Gene Transformationtechniques

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

Physical means of gene transformation

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

Loading in macro projectile

• It act as cartridge• Specified DNA beads in tubes are

loaded in it

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

• *

Other methods

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

Operating procedure

• Parameters are optimized

• 60-75 Celsius.

• Voltage of 150-160 volt

Description

Stem cell

• Applied on Stem cells

• Are totipotent

• Uses physical tools

• Direct transformation

• Embryonic stem cell mediated gene transfer

Procedure

• Isolation of Blastula cells

• Transformation by physical method

• Success depend on transformation tools

• confirmation of positive clone• Positive clones are cultures

• Re implantation of positive clones in blastula

Advantage

Advantages

Transgenic progeny

Short time production

Efficient success rate

Chemical means of gene transfer

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

Liposome

preparation

• Preparation of core

• Preparation of lipid bilayer

• Sonication

• preparation immune-protective specie

• Addition of homing peptides

• Confirmation

Applications of gene transfer

• Transgenic animals

• Transgenic crops

• Gene therapy

• Cloning

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:

https://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=19&cad=rja&uact=8&ved=0ahUKEwiD_MjS_IjTAhUHzRQKHTW2AGYQFgiAATAS&url=http%3A%2F%2Fwww.sciencepub.net%2Fnature%2F0301%2F03-mahongbao.doc&usg=AFQjCNE9aGJ4HR_y_ka0t1WvqrYXJeuLlQ

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