精子介导的基因转移法 z-bo 2021-10-14 320

　　Sperm-mediated gene transfer (sperm-mediated gene transfer, SMGT) can be pre-cultured with foreign DNA through in vitro fertilization, intracellular sperm injection or vasdeferens injection, and the sperm can be combined with the foreign DNA. To do it, the sperm that has absorbed the foreign DNA enters the egg and integrates the foreign DNA into the chromosome. This work started in Italy.

　　In 1989, Italian scientist Lavitrano and his colleagues first reported the use of mouse sperm as a carrier to combine sperm with the chlorophenol acetyltransferase (CAT) gene. After the sperm absorbs the CAT gene, the mouse egg cells are fertilized in vitro with the sperm, and then the fertilized egg cells are transferred to pseudopregnant mice. The CAT gene is detected in 30% of newborn mice. Gene transfer through sperm is also an effective method to produce genetically modified sheep, cattle and pigs, with a success rate ranging from 50% to over 80%. At present, sperm-mediated gene transfer can transfer up to 500 kb of foreign DNA into related animal genomes. Lavitrano successfully transferred the human decay promoter (hDAF) gene to the pig genome using a sperm-mediated transgenic method. Gene transfer through sperm can achieve relatively high gene transfer efficiency, which can exceed 50%.

　　The method is simple, easy to implement, and does not require an expensive micromanipulator. Gene transfer via sperm can be achieved in many ways. An important factor in the production of transgenic animals by sperm-mediated gene transfer is the effective combination of foreign DNA and sperm.

　　The following is a method to promote the combination of foreign DNA and animal sperm:

　　(1) Co-cultivation method: directly mix the semen with foreign DNA (20μg/ml) or dilute the semen to a concentration of 1000000-100000000/ml, and then incubate the DNA mixture for 20-40 minutes. Using this direct incubation method, the sperm that adsorbed foreign DNA accounted for about 6.3%, and the sperm remained active after the DNA was adsorbed. In 1991, in Horan's experiment on pig sperm, DNA and sperm were tightly bound, and each sperm was bound to about 3.8 x 100 DNA molecules, and the motile sperm captured the DNA more effectively. being shown. Linear DNA binds to sperm more easily than circular DNA. (

　　(2) Physical/chemical methods: Through physical/chemical methods such as repeated freezing and thawing and contact with detergents, the combination of foreign genes and sperm heads is greatly improved, and foreign DNA is absorbed. Can be improved.

　　(3) Antibody method: In order to promote DNA uptake, foreign DNA can bind to sperm by non-covalently binding with antibodies that recognize sperm surface proteins.

　　(4) Mix Tn5 (troponin) transposase with isolated sperm head or round sperm cell. The gene can be easily inserted into mouse chromosomes.