Grafana in space: Monitoring Japan's SLIM moon lander in real time
Basic principle of gene expression & methods of
1. Basic Principle of Gene Expression &
methods of Gene Transfer
Presented To:-
Dr. Vikram kumar Yadav
AIB
Presented by:-
Atul Chauhan
M.Sc 3rd semester
2. It is the process by which information from a
gene is used in the synthesis of a functional
gene product.
These products are often proteins, but in non-
protein coding genes such as rRNA genes or
tRNA genes, the product is a functional RNA.
What is Gene Expression?
4. Transcription is the synthesis of a single-stranded RNA
from a double stranded DNA template. RNA synthesis
occurs in the 5→3 direction and its sequence
corresponds to that of the DNA strand which is known
as the sense strand.
This is the first stage of the overall process of gene
expression and ultimately leads to synthesis of the
protein encoded by a gene. Transcription is catalyzed by
an RNA polymerase which requires a ssDNA template
as well as the precursor ribonucleotides ATP, GTP, CTP
7. Viral based vectors
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 (in vivo) or in cell culture (in vitro).
Viruses have evolved specialized molecular
mechanisms to efficiently transport their
genomes inside the cells they infect.
Delivery of genes by a virus is termed
transduction and the infected cells are described
as transduced.
8. Key properties of a viral vector
Safety - deletion of a part of the viral genome critical for
viral replication.
Low toxicity - The viral vector should have a minimal
effect on the physiology of the cell it infects.
Stability - Some viruses are genetically unstable and can
rapidly rearrange their genomes. This is detrimental to
and is avoided in their design.
Cell type specificity - Most viral vectors are engineered to
infect as wide a range of cell types as possible.
Identification - Viral vectors are often given certain genes
that help identify which cells took up the viral genes.
These genes are called Markers, a common marker is
antibiotic resistance to a certain antibiotic.
11. Single stranded.
Has two copies of the genome, which resemble eukaryotic
mRNAs.
The viral genome is reverse transcribed by reverse
transcriptase into a DNA double-strand copy inside the host
cells.
Features of RV vector
Contains gene for replication, expression and packaging (ψ
sequences).
Gene of interest may inserted in the nonessential coding
region or it may replace some essential gene (gag).
Genomes are used as vectors, generally as shuttle vectors
13. It act as a vector for in vivo gene delivery.
Long term gene expression.
They are more complicated than retroviruses,
containing an 9 virulence proteins.
Out of it 6 genes (tat, rev, vpr, vpu, nef and
vif.) will be removing without altering gene
transfer ability
15. Adeno-associated virus (AAV)
Adeno-associated virus (AAV) is a small virus which
infects humans and some other primate species.
Carries DNA as genetic material.
AAV is not currently known to cause disease and
consequently the virus causes a very mild immune
response.
AAV can infect both dividing and non-dividing cells and
may incorporate its genome into that of the host cell.
These features make AAV a very attractive candidate
for creating viral vectors for gene therapy
17. Electroporation
A method that uses short pulses of high voltage
to carry DNA across the cell membrane.
This shock is thought to cause temporary
formation of pores in the cell membrane,
allowing DNA molecules to pass through.
Electroporation is generally efficient and works
across a broad range of cell types.
However, a high rate of cell death following
electroporation has limited its use, including
clinical applications.
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20. Gene Gun/ particle bombardment
Another physical method.
In this technique, DNA is coated with gold
particles and loaded into a device which
generates a force to achieve penetration of
DNA/gold into the cells.
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22. Oligonucleotides
To inactivate the genes involved in the disease
process.
Use antisense specific to the target gene to
disrupt the transcription of the faulty gene.
23. Microinjection
The microinjection is the process of
transferring the desirable DNA into the living
cell ,through the use of glass micropipette
Glass micropipette is usually of 0.5 to 5
micrometer , easily penetrates into the cell
membrane and nuclear envelope.
The desired gene is then injected into the sub
cellular compartment and needle is removed
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25. Liposome mediated gene transfer
Liposomes are spheres of lipids which can be
used to transport molecules into the cells.
These are artificial vesicles that can act as
delivery agents for exogenous materials including
transgenes.
Promote transport after fusing with the cell
membrane.
Cationic lipids are those having a positive charge
are used for the transfer of nucleic acid.
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27. APPLICATION
Clinical gene transfer applications
Vaccine Development
Production of transgenic animals
Treatment of Cancer, AIDS
Gene Discovery
Gene Therapy