The discovery of homologous recombination in biology has laid a solid theoretical foundation for gene targeting, and the development of embryonic stem cell technology has promoted the wide application of gene targeting. Homologous recombination, also known as general recombination or nonspecific recombination, refers to the process of exchanging genetic information between similar DNA, and foreign DNA fragments can be exchanged with corresponding fragments of host genome (i.e. recombination). Gene targeting usually refers to an exogenous DNA introduction technique in which DNA fragments with known sequences are homologous recombined with genes with the same or similar sequences in the genome of the recipient cell, integrated into the genome of the recipient cell and expressed.
Main strategies:
(A) complete gene knockout strategy
PNS vector is still the most commonly used strategy for gene targeting of embryonic stem cells. With the help of positive selection marker gene, the target gene is usually inserted into the exon with the most critical function, or the most important functional domain of the target gene is deleted by homologous recombination, so that the target gene is completely eliminated.
(b) Large-scale random gene knockout-gene capture
Using gene capture to establish ES cell bank with random insertion mutation can save a lot of work and cost of screening genome library and constructing specific targeting vector, and analyze the function of mouse genome more effectively and quickly. In addition, another disadvantage of gene knockout by gene capture method is that it is impossible to carry out fine genetic modification on genes.
(3) introduction of fine variation
Many human diseases are due to the loss of gene function, and many are due to gene over-expression or function acquisition. For the latter, the corresponding disease model cannot be obtained by gene knockout. To this end, researchers have invented various methods to introduce subtle mutations, such as inserting stop codons or replacing amino acids in mouse genomes.
(1) Hit-and-go strategy, also known as advance and retreat strategy.
(2) Double replacement method
(3) "mark and replace" method
(4) introducing point mutation through Cre-LoxP system.
Main process:
Firstly, ES cell lines were obtained, and the mutant culture medium target ES cells designed by researchers were obtained by homologous recombination technology.
Genetically modified ES cells were introduced into recipient embryos by microinjection or embryo fusion.
The genetically modified ES cells still maintain the totipotency of differentiation and can develop into the germ cells of chimera animals, so that the modified genetic information can be transmitted through the germ line.
The obtained mutant animals with specific modifications provide researchers with a special research system, enabling them to study the functions of specific genes in living organisms.