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Gene Transfer Methods

Gene transfer methods can be direct or indirect. Indirect methods include Agrobacterium-mediated transfer using Ti plasmid and vir genes. Direct methods are biolistic/particle bombardment, microinjection, liposome/PEG-mediated transfer, and electroporation. These introduce exogenous DNA into cells and organisms. Natural methods are conjugation, transformation, and transduction using bacteriophages. Gene transfer has applications in generating transgenic plants and animals with desired traits.

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73 views

Gene Transfer Methods

Gene transfer methods can be direct or indirect. Indirect methods include Agrobacterium-mediated transfer using Ti plasmid and vir genes. Direct methods are biolistic/particle bombardment, microinjection, liposome/PEG-mediated transfer, and electroporation. These introduce exogenous DNA into cells and organisms. Natural methods are conjugation, transformation, and transduction using bacteriophages. Gene transfer has applications in generating transgenic plants and animals with desired traits.

Uploaded by

ayeshasattarsandhu
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Gene Transfer

Methods in plants
Gene transfer methods
•Transfer of a gene from one
DNA molecule to another DNA

•Genetic transformation

•Transient and stable


transformation

•Transgene and transgenic


Indirect gene transfer method

Agrobacterium tumeficians:

•Soil born gram negative bacteria

•Rod shaped and motile

•Belongs to bacteria family of


Rhizobiaceae

•Causes crown gall disease


Agrobacterium mediated gene transfer

Ti plasmid and Vir gene:

•T DNA region

•Virulence region

•Opine catabolism region


Transformation of cell by A.tumefaciens
1. Signal induction to
Agrobacterium
2. Attachment of
Agrobacterium to plant
cell
3. Production of virulence
protein
4. Production of single
stranded T DNA
5. Transfer of T DNA out of
Agrobacterium
6. Integration
Applications

•Generate a large number of


transgenic plants

•Introduce many new traits

•Herbicides resistance

•Disease resistance

•High growth rate


Direct gene transfer method
Biolistic method:

•Also known as particle bombardment

•Use accelerated microprojectiles

•DNA coated with gold or tungsten


filament

•Coated beads are attached to the end of


plastic bullet

•Beads pass through the cell wall


Application
•Used successfully to transform
soya bean, cotton, spruce,
sugarcane, papaya

• Transformation of Mitochondria
of plants and chloroplast of
chlamydomonas

•Manipulation of subcellular
organelles
Microinjection method

•Artificial DNA transfer to cereals plants

•Diameter of needle greater than cell


diameter

•Around 0.3 ml of DNA solution is


injected

•Timing of injection is important

•Fourteen days before meiosis.


Microinjection method
Application

•Method is effective in transforming primary cells as


well as cells in established culture

•Technique is ideally useful for producing transgenic


animal quickly

•Procedure is important for gene transfer to


embryonic cells

•Method has been successfully used with cells and


protoplast of tobacco, alfalfa etc.
Liposome mediated method

•Liposomes are spheres of lipids used to


transport molecules into the cells

•Artificial vesicles

•Cationic lipids

•Fusion

•aqueous solution of DNA molecules.

•self-organizing process
Liposome mediated method
Application

•Germline trangenises

•Transfection of ES cells

•vaccination against virus


infection

•In tumor therapy

•In multi drug resistance


PEG mediated method

•Utilized for protoplast


•Stimulates endocytosis
•Protoplast are kept in the solution containing
PEG
•Molecular weight of PEG is 8000D with final
conc. Of 15%
•Addition of cacl2
•Glucose and sucrose act as buffering agent
•Culturing of protoplast to form cells with
walls and colonies
Application
•Vaccine development

•Enhancing resistance in
plant

•Treatment of cancer

•Gene discovery

•Gene therapy
Electroporation
•It uses electrical pulse to
produce transient pores in the
plasma membrane

•Microscopic pores are induced


in biological membrane

•These pores are known as


electro pores which allow the
molecules, ions and water to
pass from one side of the
membrane to another

•Electropores reseal
spontaneously and the cell
can recover
Application
•Effective method of introducing exogenous DNA into
embryonic stem cells

•It transfer genes into cultured mammalian embryos at


defined stages of development

•Electroporation has been reported to enhance the level of


gene expression

•It significantly improve immune responses elicited to DNA


vaccines
Natural gene transfer method

Conjugation
•Require the presence of special plasmid called F plasmid
• F+ and F- plasmids
•F plasmid consist of 25 genes code for production of sex
pilli
•Conjugation event occur when the male cell extends his
sex pilli and attaches to female cell
•Attached pilus acts as a cytoplasmic bridge
•High frequency cell
Bacterial transformation

•Transformation is direct uptake of exogenous DNA from its


surrounding
•Development of competence,
•Binding of DNA to the cell surface
•Processing and uptake of free DNA
•integration of the DNA into the chromosome by
recombination.
Transduction
•Genetic transfer from one bacteria to another
through a virus
•virus uses the host machinery to make multiple
copies either directly by the lytic cycle
•Assembly of bacteriophages
• lysogenic cycle
•Specialized transduction
•General transduction
Application

•Transduction is used to insert the genes of choices in


animals and plant cells to modify the genetic constituents
and get the desired characteristics

•It can be used for gene therapy

•It has huge potential to cure genetic diseases

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