Method for producing transgenic rabbits with microscopic insemination technology
A transgenic and technical technology, which is applied in the field of directional genetic improvement of rabbit meat, can solve the problems of difficulty in obtaining transgenic embryos, low vitality of rabbit somatic cells, and affecting the economic benefits of rabbit breeding.
Inactive Publication Date: 2011-11-16
GUANGXI UNIV
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Problems solved by technology
However, the slow growth rate and low slaughter rate of domestic rabbit breeds in our country directly affect the economic benefits of the rabbit industry. It is urgent to apply modern biotechnology to improve it and cultivate new rabbit breeds with good meat quality and high meat production rate.
Transgenic rabbits are produced by transgenic cloning technology. Due to the low viability of rabbit somatic cells, it is difficult to obtain transgenic embryos by nuclear transfer after gene transfection and culture screening.
Method used
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Embodiment 1
[0026] On November 9, 2010, we produced 35 transgenic rabbit embryos through micro-insemination technology, of which 23 (65.7%) expressed GFP at the 2-cell embryo stage, and transplanted the positive embryos into the oviduct of recipient female rabbits through surgery. After pregnancy, all 3 pups survived on December 12, 2010, and 2 of them were positive transgenic rabbits detected by molecular biology methods.
Embodiment 2
[0028] On December 7, 2010, we transplanted 38 positive embryos that had split to 2 cells into the fallopian tubes of two female rabbits in estrus at the same time, and they were all pregnant. On January 6, 2011, 7 pups were born. Among them, 4 survived. Then, a series of molecular biological identification tests were carried out on these 4 survived rabbits, and the results proved that 2 were positive transgenic rabbits.
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The invention relates to a method for producing transgenic rabbits with a microscopic insemination technology. The method comprises the following steps: collecting a seminal fluid through a false vagina process or the seminal fluid from an epididymis, suspending with a PBS (phosphate buffer solution), packaging into a cryovial after centrifugation washing, and sending to liquid nitrogen for saving; taking the cryovial with sperms out of the liquid nitrogen, unfreezing at room temperature, adding an exogenous DNA plasmid seminal fluid for being fully mixed with the freeze-thaw sperms to make the final concentration of DNA plasmids to be 30 ng / mul, and incubating for 10 min at a temperature of 4 DEG C to allow the sperms to be fully combined with DNA before a microscopic operation; and then injecting the sperms which breed the DNA into rabbit oocyte cytoplasm through the microscopic insemination technology, and putting in a culture solution and culturing for 2-4 days after carrying out chemical activation processing on microscopic inseminated oocytes. Transgenic rabbit embryos are successfully produced through the method of the present invention, and the positive rate of the embryos reaches 59.65%. Female rabbits are pregnant through embryo transplant and bears live little rabbits, the little rabbits are subjected to cellular and molecular biology identification, and a case that four transgenic rabbits express the exogenous genes is proved, so the positive rate is 57.14%.
Description
technical field [0001] The invention belongs to the field of animal biotechnology, especially the directional genetic improvement of rabbit meat quality. Specifically, it is a method for producing transgenic rabbits by micro-insemination technology. Background technique [0002] Rabbit is a widely used economic animal and experimental animal. It has the advantages of small size, fast reproduction speed and short production cycle. However, the slow growth rate and low slaughter rate of domestic rabbit breeds in our country directly affect the economic benefits of the rabbit industry. It is urgent to use modern biotechnology to improve them and cultivate new breeds of rabbits with good meat quality and high meat yield. Transgenic rabbits are produced by transgenic cloning technology. Due to the low viability of rabbit somatic cells, it is difficult to obtain transgenic embryos after gene transfection, culture and screening, and then nuclear transfer. The micro-insemination t...
Claims
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IPC IPC(8): C12N15/873A01K67/027
Inventor 陆凤花石德顺张顺刘庆友罗婵李湘萍
Owner GUANGXI UNIV