Gene Transfer Lab Report

Transfection: One of the methods of gene transfer where the genetic material is deliberately introduced into the animal cell in view of studying various functions of proteins and the gene. This mode of gene transfer involves creation of pores on the cell membrane enabling the cell to receive the foreign genetic material. Transfection can be carried out using calcium phosphate (i.e. tricalcium phosphate), by electroporation, by cell squeezing or by mixing a cationic lipid with the material to produce liposomes which fuse with the cell membrane and deposit their cargo inside. The choice of methods of DNA transfer depends upon the target cells in which transformation will be performed. It also depends upon the objectives of gene manipulation.

It is an efficient process to transfer DNA into cells. Microscopic pores are induced in biological membrane by the application of electric field. These pores are known as electro pores which allow the molecules, ions and water to pass from one side of the membrane to another. The pores can be recovered only if a suitable electric pulse is applied. The electro pores reseal spontaneously and the cell can recover. The formation of electro pores depends upon the cells that are used and the amplitude and duration of the electric pulse that is applied to them. Electric currents can lead to dramatic heating of the cells that can results in cell death. Heating effects are minimized by using relatively high amplitude, a short duration pulse or by using two very short duration pulses. In terms of mammalian trans genesis, electroporation is an effective method of introducing exogenous DNA into embryonic stem (ES) cells. This technique has recently, been used to transfer genes into cultured mammalian embryos at defined stages of development. It was reported that there is an increase from 12 to 19% of transgenic bovine blastocysts when electroporation was included in an otherwise passive sperm-DNA uptake protocol. Similar findings were reported, again with transgenic bovine blastocysts. Fish species were also reported to be genetically manipulated in

1. Introduction of exogenous DNA into animal cell lines, plant protoplast, yeast protoplast and bacterial protoplast.

2. Electroporation can be used to increase efficiency of transformation or transfection of bacterial cells.

3. Wheat, rice, maize, tobacco have been stably transformed with frequency up to 1% by this method.

4. Genes encoding selectable marker may be used to introduce genes using electroporation.

5. To study the transient expression of molecular constructs.

6. Electroporation of early embryo may result in the production of transgenic animals.

7. Hepatocytes, epidermal cells, haematopoietic stem cells, fibroblast, mouse T and B lymphocytes can be transformed by this technique.

8. Naked DNA may be used for gene therapy by applying electroporation device on animal