BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Methods for Introducing Recombinant DNA into Cells

There are 4 main groups of Methods for introducing recombinant DNA:

— chemical methods (using Liposomes, Metal Ions, and other chemical Reagents);

- physical methods (electroporation, laser irradiation);

— mechanical methods (microinjection, biolistic transformation);

— biological methods (Gene transfer via viral infection, or transfection).

The process by which Cells take up exogenous DNA is known as transformation.

Transformation efficiency is defined as the number of transformants per 1 μg of introduced DNA. Modern Bacterial Transformation methods allow researchers to obtain up to 10⁷ – 10⁸ transformants per 1 μg of plasmid DNA.

Cells capable of taking up foreign DNA are referred to as competent cells. The proportion of competent cells can be increased by using special nutrient media or optimized culture conditions.

The frequency of Cell transformation and transfection in E. coli can be enhanced through various techniques, such as heat Shock and exposure to metal ions (Ca²⁺, Rb⁺).

When E. coli cells are treated with CaCl₂ at 0 °C, followed by a Temperature spike to 42 °C for 1.5 minutes, localized damage to The Cell wall occurs. This method yields a transformation frequency of 10³ (1 transformant per 1,000 cells).

Liposomes are used to introduce recombinant DNA into animal and human cells. They penetrate The Cell interior via endocytosis.

Electroporation (a brief exposure to an electric field for 10 microseconds to 100 milliseconds) makes it possible to obtain 10⁹ – 10¹⁰ transformants per 1 μg of DNA. The electric current increases the permeability of cell membranes. Electroporation is utilized to introduce bacterial artificial Chromosomes (BACs) into E. coli. The optimal electroporation conditions vary among different bacterial species. The efficiency

of transformation is 10⁹ for small Plasmids, and 10⁶ for large ones (135 kbp).

Laser irradiation is employed to create micropores (2–120 nm in diameter) in the membranes of recipient cells receiving recombinant DNA.

Microinjection of recombinant DNA is performed using micromanipulators equipped with Glass microcapillaries, which puncture the cell membranes. This technique is extensively used in the generation of Transgenic Animals. Large DNA fragments are injected directly into the cell nuclei. Among all transformation methods, microinjection is the only one that allows a precisely controlled dosage of DNA molecules to be introduced into cells.

Bombarding cells with microparticles of tungsten, gold, or ice coated with recombinant DNA on their surface is called biolistic transformation. This method enables DNA delivery not only into nuclei, but also into Mitochondria and METABOLISM/14.html">Chloroplasts. Biolistic transformation is applied in Gene Therapy, DNA vaccine immunization, and The production of Transgenic Plants and animals.

Biological methods, in which gene transfer occurs via viral infection, are the most commonly used approaches for introducing recombinant DNA in Introduction/32.html">Genetic Engineering.

The following methods are used to identify transformed cells:

- testing for resistance to specific Antibiotics;

- Hybridization with a probe complementary to a specific region of the target gene;

- immunological assays to detect the specific protein product of the cloned gene.

To verify the expression of inserted genes, so-called reporter genes are employed, the products of which are easily detectable. Most frequently, the green fluorescent protein (GFP) gene or the β-galactosidase gene is used as a reporter. β-galactosidase cleaves the artificial substrate X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) to produce a blue reaction product. Bacterial colonies containing recombinant plasmids with the β-galactosidase gene appear blue in color.