Transfection of DNA to host cell can be done by various methods in lab scale.Gene gun,electroporation,lipofection .These methods are used to transfer DNA to the host cell.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
An overview of the Agrobacterium-mediated gene transfer process. Moreover, studied different kinds of Agrobacterium species are involved in this mechanism.
Agrobacterium is a rod-shaped, Gram-negative bacteria found mostly in the soil. It is a plant pathogen that is responsible for causing crown gall disease in them. This bacteria is also known as the natural genetic engineer because of it's the ability to integrate its plasmid Gene into the plant genome.
Agrobacterium tumefaciens transfer of their genetic material T-DNA of Ti-plasmid into the plant cell: A: Agrobacterium tumefaciens; B: Agrobacterium genome; C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes; D: Plant cell
A Ti-Plasmid (tumor-inducing plasmid) is a ds, circular DNA that often, but not always. It's a piece of genetic equipment that transfers genetic material from bacterial cells means Agrobacterium tumefaciens into plant cells used to induce tumors in the plant. The Ti-plasmid is damage when Agrobacterium is grown above 28 °C. Such cured bacteria don't induce crown gall disease in the plant due to they are avirulent. The Ti-Plasmid are classified into two types on the basis of opine genes are present in T-DNA.
The Plasmid has 196 genes that code for 195 proteins. There is no one structural RNA. The plasmid is 206.479 nucleotides long. the GC content is 56% and 81% of the genetic material is coding genes.
The modification of this plasmid is a very important source in the production of transgenic plants.
The T-DNA must be cut out of the circular plasmid. A VirD1/D2 complex nicks the DNA at the left and right border sequences. The VirD2 protein is covalently attached to the 5' end. VirD2 contains a motif that leads to the nucleoprotein complex being targeted to the type IV secretion system (T4SS).
In the cytoplasm of the recipient cell, the T-DNA complex becomes coated with VirE2 proteins, which are exported through the T4SS independently from the T-DNA complex. Nuclear localization signals, or NLS, located on the VirE2 and VirD2 are recognized by the importin alpha protein, which then associates with importin beta and the nuclear pore complex to transfer the T-DNA into the nucleus. So that the T-DNA can integrate into the host genome.
We inoculate Agrobacterium containing our genes of interest, onto wounded plant tissue explants. The Agrobacterium then transfers the gene of interest into the DNA of the plant tissue.
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
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2. METHODS
Different methods of introducing genes to the eukaryotic host
cell
• Chemical
transfection
• Lipofection
Chemical
method
• Electroporation
• Microinjection
• Gene gun
Physical
method
4. Transfection
Transfection is a method of transporting DNA, RNA
and/or various macromolecules into an eukaryotic
cell
Using chemical, lipid or physical based methods
Neutralize negative charges on DNA
5. TYPES
Chemical method:
Calcium phosphate creates
precipitates that settle on cells
and are taken in
Lipid or Polymer method:
Interact with DNA, promotes
binding to cells and uptake via
endocytosis
11. Applications
Direct transfer of plasmid between cells
Transdermal drug delivery
Cancer tumour electro chemotherapy
Gene therapy
12. Microinjection
Use of a fine needle
Use to transfer of DNA into embryonic stem cells
Injection of linear DNA
Random integration
Inefficient
13.
14. Advantages
No requirement of marker gene
Introduction of the target gene directly into a single cell
Easy identification of transformed cells
No requirement of selection of transformed cells
(antibiotic resistance or herbicide resistance markers)
Can be used for transgenic animals
15. Gene gun
Biolistic Particle delivery
Uses high velocity for delivery of nucleic acids & penetration of cell wall
Also termed as particle bombardment ,particle gun, microprojectile
16.
17. Advantages
Simple and convenient method involving coating DNA/RNA on to gold
micro-carrier, loading sample cartridges, pointing the nozzle and firing the
device.
Eliminates the use of potentially harmful viruses or toxic chemical
treatment as gene delivery vehicle.
18. Advantages & Disadvantages
Advantages
Provides the ability to transfer
in negatively charged
molecules into cells with a
negatively charged membrane
Liposome-mediated transport
of DNA has high efficiency.
Good for long-term studies
requiring incorporation of
genetic material into the
chromosome
Disadvantages
Chemical Reagents: not useful
for long-term studies
Transfection efficiency is
dependent on cell health, DNA
quality, DNA quantity,
confluency (40-80%)
Direct Microinjection and
Biolistic Particle delivery is an
expensive and at times a
difficult method