Genetic modification of embryonic stem cells: efficiency increases
Biologists at the University of California at San Diego, working under the guidance of Professor Yang Xu, have developed an effective method of genetic modification of human embryonic stem cells using artificial bacterial chromosomes that deliver defective gene variants into cells. The cell lines obtained using this method can be used both as models for studying human genetic diseases and for screening potential therapeutic agents.
The results of the work are published in the January issue of the journal Cell Stem Cell in the article "Modeling Disease in Human ESCs Using an Efficient BAC-Based Homologous Recombination System".
The effectiveness of most existing methods of changing cell genotypes is extremely low and ensures the appearance of genetic modifications in less than 1% of the treated cells. To solve this problem, the authors used artificial bacterial chromosomes – synthetic ring DNA replicated by bacteria along with their own genetic material. Commercially available artificial bacterial chromosomes can be modified inside bacterial cells, for example, by embedding modified copies of the studied genes in them. When introduced into human cells, such chromosomes can attach to human DNA and enter into the process of homologous recombination – the exchange of complementary sites.
The advantage provided by the use of artificial bacterial chromosomes is due to the presence of long sequences extending both end sections of the studied gene and increasing the probability of interaction of chromosomes with human DNA and gene exchange by up to 20%. The effectiveness of other genetic approaches is limited to shorter DNA segments.
As a result of the two-fold application of the proposed approach, the authors created a line of human embryonic stem cells carrying two defective copies of the p53 tumor suppressor gene.
They also successfully integrated the ATM gene into the genome of cells, the mutation of which leads to the development of ataxia-telangiectasia in humans – a disease characterized by a complex of systemic defects, including a high risk of cancer, degeneration of certain types of brain cells and degrading telomeres (protective end sections of chromosomes, the length of which determines the number of cell divisions).
Genetically modified mice with two damaged copies of the ATM gene are characterized by some symptoms of ataxia-telangiectasia, but they do not have degeneration of neurons and shortening of telomeres. The authors believe that the embryonic stem cells of the line they obtained are a more adequate model for studying this disease. They have already established that such cells have degraded chromosomes. The study of other symptoms of the disease, such as the degradation of certain types of neurons, is the purpose of their further work.
The researchers believe that their proposed approach can be used to modify other genes in other human embryonic stem cell lines. Initially, they used a line for experiments that quickly forms new colonies from individual cells, but they managed to repeat the procedure on another H9 cell line, which is difficult to manipulate.
Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on the materials of the University of California, San Diego: Biologists Develop Efficient Genetic Modification of Human Embryonic Stem Cells.
15.01.2010