Download - Gene transformation techniques
Introduction:
• Gene alteration or modification is known as gene transformation.
• Frederick Griffith 1928 revealed genetic transformation in bacterial strains .
• Gene transformation are of two types• Natural transformation• Artificial transformation
Natural transformation
• Conjugation Genetic exchange by cellular contacts
TransductionGenetic exchange through a vehicle Like plasmid or vector
• Agrobacterium mediated gene transfer
Why gene transformation
• science of transformation starts in 1970 with discovery of restriction enzymes by H-Smith
• main purpose includes
• Cloning of gene• Production of desired proteins• Enhancement of products• GMOs
Types
• Stable transformation
integration of gene permanently.
e.g. agrobacterium mediated
• Transient
Temporary transformation
Liposome mediated
Genetic transformation techniques
• Three categories
• Biological means
• Physical means
• Chemical means
Modes of gene transformation
• Indirect method
It uses carriers
e.g. plasmids, vectors, liposome's,
• Direct
carrier free method
e.g. microinjections
Gene gun
Biological Means of transformation
• This technique uses biological systems for gene transfer.
• E.g. • Plasmids• Vectors • Cosmid• Phagemids• BACs and YACs• Modified Replication deficient viruses
Introduction
• It uses physical tools
• Operates direct transformation
• Auto regulation of genes i.e. auto integration
• It overcomes the work-burden unlike biological means
• No side effect and pathogenicity
Types
• The well known physical means of gene transformation techniques include,
• Gene Gun , Biolistic / Ballistic or Golden Gun method
• Microinjections• Electroporation.• Laser beam technique• Ultrasound / Sonication etc
Gene gun/Golden gunBiolistics
• Gene gun is an instrument used to shoot the DNA on target sites.
• Invented by John c Sanford in 1985• Also known as• Golden gun• Ballistics or Biolistics• Particle bombardment method
Gene gun
Construction• Firing pin• Macro projectile• Micro projectile• Safety vents• Plates containing
target cells.
Operating procedure
The gene transfer is followed by four basic operating steps.
• Preparation of DNA loaded beads• Preparation of plastic tubes.• Loading of Macro projectile• Shooting or Transformation
DNA Beads formation
• 250mg gold particles in tube that are 1.6 micro meter in size.
• Add spermadine
• Prepare DNA solution
• Mix (adhering occur)
• Add IM Cacl2
• Centrifugation
Plastic tube preparation
• Add polyvinyl pyridine to solution• Inert, dried tubes are used• Solution is transferred in to tube• Adhering occur• Rotation and refurnished• Incubation • Cutting of tubes
Shooting
• Pressurized gas is used• Distance and pressure ere optimized• Transfection of cell is done by shooting.• Ready for transfection.
Success rate is 30 to 50 %
Examples
Nerve cells
Liver cells were transformed
Micro injection
• Introduced by Dr Marshall Barber
• Physical technique of transformation
• Direct transformation of both plant and animal cells
• It uses micromanipulators
• Syringes of 0.5µm-5µm in diameter size
Operating mechanism
• Operate under observing lens
• Micromanipulators hold plates containing target cells and injection pipettes.
Application
Can be transferred to fertilized ovum e.g ;as in Dolly fish.
Simple and Cheaper Method .Easy Handling Feasible Visible
Ultrasound
• In 1988 Weber
• It uses ultraviolet micro laser
• (200-400nm)
• Laser induce permeability in cell
• Uptake of DNA
• Example
• Brassica napus
• Less efficient
• Advantages and Disadvantages,
• Physical methods of transformations Direct method
• Efficient success rate. 30-40 percent.
• Quick gene transfer.
• The limitation are
• Availability
• cost effective tools.
• requires expertise.
Electroporation
• Physical mean
• Dr Neumann 1982
• Uses electric pulse
• Observed under microscope
• known as electro-permeabilization
• Usually applied for protoplast transformation
Stem cell
• Applied on Stem cells
• Are totipotent
• Uses physical tools
• Direct transformation
• Embryonic stem cell mediated gene transfer
• Success depend on transformation tools
• confirmation of positive clone• Positive clones are cultures
• Re implantation of positive clones in blastula
Introduction
• Uses chemical reagents
• targeted delivery
• Less side effects
• Works on binding affinity
Examples
• DEAE dextran
• Calcium phosphate
• Silicon carbide whiskers method.
• Poly-l-ornithine
• Polyethyelenimine
• Polyethylene glycol method
• Liposome’s mediated gene transfer
PEG
• Used for plant transformation
• Fusiogenic chemical
• 40% w/V
• Medium of mannitol
• Chloroplast and protoplast transformation
Liposome's
• Lipid in nature
• Small in size 5µm-10 µm
• Use for target delivery
• Significance is bilayer
• Lipid soluble layer
• Core i.e. water soluble layer
preparation
• Preparation of core
• Preparation of lipid bilayer
• Sonication
• preparation immune-protective specie
• Addition of homing peptides
• Confirmation
Examples
• Onions : Agrobacterium
• Barely (Hordeum vulgare) electroporation
• Insulin by plasmids
• Sorghum by biolistics
• Sunflower by PEG
• . Bibliography
The given facts and figure are sort out from different sources which are given below accordingly
• GENETIC ENGINEERING principles, procedure and consequences Author: Gurbachan S.Migalani , published by Narosa
• Genetic Transformation of Plants
Edited by J.F.Jackson and H.F.Linksens by Springer (chapter 2)• Gene Transfer techniques by Hongbao Ma, Guozhong Chen
[Nature and Science. 2005;3(1):25-31]• URL:
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