BIOTECHNOLOGY - Inshyna N.M. - 2009

CHAPTER 1. GENETIC ENGINEERING

Animal Genetic Engineering. Technology for Producing Transgenic Animals

In 1973, the term "transgenesis" was introduced to describe The transfer of genes from one Organism to another, including evolutionarily distant species. Transgenesis makes it possible to improve the genotype of existing livestock breeds and develop breeds with novel traits. Of particular current relevance is the creation of domestic animals with hereditary resistance to bacterial and viral infections, as well as parasitic infestations.

Rudolf Jaenisch produced the first Transgenic Animals in 1974 in Cambridge (USA) by microinjecting the DNA of the simian virus SV40 into mouse embryos.

In 1980, American scientist Gordon proposed using DNA microinjection into the pronucleus of a zygote to create transgenic animals. This approach marked the beginning of the widespread development of transgenic animal technology. In 1985, the first transgenic farm animals—rabbits, sheep, and pigs—were obtained in the USA using DNA microinjection into the zygote pronucleus. In Russia, the first transgenic animals appeared in 1982.

To date, alongside microinjection, various other Methods are used to produce transgenic animals: infection of Cells with recombinant Viruses, electroporation, and biolistic transfection (bombardment of cells with gold or tungsten particles coated with recombinant DNA).

Genetic modification of animals using Recombinant DNA technology is based on introducing a cloned Gene into The Genome of a Cell capable of contributing to the germline. By crossing transgenic offspring, homozygous lines of transgenic animals can be established.

The technology for producing transgenic animals involves the following stages:

- introducing the cloned gene into The Nucleus of a fertilized egg;

- implanting the egg into the Uterus of a recipient animal;

- screening for offspring that carry the cloned gene in all cells;

- crossing animals carrying the cloned gene to establish a new genetic line.

An alternative approach involves isolating Embryonic Stem Cells and transfecting them with the cloned gene. Cells containing the target gene are selected and cultured, and then introduced into embryos at early developmental stages. Embryonic stem cells can give rise to any cell type, including germline cells. Artificial Chromosomes based on Yeast are used to transform embryonic stem cells. This method has been successfully used to produce mice that synthesize human Antibodies.

Transgenic laboratory animal models are used to investigate various gene Functions, The regulation of their expression, phenotypic manifestations, and mutagenesis. Transgenic animals with targeted gene disruptions can serve as models for studying human diseases at THE MOLECULAR LEVEL. For instance, the "knockout" (targeted disruption) of the rhodopsin gene in mice inactivates retinal rods, mimicking the human condition known as retinitis pigmentosa. Such animals allow researchers to study retinal degeneration processes as well as the therapeutic effects of pharmacological agents. Over 250 knockout mouse lines have been created and are widely used as models for studying various human pathologies.

Certain transgenic animals (such as cows, sheep, and goats) can serve as unique "bioreactors" for producing cloned gene products secreted into their milk. One liter of milk from such animals can contain anywhere from tens of milligrams to several grams of biologically active human Proteins. Milk from transgenic animals can be a source of therapeutic drugs and Enzymes. For example, transgenic goats produce human alpha-1 antitrypsin and antithrombin III. In Russia, transgenic sheep have been developed that synthesize the enzymes chymosin and rennin. The concentration of chymosin in the milk of these transgenic animals reaches 300 mg/L. Chymosin and rennin are widely used in cheesemaking. A herd of 20 transgenic cows can yield approximately 100 kg of recombinant protein C per year, which is used for thrombosis Prevention. A single transgenic cow is sufficient to meet the annual demand for Blood clotting factor IX, administered to hemophilia patients. However, generating transgenic cows is time-consuming: raising a sexually mature animal from a fertilized egg takes approximately 2 years.

Stages of producing transgenic cows:

- oocyte collection;

- in vitro oocyte maturation;

- in vitro oocyte Fertilization;

- centrifugation of fertilized eggs (to visualize the male pronucleus and concentrate the yolk);

- DNA microinjection into the male pronucleus;

- in vitro embryo development to the blastocyst stage;

- embryo transfer to recipient females;

- DNA screening of offspring for the presence of the transgene.

In test experiments involving 2,470 oocytes, two transgenic calves were obtained. This indicates the low efficiency of the method. Currently, work is underway to improve the transgenesis Procedure.

Modern gene transfer methods are not sufficiently efficient: obtaining a single transgenic animal requires DNA microinjection into 40 mouse zygotes, 90 goat zygotes, 100 pig zygotes, 110 sheep zygotes, and 1,600 cow zygotes. Out of 1,000 implanted fertilized eggs, 30 to 50 transgenic animals develop. The mechanisms of exogenous DNA Integration and The formation of autonomous replicons during transgenesis remain unknown. The integration of transgenes in each transgenic animal occurs at different chromosomal sites, and may involve the insertion of either a single copy or multiple copies of the transgene.