Direct Gene Transfer Methods

-Saugat Bhattacharjee

Dept. of Genetics

Genetic transfer is the mechanism by which DNA is transferred from a donor to a recipient.

Once donor DNA is inside the recipient, crossing over can occur.◦ The result is a recombinant cell that has a genome different from

either the donor or the recipient.

Gene transfer- Introduction

GENE TRANSFER TECHNOLOGIES

Gene transfer technology provides the ability to genetically manipulate the cells of higher animals.

During the 1970s it became possible to introduce exogenous DNA constructs into higher eukaryotic cells in vitro.

Mammalian (germline) transgenesis was first achieved in the early 1980s.

The model used in this study was mice.

DNA can be introduced into cells or protoplast with the help of very fine needles or glass micropipettes having the diameter of 0.5 to 10 µm.

Computerized control of holding pipette, needle, microscope stage and video technology has improved the efficiency of this technique.

Microinjection

Advantages of Microinjection

Gene Gun Simplest method of direct introduction of therapeutic DNA into target

cells Looks like a pistol but works more like a shotgun Golden Pellets. First described as a method of gene transfer into plants John Sanford

at Cornell University in 1987. This device is able to transform almost any type of cell, including

plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plastids.

Applying Gun Technique

DNA (or RNA) become “sticky,” adheres to biologically inert particles such as metal atoms (usually tungsten or gold)

accelerating this DNA-particle complex in a partial vacuum and placing the target tissue within the acceleration path gathers the DNA

cells that take up the desired DNA, identified through the use of a marker gene are then cultured to replicate the gene and possibly cloned

most useful for inserting genes into plant cells such as pesticide or herbicide resistance

The process of transfection involves the admixture of isolated DNA (10-100ug) with solution of calcium chloride and potassium phosphate under condition which allow the precipitate of calcium phosphate to be formed.

Cells are then incubated with precipitated DNA either in solution or in tissue culture dish.

A fraction of cells will take up the calcium phosphate DNA precipitate by endocytosis.

Calcium Phosphate Method

Limitations of calcium phosphate mediated DNA transfer:

Frequency is very low. Integrated genes undergo substantial modification. Many cells do not like having the solid precipitate

adhering to them and the surface of their culture vessel.

Integration with host cell chromosome is random. Due to above limitations transfection applied to

somatic gene therapy is limited

It uses electrical pulse to produce transient pores in the plasma membrane thereby allowing macromolecules into the cells.

Microscopic pores are induced in biological membrane by the application of electric field.

These pores are known as electropores which allow the molecules, ions and water to pass from one side of the membrane to another.

Electroporation

Thus there are a number of ways by which the gene can be introduced into the cells. With the advent of molecular tools and technologies it is now comparatively easy to introduce gene into cells without loosing its integrity and biological activity. Moreover the recent development in molecular biology has made the transfer of gene with great accuracy to the target cell. The transfer of gene through different gene transfer technologies has cured a number of diseases. Research is on progress to cure those diseases which cannot be cured by using drugs. Moreover the treatment of diseases by gene transfer provides better result for a prolong period of time. It is the need of hour to discover new and cheap method of gene transfer technologies so to make the treatment of the diseases a little easier and affordable.

CONCLUSION