidy associated with Down syndrome. There have also been instances when they have been used to study specific oncogenes associated with cancers. They are transferred over to these genetic disease models by electroporation/transformation, transfection with a suitable virus or microinjection. BACs can also be utilized to detect genes or large sequences of interest and then used to map them onto the human chromosome using

Cloning Vectors
BAC arrays. BACs are preferred for these kind of genetic studies because they accommodate much larger sequences without the risk of rearrangement, and are therefore more stable than other types of cloning vectors.
Vectors Retrovirus Characteristics Relatively high titers (10 -10 cfu/ml). Broad cell tropism. Stable gene expression. No toxic effect on infected cells. Total insert capacity is in the range of 10 kb. Only infect dividing cells. Lentivirus Can infect nondividing cells. Can be pseudotyped with retroviral or VSV G envelopes, therefore, they also have broad cell tropism. Serum conversion to HIV-1. Possible proviral insertional mutagenesis in target cells. Presence of tat and rev regulatory proteins,(the early lentiviralvectors also have some HIV-1 accessory proteins).
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Disadvantages Random insertion of viral genome, may possibly result in mutagenesis. Possibility of replication competent virus formation by homologous recombination.

Stable gene expression. Total insert capacity is in the range of 10kb. Adenovirus Very high titers (1010 pfu/ml). Transiently high levels of gene expression. Can also infect non-dividing cells. Complicated vector genome. Adenoassociated virus Large DNA inserts can be accomodated (7-8 kb). Wide range of cells can be infected, including nondividing cells. Ability of the virus to establish latent infection by viral genome integration into cell genome. Viral integration specific for human chromosome 19 (only for wild-type AAV). Nonpathogenic, nontoxic. Small genome (5 kb). Cationic liposomes Not infectious. Theoretically, there is no limit to the size of DNA. Low degree of toxicity. Targeting is not specific. Low transfection efficiency. Only transient expression. Difficult in vivo applications. Not suitable for long-term expression due to lack of integration into host genome. High titers of pure virus are difficult to obtain. Still not well characterized. Limited capacity for foreign genes (about 4 kb). Lack of specific integration for recombinant AAV vectors. Requires a helper adeno- or herpes virus for replication. Host immune response.