In
molecular biologyMolecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, a
vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. Common to all engineered vectors are an
origin of replicationThe origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
, a
multicloning siteA multiple cloning site , also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during...
, and a
selectable markerA selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a...
.
The vector itself is generally a
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information...
sequence that consists of an
insertIn Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism....
(
transgeneA transgene is a gene or genetic material that has been transferred naturally or by any of a number of genetic engineering techniques from one organism to another....
) and a larger sequence that serves as the "backbone" of the vector.
In
molecular biologyMolecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
, a
vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes. Common to all engineered vectors are an
origin of replicationThe origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
, a
multicloning siteA multiple cloning site , also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during...
, and a
selectable markerA selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a...
.
The vector itself is generally a
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information...
sequence that consists of an
insertIn Molecular biology, an insert is a piece of DNA that is inserted into a larger DNA vector by a recombinant DNA technique, such as ligation or recombination. This allows it to be multiplied, selected, further manipulated or expressed in a host organism....
(
transgeneA transgene is a gene or genetic material that has been transferred naturally or by any of a number of genetic engineering techniques from one organism to another....
) 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. Vectors called
expression vectorAn expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
s (expression constructs) specifically are for the expression of the transgene in the target cell, and generally have a
promoterIn genetics, a promoter is a region of DNA that facilitates the transcription of a particular gene. Promoters are typically located near the genes they regulate, on the same strand and upstream .-Overview:...
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
transfectionTransfection is the process of introducing nucleic acids into cells by non-viral methods. The term "transformation" is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells; "transduction" is often used to describe virus-mediated DNA transfer.Transfection of...
, although insertion of a viral vector is often called
transductionTransduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector...
.
Plasmids
PlasmidA plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded...
s 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 replicationThe origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
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 enzymeA restriction enzyme is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
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
polyadenylationPolyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA which only has As. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
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 markerA genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci, that can be observed...
s in the host genome after incorporating the transgene. For example,
retrovirusA retrovirus is an RNA virus that is replicated in a host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA...
es 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
dexamethasoneDexamethasone is a potent synthetic member of the glucocorticoid class of steroid hormones. It acts as an anti-inflammatory and immunosuppressant...
application and the
Drosophilia heat shock promoter only initiates 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
Polyadenylation is the addition of a poly tail to an RNA molecule. The poly tail consists of multiple adenosine monophosphates; in other words, it is a stretch of RNA which only has As. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA for translation...
: Creates a polyadenylation tail at the end of the transcribed pre-mRNA that protects the mRNA from exonucleaseExonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction occurs that breaks phosphodiester bonds at either the 3’ or 5’ ends. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle of a...
s 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
Ribosomes are complexes of RNA and protein that are found in all cells. The ribosome is part of the mechanism that translates the DNA sequence into the protein sequence. Ribosomes from bacteria, archaea and eukaryotes , have significantly different structure and RNA...
for translation of the mRNA.
Features
Modern vectors may encompass additional features besides the transgene insert and a backbone:
- Promoter
In genetics, a promoter is a region of DNA that facilitates the transcription of a particular gene. Promoters are typically located near the genes they regulate, on the same strand and upstream .-Overview:...
: Necessary component for all vectors: used to drive transcription of the vector's transgene.
- Genetic marker
A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci, that can be observed...
s: Genetic markers for viral vectors allow for confirmation that the vector has integrated with the host genomic DNA.
- Antibiotic
In common usage, an antibiotic is a substance or compound that kills or inhibits the growth of bacteria...
resistance: Vectors with antibiotic-resistance open reading frameIn molecular genetics, an open reading frame is a portion of an organism's genome which contains a sequence of bases that could potentially encode a protein. The start-points and end-points of a given ORF are not equivalent to the ends of the messenger RNA , but the ends of the ORF are usually...
s allow for identification of which cells have uptaken the vector through antibiotic selection.
- Epitope
An epitope, also known as antigenic determinant, is the part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope...
: 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
Galactose is a type of sugar which is less sweet than glucose. It is considered a nutritive sweetener because it has food energy. Its name comes from the Ancient Greek word for milk, γάλακτος ....
, 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 NOT have the coding sequence for β-galactosidase and be unable to digest galactose, and a subsequent color dyeThe blue-white screen is a molecular technique that allows for the detection of successful ligations in vector-based gene cloning. DNA of interest is ligated into a vector. The vector is then transformed into competent cell . The competent cells are grown in the presence of X-gal...
for galactose (X-galX-gal is an organic compound consisting of galactoside linked to indole.-Cloning:...
) 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
- Plasmid
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded...
- Viral vector
Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture . Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect...
- 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. There are many types...
- Expression vector
An expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
- Recombinant DNA
Recombinant DNA is a form of DNA that does not exist naturally, which is created by combining DNA sequences that would not normally occur together...
- Naked DNA
Naked DNA is histone-free DNA that is passed from cell to cell during a gene transfer process called transformation or transfection. In transformation , purified or naked DNA is taken up by the recipient cell which will give the recipient cell a new characteristic or phenotype...
- Vector (epidemiology), an organism that transmits disease
External links