Vectors

Vectors can be defined in three ways:
In epidemiology:
An organism or vehicle that transmits the causative agent or disease-
causing organism from the reservoir to the host.
In molecular biology:
A vehicle (e.g. a plasmid) used to transfer the genetic material such as DNA
sequences from the donor organism to the target cell of the recipient
organism.
In biology:
A biotic agent that disperses reproductive structures of another organism,
as a bee transmitting pollen to the stigma of a flower.
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Origin of
replication
Origin of
replication
Promoter
Cloning
site
Protein
purification
tags
Reporter
genes:
Reporter
genes:
Targeting
sequence
Antibiotic
resistance
Epitope
Genetic
markers

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Origin of
replication
Promoter
Cloning
site
for replication and maintenance of vector
in host cell.
•to drive transcription of vector's transgene
•Also to drive transcription of other genes in vector
such as the antibiotic resistance gene
•allow for the insertion of foreign DNA into the
vector through ligation

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Protein
purification
tags
Some expression vectors include proteins or peptide
sequences that allows for easier purification of
expressed protein.
Reporter
genes:
Targeting
sequence
that allow for identification of plasmid that
contains inserted DNA sequence.
that directs the expressed protein to a specific
organelle in the cell or specific location

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Antibiotic
resistance
EpitopeEpitope
allow for survival of cells that have taken up
the vector in growth media containing
antibiotics through antibiotic selection.
allows for antibody identification of cells
expressing the target protein.
Genetic
markers
Genetic
markers
allow for confirmation that the vector has
integrated with the host genomic DNA.

Vectors perform their functions in two ways mostly:
Transcription:
Expression:
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2)Expression:
• there are two types of expression vector:
Prokaryotes expression vector:
Eukaryotes expression vector:
Prokaryotes expression vector:
• Promoter
• Ribosome Binding Site (RBS)
• Translation initiation site -
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2)Expression:
Eukaryotes expression vectors:
These require sequences that encode for:
Polyadenylation tail:
Minimal UTR length:
Kozak sequence:

In molecular biology, a vector may refer to
oBinary vector
oCloning vector
oShuttle vector
oExpression vector
o Plasmid vector
oViral vector
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Plasmid:
Plasmid can be defined as:
An extra chromosomal self-replicating structure found in
bacterial cells that carries genes for a variety of functions not
essential for cell growth.
OR
A circular, double-stranded unit of DNA that replicates within a
cell independently of the chromosomal DNA is called plasmid.
Plasmids are most often found in bacteria and are used in
recombinant DNA research to transfer genes between cells.

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What are the main parts of a
Plasmid?
DNA sequence which allows
initiation of replication within a
plasmid by recruiting
transcriptional machinery
proteins
Allows for selection of
plasmid-containing
bacteria.
Allows for selection of
plasmid-containing
bacteria.
Short segment of DNA which
contains several restriction sites
allowing for the easy insertion of
DNA. In expression plasmids, the
MCS is often downstream from a
promoter.
Short segment of DNA which
contains several restriction sites
allowing for the easy insertion of
DNA. In expression plasmids, the
MCS is often downstream from a
promoter.
 Drives transcription of
target gene.
 Vital component for
expression vectors:
 determines which cell types
the gene is expressed in and
amount of recombinant
protein obtained.
 Drives transcription of
target gene.
 Vital component for
expression vectors:
 determines which cell types
the gene is expressed in and
amount of recombinant
protein obtained.
Antibiotic resistance gene
allows for selection in
bacteria.
However, many plasmids
also have selectable
markers for use in other
cell types.
Gene, promoter or other DNA
fragment cloned into the MCS
for further study.
Gene, promoter or other DNA
fragment cloned into the MCS
for further study.
A short single-stranded DNA
sequence used as an initiation point
for PCR amplification or sequencing.
Primers can be exploited for
sequence verification of
plasmids.
A short single-stranded DNA
sequence used as an initiation point
for PCR amplification or sequencing.
Primers can be exploited for
sequence verification of
plasmids.

What is mode of action of a
plasmid vector?
 Plasmids are pieces of DNA that can be
manipulated through the use of restriction
enzymes. Since these plasmids are already in
the bacteria, the restriction enzymes are able to
act as "molecular scissors" and cut up the part
of the plasmid that is desired to be changed.
 Then, the gene of interest is inserted as a new
part of the plasmid, allowing the bacteria to
now demonstrate this gene.
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What is mode of action of a
plasmid vector?
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 sites can be cut by restriction
enzymes to excise the hundredfold or thousand
fold 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.

What are the types of plasmid
vectors?
Plasmid vectors may be:
Conjugative/transmissible vectors:
They mediate DNA transfer through conjugation and therefore spread rapidly
among the bacterial cells of a population; e.g., F plasmid, many R and some col
plasmids.
Non-conjugative vectors:
They do not mediate DNA through conjugation, e.g., many R and col plasmids.
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1)Ti plasmid:
A Ti or tumor inducing plasmid is a circular plasmid is often, a part of the genetic
equipment of Agrobacterium tumefaciens and Agrobacterium rhizogenes.
Use to transduce its genetic material to plants.
•Classification of Ti plasmid:
•Based on the type of opine produced by their genes. The different opines
specified by pTi are octopine, nopaline, succinamopine and leucinopine.
•The plasmid has 196 genes that code for 195 proteins. There are no one
structural RNA. The plasmid is 206,479 nucleotides long; the GC content is 56%
and 81% of the material is coding genes. There are no pseudo genes.
• Important in the creation of transgenic plants.06/27/15 17
vectors?

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vectors?
2)Col plasmid:
Any plasmid that carries genetic information for the production of a colicin;
Involved in killing the other harmful strains of bacteria also called bacteriocinogenic
plasmid.
They produce proteins that are sensitive to bacteria and inhibit its essential processes
producing a clear area called lacuna.
3)Virulence Plasmid:
Virulence plasmid turns the bacterium into a pathogen.
So they are responsible for carrying the genes which cause diseases.

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vectors?4)F Plasmid:
 The Fertility factor allows genes to be transferred from one bacterium to another by
conjugation.
F plasmid a conjugative plasmid found in F+
(male) bacterial cells that leads with high
frequency to its transfer.
A cell possessing the F plasmid (F+
, male) can form a conjugation bridge (F pilus) to a cell
lacking the F plasmid (F−
, female), through which genetic material may pass from one cell to
another.

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5)R (Resistance) Plasmid:
vectors?
• R plasmid is a conjugative factor in bacterial cells.
Promotes resistance to agents such as antibiotics, metal ions, ultraviolet radiation,
and bacteriophage.
• Many R-factors can pass from one bacterium to another and through which antibiotic
resistance spreads between bacterial species, genera and even families.
For example:
RP1, a plasmid that encodes resistance to ampicillin, tetracycline and kanamycin
originated in a species of Pseudomonas, but also in bacteria such as Escherichia coli.

6)Degradative Plasmid:
These plasmid are types of plasmids present in certain
bacteria’s such as Pseudomonas putida which impart the ability
of degrade xenobiotic compounds.
For example: salicylic acid, 2-4D etc.
There are 3 such plasmids:
• CAMplasmid- which degrades camphor.
• XYL, - , xylene.
• NAH, - , naphthalene.
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vectors?

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vectors?
7)Yeast plasmids:
Yeast naturally harbors plasmids.
These are 2µm plasmid - small circular plasmid often used for genetic engineering of
yeast and linear pGKL plasmids from kluyveromyces lactis that are responsible for killer
phenotype. Yeast cloning vectors include:

What is importance of Plasmids?
Easy to work with - Plasmids are a convenient size (generally 1,000-20,000 base pairs).
With current cloning technology, it is easy to create and modify plasmids containing the
genetic element that you are interested in.
Self-replicating - Once you have constructed a plasmid, you can make an endless
number of copies of the plasmid by growing the plasmid in bacteria.
Stable - Plasmids are stable long-term either as purified DNA or within bacteria (as
glycerol stocks).
Functional in many species and can useful for a diverse set of applications -
Plasmids can drive gene expression in a wide variety of organisms, including plants, worms,
mice and even cultured human cells.
used to understand gene function,
they can also be used to investigate promoters, small RNAs, or other genetic elements.
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A viral vector is a virus which has been modified in a laboratory environment for
purpose of introducing genetic material into a cell.
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What are viral vectors?What are viral vectors?
 Viruses are highly evolved natural vectors for the transfer of foreign genetic
information into cells. But to improve safety, they need to be replication
defective.
 So the viruses can be used as vehicles to carry 'good' genes into a human cell.
 To form a viral vector, remove the genes in the virus that cause disease. Then
replace those genes with genes encoding the desired effect (for instance,
insulin production in the case of diabetics). This procedure must be done in
such a way that the genes which allow the virus to insert its genome into its
host's genome are left intact.

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What are viral vectors?What are viral vectors?
 Development of the viral vector dates to 1960s, when a number of researchers
recognized that since viruses operate by inserting genetic material into cells,
surely researchers could harness this trait by modifying viruses to change the
genetic material they are inserting.
 The process of delivering genetic material with the use of a virus is known as
transduction, with the first successful attempt occurring in 1968 with plant
cells.

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What are Key properties for
a viral vector?
Safety
Low toxicity
Stability
Cell type
specificity
Identification

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a viral vector?
Safety
 Viral vector should be modified in such a way
as to minimize the risk of handling them.
 Usually involves the deletion of a part of the
viral genome critical for viral replication
Low toxicity
viral vector should have a minimal effect
on the physiology of the cell it infects.
Stability
 Some viruses are genetically unstable & can
rapidly rearrange their genomes.
 This is detrimental to predictability and
reproducibility of work conducted using a viral
vector and is avoided in their design.

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a viral vector?
Cell type
specificity
 Most viral vectors are engineered to infect as
wide a range of cell types as possible.
 viral receptor can be modified to target the
virus to a specific kind of cell. Viruses modified
in this manner are said to be pseudo typed.
Identification
Viral vectors are often given certain genes
that help identify which cells took up the viral
genes. These genes are called Markers

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What are main types of viral vectors?
Retrovirus
Adenovirus
Lentivirus
Adeno-Associated virus (AAV)
Herpes virus

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 In Gene therapy
 In vaccines production:
Main applications of viral vectors?

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 In Gene therapy:
 Gene therapy is a technique for
correcting defective genes responsible
for disease development.
 There are following delivery system for
gene therapy:
• Physical methods
• Non-viral vectors
• Viral vectors
 Virus is usually “crippled” to disable its
ability to cause disease and viral
methods have proved to be the most
efficient to date

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 In Gene therapy:
 If the pathogenicity of a
specific virus, such as
adenovirus, can be
eliminated while the
efficiency of gene
transfer and expression
is retained, the gene
may be well suited for
gene therapy.

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 Viruses expressing pathogen proteins are currently being developed as vaccines
against these pathogens, based on the same rationale as DNA vaccines.
 T-lymphocytes recognize cells infected with intracellular parasites based on the
foreign proteins produced within the cell. T cell immunity is crucial for protection
against viral infections and such diseases as malaria.
 A viral vaccine induces expression of pathogen proteins within host cells. Since viral
vaccines contain only a small fraction of pathogen genes, they are much safer and
sporadic infection by the pathogen is impossible.
 Adenoviruses are being actively developed as vaccines.

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This vaccine approach involves:
 inserting influenza virus genes into a
different carrier virus, or vector, that is used
as a vaccine.
DNA or RNA-encoding influenza proteins,
such as hem agglutinin, are engineered into
a vector that infects humans but does not
cause disease.
With a microbial vector vaccine, the vector
itself, including the influenza genetic
material, is injected directly into a person.
The harmless vector virus can then express
the proteins necessary to prompt an
immune response.

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Retroviral Vectors:
 Retrovirus Structure:
 Outer coat:
 Inner core:

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Lentivirus distinguishing features
Presence of:
Accessory genes ( regulation of transcription,
RNA transport)
Transduction in both dividing and non dividing
cells
Helper plasmids

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Retroviral mediated gene transfer:
Transfection of packaging cell line:
 Transfection of packaging cell line
 Virus Collection
 Transduction in target cells:
 The production of replication defective virus is accomplished when packaging cells are
transfected with plasmids that express all of the viral proteins necessary to generate
infectious particles,
 as well as the nucleic acid sequence of interest that will be packaged within them for
delivery.
 retroviral vectors produce replication defective, or self-inactivating, particles.
 This allows for delivery of the desired sequence, without continued viral replication in the
target cells.

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 The packaging cells produce infectious particles, whose genome only encodes
sequences from the transfer plasmid, which can be used to transduce the target
cells.
 The highest concentration of virus is typically produced between 48-72 hours.
 To collect the virus, remove the supernatant from the packaging cell line.
 The purified virus containing the desired gene can be stored at -80 °C until needed.
2-Virus Collection

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2-Virus Collection

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3-Transduction in target cells
 Viral Envelope glycoprotein protein interacts with the cellular receptor and enters
the actively dividing cells.
 The viral dsDNA of retroviruses is not capable of passing through the nuclear pore
complex and requires breakdown of the nuclear membrane during mitosis for
integration of nucleic acid.
 The viral genome enters the nucleus and desired gene sequence is integrated into
the host genome through the activity of restriction enzymes and Integrase while the
other portion of viral genome is self inactivated and replication defective.
 Some transfer vectors also contain a marker such as a fluorescent reporter or drug
selection marker for the selection of transduced cells.

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3-Transduction in target cells

Comparison between bacterial &
viral mediated gene transfer:
Viral mediated
 DNA insert length is upto
30kbp
 More transfection
effeciency
 Long term persistence and
stable gene transfer
Bacterial mediated
 DNA insert length is upto 10
kbp
 comparitively less
transfection effeciency
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