Transformation methods in animals in rDNA technology

Several approaches have been used for the introduction of DNA into animal cells/ embryos, which are listed as follows:

1) Calcium phosphate Precipitation method

2) DEAE-Dextran mediated transfection

3) Lipofection

4) Electroporation

5) Microinjection method

6) Retroviral infection method

1) Calcium Phosphate method:

The DNA preparation to be used for transfection is first dissolved in a phosphate buffer, calcium chloride solution is then added to the DNA solution; this leads to the formation of insoluble calcium phosphate which co-precipitates with the DNA. The calcium-phosphate DNA precipitate is added to the cells to be transfected. The cells take in the precipitate particles by “Phagocytosis”. Initially, 1-2% of the cells was transfected by this approach. In a small proportion of the transfected cells, the DNA becomes integrated into the cell genome producing stable (or) permanent transfection.

2) DEAE-Dextran mediated transfection method:

DEAE-dextran (Diethyl amino ethyl – dextran) is water soluble and polycationic i.e., has a multiple positive charge. It is added to the transfection solution containing the DNA. DEAE-dextran brings about DNA uptake by the cells through “Endocytosis”. Posing its interaction with the negatively charged DNA molecules and with the components of the cell surface plays an important role.

3) Lipofection:

The delivery of DNA into cells using liposomes are “Lipofection”. Liposomes are small vesicles prepared from a suitable lipid. Initially, nonionic lipids were usd for preparing liposomes so that DNA had to be introduced within the vesicles following specific encapsidation procedures.

• Usually liposomes are prepared by dispersion of a Phospholipid like Phosphotidyl choline (PC) in water by mechanical methods like Sonication, which tend to destroy DNA. DNA of up to 1 kb has been incorporated into sonicated liposomes.
• Use of choline cationic liposomes to which DNA binds on the outside by electrostatic attraction. These liposomes cause perturbations in plasma membrane due to which they fuse and the DNA enter into the cytoplasm. Cationinc liposomes are available commercially (marketed as “LIPOFECTIN” by “Gibco-BRL”).

The positively charged liposomes not only complex with DNA, but also bind to cultured animal cells and are efficient in transforming them, by fusion with the plasma membrane. The use of liposomes as a transformation (or) transfection system is called “Lipofection”.

4) Electoporation:

Transfection mixture containing cells and DNA is exposed to very brief period (few milliseconds) to a very high voltage gradient (eg: 4000 to 8000v/cm). This includes transient pores in the cell membranes through which DNA seems to enter the cells. Linearized DNA is far more efficient in transfection than circular supercoiled DNA.

5) Microinjection:

DNA solution is injected directly into the nucleus of a cell (or) into the male pronucleus of a fertilized one-to –two-cell ovum. The general procedure for microinjection is as follows. Donors’ females are induced to superovulated using appropriate hormone treatments. Female mice are subjected to a regime of pregnant mare serum gonadotrophin (PMSG), which stimulate growth, and development of follicles, which contain the developing oocytes. The ovulation induced by the subsequent treatment with human chronic gonadotropin (HCG). The superovulated females re then mated with fertile males, and collect fertilized eggs surgically.

The transgene construct is prepared in a buffer solution and is injected into the male pronuclei of fertilized eggs using a microinjection assembly. The linearized transgene constructs is injected into the cytoplasm of a single ovum. The microinjection embryos are cultured invitro up to the morula (or) blastocyst stage. The surviving embryos are then transferred into the uterus of surrogate mothers. These embryos develop to full term and give rise to normal mice. The microinjection method is frequently using in “transgenic biology” for producing “transgenic animals” and “Transgenic plants”.

6) Retroviral infection:

Recombinant retroviruses produce virions, which are used to infect animal cells and mice embryos. Generally, early 4 to 16 celled embryos are used for this method. The recombinant retrovirus RNA genome is copied by “Reverse transcriptase” to yield a DNA copy (reverse transcription) which becomes integrated into the cell’s genome. The reverse transcriptase is encoded by the retrovirus. And is produced immediately after infection. The recombinant retrovirus integrates into the cellular genome at random sites & usually is not accompanied with dilations (or) rearrangements.