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Molecular Biology Sabah Linjawi ١
Lecture 13
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Molecular Biology Sabah Linjawi ٢
Eukaryotic vectors
Most cloning experiments are carried out with E. coli as the host
The widest variety of cloning vectors are available for this organism
E. coli is particularly popular when the aim of the cloning experiment is to study gene structure and function.
In biotechnology It may use a different host for cloning experiment.
where the aim to use cloning to control or improve synthesis of an important metabolic product (e.g. a hormone such as
insulin), or to change the properties of the organism
We must therefore consider cloning vectors for organisms other than E. coli.
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The pGEX-3x plasmid is a popular
cloning vector
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Molecular Biology Sabah Linjawi ٤
Transfection of eukaryotic cells
Transfection is the process of introducing nucleic acids into cells by non-viral methods
Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material.
Transfection can be carried out using calcium phosphate, by electroporation, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and
deposit their cargo inside.
Transfection of DNA into eukaryotic cells may require the digestion of the cell wall (yeast and plants)
Or the precipitation of the DNA on the cells (animal cells)
Take-up of DNA may also be promoted by electroporation
Injection by needle and bombardment with solid particles have also been used
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Molecular Biology Sabah Linjawi ٥
Shuttle vectors
A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species
Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types.
The main advantage of these vectors is they can be manipulated in E. coli and then used in a system which is more difficult or
slower to use (e.g. yeast, other bacteria).
Shuttle vectors include plasmids that can propagate in
eukaryotes and prokaryotes or in different species of bacteria
Shuttle vectors are frequently used to quickly make multiple copies of the gene in E. coli (amplification).
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Molecular Biology Sabah Linjawi ٦
Illustration of a bacterium with plasmid enclosed chromosomal DNA
and plasmids
Chromosomal DNA Plasmids
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Overview of Bacterial conjugation
There are two types of plasmid
integration into a host bacteria: Non- integrating plasmids replicate as with the top instance;
whereas episomes,
the lower example,
integrate into the
host chromosome.
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Molecular Biology Sabah Linjawi ٨
Gene therapy
The success of gene therapy depends on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome Without causing cell injury, oncogenic mutations (cancer) or an immune response.
Plasmid vectors could be used for this purpose.
Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double strand break to the DNA genome and cause homologous recombination.
This makes targeted gene correction a viable option in human cells.
Plasmids encoding ZFN could be used to deliver a therapeutic gene to a pre-selected chromosomal site.
This approach to gene therapy could be less problematic to the alternative viral-based delivery of therapeutic genes
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Molecular Biology Sabah Linjawi ٩
Yeast Plasmids
Other types of plasmids, often related to yeast cloning vectors include:
Yeast integrative plasmid (YIp), yeast vectors that rely on
integration into the host chromosome for survival and replication
And are usually used when studying the functionality of a solo gene or when the gene is toxic.
Also connected with the gene URA3, that codes an enzyme related to the biosynthesis of pyrimidine nucleotides (T, C)
Yeast Replicative Plasmid (YRp), which transport a sequence of chromosomal DNA that includes an origin of replication.
These plasmids are less stable, as they can "get lost" during the budding.
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Mammalian viral vectors
Vectors for the transfer of genes into
mammalian cells have also been based on viruses
One of the first to be used in this way was SV40, which infects a number of mammlian species
The genome of SV40 is only 5.2 kb in size and it suffers from packaging constraints similar to
phage λ
So its utility for transferring large fragments is
limited
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pRL-SV40 Vector
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Retroviruses
A 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.
Retroviruses are enveloped viruses that
belong to the viral family Retroviridae
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Molecular Biology Sabah Linjawi ١٣
Retroviruses
The virus itself stores its nucleic acid in the form of a +mRNA genome and serves as a means of delivery of that genome into cells it targets as an obligate
parasite, and constitutes the infection.
Once in the host's cell, the RNA strands undergo reverse transcription in the cytosol and are
integrated into the host's genome, at which point the retroviral DNA is referred to as a provirus.
It is difficult to detect the virus until it has infected
the host.
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Cytomegalovirus
Schematic of a
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References
Molecular Biology. P.C. Turner, A.G. Mclennan, A.D.
Bates & M.R.H. White.School of Biological
Sciences, University of Liverpool, Liverpool, UK.
Second edition. BIOS Scientific Publishers, 2000.
www.stanford.edu/dept/EHS/.../Working_with_Viral_
Vectors.pdf
www.markergene.com/.../image002.jpg
en.wikipedia.org/wiki/Retrovirus
bioweb.wku.edu/courses/biol350/.../Review.html
