METHODS OF GENE TRANSFER

DANDE SWAPNA SREE

Assistant Professor in Botany

KVRGCW(A) , Kurnool

DEFINITION

A technique to efficiently and stably

INTRODUCE FOREIGN GENES

INTO

THE GENOME OF TARGET CELLS.

TERMINOLOGYNUCLEUS CHROMOSOME DNA

GENE GENOME FOREIGN GENE

GENETIC TRANSFORMATION

TRANSGENE TRANSGENICS

TRANSIENT EXPRESSION

STRUCTURAL GENE

CODES FOR

RNA OR PROTEIN

REGULATORY GENE

TO EXPRESS

FOREIGN GENE

CORRECTLY

MARKER GENE

TO IDENTIFY TRANSFORMED CELLS

• GENE TRANSFER- UPTAKE OF DNA

• DESIRABLE GENE- GENE OF INTEREST

• TRANSFORMATION-INTEGRATION AND EXPRESSION OF FOREIGN GENEINTO GENOME

• TRANSGENE- TRANSFORMED GENE

• TRANSGENIC (TRANSFORMED) PLANTS-- PLANTS THAT CARRY THE

STABLE INTEGRATED FOREIGN GENE

• TRANSIENT EXPRESSION- TRANSGENE EXPRESSED ONLY FOR A SHORT

PERIOD OF TIME

• STABLE TRANSFORMATION- OCCURRENCE OF TRANSGENE IN

REGENERATED PLANT AND IS INHERITED TO

SUBSEQUENT GENERATIONS

Horizontal gene transfer • The natural transfer of genetic material from one organism to

another is referred to as horizontal gene transfer or lateral genetransfer

• The foreign DNA is either randomly inserted into the host genome orrecombines if there is sequence homology between the twogenomes.

• Horizontal gene transfer plays important role in evolution of bothprokaryotes and eukaryotes.

• Horizontal gene transfer is facilitated by various mechanisms.

PROKARYOTES• transformation (intake of genetic material from

surrounding)

• conjugation (exchange of genetic material with the physical union of two cells) and

• transduction (transmission of DNA through bacteriophages from one cell to another)

are responsible for the transfer of the gene within organisms.

•

EUKARYOTES

the presence of the

outer cell membrane and

nuclear membrane

makes transfer of DNA difficult between organisms.

Vertical gene transfer

the genetic material is transferred from the parents to the off springs, through sexual reproduction.

Plant

Explant(leaf, embryo, meristem, callus, cells, protoplasts)

DNA Delivery

Transformed plants

(Transgenics)

Testing for transgenics

plant by PCR, Southern

Hybridization)

Transformed cell / tissue

Transgenic seeds

Transformed plants screened in next

generation

DIRECT/INDIRECT methods

Plant transformation system- STEPS

• Introduction of a DNA segment into totipotent cells.

• Integration into host cells genome.

•Regeneration from transformed cell to produce whole plant.

•The DNA segment introduced in this process contains the gene of interest and a cassette containing additional genetic material.

ADDITIONAL GENETIC MATERIAL

A promoter gene (to determine the site andtiming of expression of the introduced gene)

• A terminator gene (to identify the end oftranscription) and

• A marker gene (to allow selection of plants having the introduced gene).

METHODS OF GENE TRANSFER

Different methods are available to achieve genetictransformation of plants i.e. the delivery of the foreign DNAinto the host plant. These are divided into two main groups

• Indirect method: In this case vector is needed forinsertion of the foreign DNA into the host genome.

• Direct method: This method is vector independent.The DNA is directly inserted into the host genome.

INDIRECT

AGROBACTERIUM MEDIATED

ELECTROPORATION

DIRECT

MICROINJECTION

PARTICLE BOMBARDMENT /GENE GUN/ MICROPROJECTILE

METHODS OF GENE TRANSFER

Direct Gene Transfer• Vectorless

• Rely on large amount of naked DNA whilst plant is transiently permeabilised

Physical Methods

Microinjection

Biolistics-Gene gun/ Particle Bombardment

Electroporation

Electroporation

Method of transformation via direct gene transfer.

In this technique mixture containing cells and DNA is exposed to very high voltage electrical pulses (4000 – 8000 V/cm) for very brief time periods (few milliseconds). It results in formation of transient pores in the plasma membrane, thorough which DNA seems to enter inside the cell and then nucleus.

Electroporation

• Electroporation produces electric pulse to create transient pores called electroporesin the plasma membrane of the target cells there by allowing the entry of foreign DNA into the cells.

• Cells which are arrested at metaphase stage of cell cycle are especially suitable for electroporation as these cells have absence of nuclear envelope and an unusual permeability of the plasma membrane.

• Protoplasts are used for electroporation of plant cells as thick plant cell walls restrict movement of DNA.

• The electroporation method was originally developed for protoplasts, but has given equally good results with cells and even tissues with easy recovery of regenerated plantlets.

• Immature zygotic embryos and embryogenic calli have also been used for electroporation to produce transgenic maize.

•Transformation of protoplast is associated withlow transient expression of transgenes ascompared to organized tissues and lowregeneration frequency especiallyin monocotyledonous plants.

•The electrical field and chemical substancesapplied to disorganize cell walls strongly reducethe viability and capability of divisionof protoplasts.

Electoporation as a transformation method is

• Fast

•Convenient,

•Simple, and

• Inexpensive and

•Has low cell toxicity.

The disadvantage associated with this technique is difficulty in regenerating plants from protoplasts, if protoplast is used for electroporation.

ADVANTAGES:1.Fast method2. Less Cost3. Applicable for a number of

cell types4. A large number of cells

can be producedsimultaneously

5.High percentage of stabletransformants can be

produced

BIOLISTICS (Gene Gun or Particle Delivery system)

•Micro projectile : An elemental particle of a heavy metal (gold or Tungsten) •Payload :Microprojectile coated with plasmid DNA

Biolistics• a method where cells are physically impregnated with nucleic acids or

other biological molecules.

• biolistic particle delivery system is a device for plant transformation where cells are bombarded with heavy metal particles coated with DNA/RNA.

• This technique was invented by John Stanford in 1984 for introduction of DNA into cells by physical means to avoid the host-range restrictions of Agrobacterium.

• Agrobacterium-mediated genetic transformation system works well for dicotyledonous plants but has low efficiency for monocots.

• Biolistic particle delivery system provides an effective and versatile way to transform almost all type of cells.

• It has been proven to be a successful alternative for creating transgenic organisms in prokaryotes, mammalian and plant species.

• In this process, • construct having gene of interest is coated on the surface of tiny particles

of gold or tungsten (0.6 – 1 mm in size). • Prior to coating, DNA is precipitated with calcium chloride, spermidine and

polyethylene glycol. These coated microparticles are loaded on to the macrocarrier and accelerated to high speed by using pressurized helium gas.

• Plant cell suspensions, callus cultures, or tissues could be used as the target of these microprojectiles.

• As the microprojectiles penetrate the plant cell walls and membranes to enter the cells, coated DNA is released from its surface and incorporated into the plant’s genome.

Particle bombardment method of Plant transformation

(1) Isolation of protoplasts. (2)Injection of DNA-coated particles using particle gun.

(3) Regeneration of transformed protoplasts into plantlets.

(4) Acclimatization of regenerated plantlets in a greenhouse.

Parameters to be considered before using particle bombardment.

Physical

Environmental

Biological

Physical parameters

•Nature, chemical and physical

properties of the metal particles

utilized to carry the foreign DNA.

•The nature and preparation of DNA

•Binding of DNA on the particles and

target tissues

Environmental parameters

•Temperature

•Photoperiod and humidity of

donor plants, explants, and

bombarded tissues

•affect physiology of tissues and influence

receptiveness of the target tissue.

Biological parameters

•Choice and nature of explants

• pre- and post bombardment culture

conditions

• osmotic pre- and post-treatment of

explants.

Advantages of particle bombardment over Agrobacterium-mediated DNA transfer:

species independent and can been used

successfully for a wide range of organisms.

Many species which are recalcitrant to other

direct transfer methods or are not readily

amenable to Agrobacterium-mediated

transformation have been transformed by this

technique.

…•Introduced DNA does not need sequences necessary for T-DNA replication and transfer as complex interaction between bacterium and plant tissue does not take place

•Multiple genes can be introduced in a single plant.

…Transformation of organelle DNA

(mitochondria and chloroplasts) has also

been achieved by this method.

Particles can be coated with

DNA/RNA/large fragments of nucleic

acids.

…

•This method is especially important for monocots, for which efficiency of other transformation methods is not satisfactory.

•A wide range of tissues such as apical and floral meristems, embryos, seedlings, leaves, cultured cells and floral tissues could be used as target in this method.

Limitations

• Limited regeneration capacity of tissue being

bombarded

• Efficiency of stable integration of DNA.

• Integration of rearranged and/or truncated DNA

sequences

• Damage to the cellular tissue.

• Specialized and expensive equipments are

required

MICROINJECTION

Requirements:

A holding pipette – for immobilizing protoplast

An injecting pipette- to inject the macromolecule

About 1-5 days old Cultured protoplasts- for partial regeneration of cell wall

Useful for transformation of protoplasts with

exogenous genes

Microinjection

•The process of using a fine glass micropipette to manually inject transgene at microscopic or borderline macroscopic level is known as microinjection.

•The transgene, in the form of plasmids, cosmids, phage, YACs, or PCR products, can be circular or linear and need not be physically linked for injection.

• Microinjection involves direct mechanical introduction of DNA into the nucleus or cytoplasm using a glass microcapillary injection pipette.

• The protoplasts are immobilized in low melting agar, while working under a microscope, using a holding pipette and suction force.

• DNA is then directly injected into the cytoplasm or the nucleus.

• The injected cells are then cultured invitro and regenerated into plants.

• Successful examples of this process has been shown in rapeseed, tobacco and various other plants.

• In this technique a traditional compound microscope (around 200X magnification) or aninverted microscope (around 200x magnification) or a dissecting stereomicroscope (around 40-50x)is used.Under the microscope target cell is positioned and cell membrane and nuclear envelope are penetrated with the help of two micromanipulators.Onemicromanipulator holds the pipette and another holds the microcapillary needle.

TYPES OF MICROINJECTION SYSTEMS

pulsed flow system constant flow system

the amount of sample injected is determined by the duration for which needle remains in the cell.The constant flow system is relatively simple and inexpensive but outdated.

has greater control over the volume of substance delivered, needle placement and movement and has better precision.This technique results in less damage to the receiving cell, however, the components of this system are quite expensive.

• Stable transformants can be achieved through this method

• but it requires technical expertise and is

• a time consuming process.

• Microinjection has achieved only limited success in plant transformation due to the thick cell walls of plants and a lack of availability of a single-cell-to-plant regeneration system in most plant species.

Other Methods

•Chemical mediated gene transfer

•Cells or protoplasts can be stimulated to take up foreign DNA using some chemicals.

• Polyethylene glycol (PEG) is the most commonly used chemical for this purpose.

• It helps in precipitation of DNA, which can then be taken up by the calls through the process of endocytosis.

•Liposome mediated gene transfer

Plasmid containing foreign desired gene canbe enclosed in small lipid bags calledlipososmes, which can then be fused withprotoplasts using chemicals like PEG.

Silicon carbide method

In this method, fibres of organic material like silicon carbide are used for gene transfer.

These fibres, when mixed with plasmid DNA and plant tissue or cells, help in penetration of the foreign DNA into the plant tissue.

FAQs

•Write a short note on microinjection.

•Discuss the merits and demerits of particle bombardment method of genetic transformation.

•What is Electroporation? How it is different from microinjection?

•Why do we need a marker gene in planttransformation experiment?

•Differentiate between selectable andscreening marker? Give examples.

REFERENCES

• Narusaka, Yoshihiro, et al. "Methods to Transfer Foreign Genes toPlants." IN: Transgenic Plants–Advances and Limitations, Yelda OzdenÇiftçi (Ed.), ISBN (2012): 978-953.

• http://www.intechopen.com/books/genetic-transformation/genetic-transformation-of-forest-trees