StemCell Therapy

In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g.- plasmid, cosmid, Lambda phages). A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids.[1] Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. All vectors may be used for cloning and are therefore cloning vectors, but there are also vectors designed specially for cloning, while others may be designed specifically for other purposes, such as transcription and protein expression. Vectors designed specifically for the expression of the transgene in the target cell are called expression vectors, and generally have a promoter sequence that drives expression of the transgene. Simpler vectors called transcription vectors are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed, unlike expression vectors. Transcription vectors are used to amplify their insert.

The manipulation of DNA is normally conducted on E. coli vectors, which contain elements necessary for their maintenance in E. coli. However, vectors may also have elements that allow them to be maintained in another organism such as yeast, plant or mammalian cells, and these vectors are called shuttle vectors. Such vectors have bacterial or viral elements which may be transferred to the non-bacterial host organism, however other vectors termed intragenic vectors have also been developed to avoid the transfer of any genetic material from an alien species.[2]

Insertion of a vector into the target cell is usually called transformation for bacterial cells,[3] transfection for eukaryotic cells,[4] although insertion of a viral vector is often called transduction.[5]

Plasmids are double-stranded extra chromosomal and generally circular DNA sequences that are capable of replication using the host cell's replication machinery.[6] Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host. Plasmids are found widely in many bacteria, for example in Escherichia coli, but may also be found in a few eukaryotes, for example in yeast such as Saccharomyces cerevisiae.[7] Bacterial plasmids may be conjugative/transmissible and non-conjugative:

Plasmids with specially-constructed features are commonly used in laboratory for cloning purposes. These plasmid are generally non-conjugative but may have many more features, notably a "multiple cloning site" where multiple restriction enzyme cleavage sites allow for the insertion of a transgene insert. The bacteria containing the plasmids can generate millions of copies of the vector within the bacteria in hours, and the amplified vectors can be extracted from the bacteria for further manipulation. Plasmids may be used specifically as transcription vectors and such plasmids may lack crucial sequences for protein expression. Plasmids used for protein expression, called expression vectors, would include elements for translation of protein, such as a ribosome binding site, start and stop codons.

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. For example, retroviruses leave a characteristic retroviral integration pattern after insertion that is detectable and indicates that the viral vector has incorporated into the host genome.

Artificial chromosomes are manufactured chromosomes in the context of yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or human artificial chromosomes (HACs). An artificial chromosome can carry a much larger DNA fragment than other vectors.[8] YACs and BACs can carry a DNA fragment up to 300,000 nucleotides long. Three structural necessities of an artificial chromosome include an origin of replication, a centromere, and telomeric end sequences.[9]

Transcription of the cloned gene is a necessary component of the vector when expression of the gene is required: one gene may be amplified through transcription to generate multiple copies of mRNAs, the template on which protein may be produced through translation.[10] A larger number of mRNAs would express a greater amount of protein, and how many copies of mRNA are generated depends on the promoter used in the vector.[11] The expression may be constitutive, meaning that the protein is produced constantly in the background, or it may be inducible whereby the protein is expressed only under certain condition, for example when a chemical inducer is added. These two different types of expression depend on the types of promoter and operator used.

Viral promoters are often used for constitutive expression in plasmids and in viral vectors because they normally force constant transcription in many cell lines and types reliably.[12] 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.

Some vectors are designed for transcription only, for example for in vitro mRNA production. These vectors are called transcription vectors. They may lack the sequences necessary for polyadenylation and termination, therefore may not be used for protein production.

Expression vectors produce proteins through the transcription of the vector's insert followed by translation of the mRNA produced, they therefore require more components than the simpler transcription-only vectors. Expression in different host organism would require different elements, although they share similar requirements, for example a promoter for initiation of transcription, a ribosomal binding site for translation initiation, and termination signals.

Eukaryote expression vectors require sequences that encode for:

Modern artificially-constructed vectors contain essential components found in all vectors, and may contain other additional features found only in some vectors:

See original here:
Vector (molecular biology) - Wikipedia

This entry was posted on October 13, 2018 at 1:41 am and is filed under Molecular Genetics. You can follow any responses to this entry through the RSS 2.0 feed. Both comments and pings are currently closed. |