Gene Delivery Methods
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Lecture 26
Gene Delivery methods –Agrobacterium and particle gun
Requirements for plant genetic transformation
Source Tissues for Plant Gene
Transfer
Gene to be
transferred
Gene Transfer Methods
Regeneration of whole
Transformed Plant
Plant Transformation Vector
Effecting plant gene transfer
Gene transfer methods
DirectIndirect
Indirect Gene Transfer Methods
Whole Plant Infection
In vitro infection of tissues
Co-cultivation of protoplasts and A.tumifaciens
Leaf disc transformation
Agrobacterium mediated gene transfer
Agrobacterium tumifacienswith vector DNA having the gene of interest
Plant cell
T DNA getting into the cell
Agrobacterium tumifaciens: A natural genetic engineer
This opportunistic soil phytopathogen
causes crown gall tumours in wounded
gymnosperms and dicotyledonous
angiosperms (dicots).
Oncongenic strains contain a single
copy of a large (150-250 kb) tumour-
inducing (Ti) plasmid.
Part of this plasmid DNA (the ‘transfer’
or T-DNA) is transferred to the
wounded plant cell and stably
integrated into the genome.
Agrobacterium mediated gene transfer
Direct Gene Transfer Methods
Electroporation
Liposome mediated gene transfer
Microinjection
Electroinjection
Sonication method
Microprojectile bombardment
Gene transfer can be effected by certain means that do not usevectors (Direct gene transfer). These include
a. Electroporation: Temporary holes are formed in the plasmamembrane of the host cell by applying a high voltage. Thesepores permit entry of foreign DNA.
b. Chemical mediated genetic transformation some chemicals,such as polyethylene glycol, help foreign DNA to enter thehost cell.
c. Micro-injection: The host cell is immobilized by applying amild suction with a blunt pipette. The foreign gene is theninjected with a micro-injection needle.
When the electric field is applied, the ions move accordingto their charge.
Pathways are formed across the cell membrane allowingDNA to enter.
When the electric field is deactivated, the membrane heals.
ElectroporationTransformation is stimulated by physically disrupting the cell membranes sothat the genetic material may enter.
This method was introduced by Fromm and his coworkers in 1986
The voltage range varies from 250-2000V.
This method was reported by Krens and his colleagues in l982
Non-biological methodsPolyethylene Glycol (PEG) method:
In fusion conditions, PEG is usually present at a concentration of40 percent for several seconds whereas for DNA transformation the optimalPEG concentration is 13.3 percent for an incubation of 30 mins.
Microinjection method:Crossway and Reich in l986
Using the holding pipette, the protoplast has to be held and the DNA to beinjected into the nucleus using the injection pipette. After the micro injectionthe injected cells are cultured by hanging droplet culture method.
Micro projectile bombardment method:In 1987 Klein and his colleagues evolved a method by which the deliveryof DNA into cells of intact plant organs or cultured cells is done by aprocess called Projectile Bombardment
These particles with high kinetic energy penetrate the cells and membranesand carry foreign DNA inside of the bombarded cells. This method isotherwise called as "Biolistics Method”
Biolistic Transformation
Some of the particles will pass through the cell wall and enter individual cells.
Sonication method:
• This is a simple technique formulated by Xu and his co-workers.
• In this method the explants (especially leaves) are excised and cutinto segments, immmersed in sonication buffer containing plasmidDNA and carrier DNA in a sterile glass Petri dish. Then the sampleswere sonicated with an ultrasonic pulse generator at 0.5 c/cm2
acoustic intensity for 30 mins.• After 30 mins., the explants were rinsed in buffer solution without
DMSO and transferred to the culture medium.
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