Vector (molecular biology)

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]


Overview

In molecular biology, a vector is any vehicle used to transfer foreign genetic material into another cell.

The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves of the "backbone" of the vector. The purpose of a vector to transfer genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell. Vectors called expression vectors (expression constructs) specifically are for the expression of the transgene in the target cell, 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.

Insertion of a vector into the target cell is generally called transfection, although insertion of a viral vector is often called transduction.

Characteristics

Two common vectors are plasmids and viral vectors.

Plasmids

Plasmid Vectors

Plasmids are double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host and also the transgene insert. 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 restricted 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 expression of transcription vectors impossible.

Viral vectors

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. 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.

Transcription

Transcription is a necessary component in all vectors: the premise of a vector is to multiply the insert (although expression vectors later also drive the translation of the multiplied insert). Thus, even stable expression is determined by stable transcription, which generally depends on promoters in the vector. However, expression vectors have a variety of expression patterns: constitutive (consistent expression) or inducible (expression only under certain conditions or chemicals). This expression is based on different promoter activities, not post-transcriptional activities. Thus, these two different types of expression vectors depend on different types of promoters.

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 Drosphilia heat shock promoter only iniates after high temperatures.

Expression

Expression vectors require not only transcription but translation of the vector's insert, thus requiring more components than simpler transcription-only vectors. Expression vectors require sequences that encode for:

  • Polyadenylation tail: Creates a polyadenylation tail at the end of the transcribed pre-mRNA 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.

Features

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.
  • 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 which cells have uptaken the vector through antibiotic selection.
  • 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.
  • β-galactosidase: Vector's multiple cloning site contains sequence for β-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 β-galactosidase and digest galactose. However, cells that express a vector with a transgene will have the coding sequence for β-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.
  • 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.

See also

References

  • Freshney, Ian R. Culture of Animal Cells: A manual of basic technique. John Wiley & Sons, Inc., Hoboken, New Jersey. ISBN 13 978-0-471-45329-1

External links

it:Vettore (biotecnologie)



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