Method for cloning buffalo somatic cell

A technology of somatic cell cloning and donor cells, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., which can solve the problems of low developmental potential and failure of eggs

Inactive Publication Date: 2006-02-15
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although somatic cell cloning has been successfully performed in various animals such as cattle, mice, rats, pigs, goats, horses, and mules, buffalo clones of this species ( Buffaloes, including embryonic cell cloning) have so far not been internationally successful

Method used

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  • Method for cloning buffalo somatic cell
  • Method for cloning buffalo somatic cell
  • Method for cloning buffalo somatic cell

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Experimental program
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Effect test

Embodiment 1

[0014] On December 5, 2003, we used the method of the present invention to subculture (3 generations) female buffalo fetal fibroblasts with 0.1 μg / ml Aphidicolin culture treatment for 24h, then starved culture with the culture solution containing 0.5% FCS 5d. On December 11, 2003, these cultured cells were transplanted into enucleated local buffalo oocytes to construct recombinant embryos. The recombinant embryos were cultured for 6 days to form blastocysts, which were transplanted into the recipient buffalo uterus on December 17, 2003. After 343 days of gestation, a female cloned buffalo was obtained by caesarean section on November 19, 2004. The cloned cow weighed 29kg and had the same sex as the donor cell. According to the microsatellite DNA identification, the cloned buffalo was completely identical to the donor cell.

Embodiment 2

[0016] On March 31, 2004, we treated subcultured (fifth generation) adult buffalo ovarian granulosa cells with 0.1 μg / ml Aphidicolin for 24 hours, and then starved them for 3 days with a medium containing 0.5% FCS. On April 4, 2004, these cultured buffalo ovary granulosa cells were used as donor cells to recombine with enucleated native buffalo oocytes to form reconstituted embryos, which were cultured in vitro for 6 days to form cloned blastocysts. On April 10, 2004, the cloned blastocyst was non-surgically transplanted into the uterus of a recipient buffalo. After 348 days of gestation, a female cloned buffalo was born naturally on March 17, 2005. The cloned buffalo weighs 23kg, has a body length of 86cm, a body height of 62.5cm, and the sex is the same as the donor cell. Microsatellite DNA identification confirmed that the cloned buffalo was indeed derived from the donor cell.

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Abstract

Disclosed is a method for cloning buffalo's body cells comprising the following steps: charging calf follicular fluid and epithelium cell growth factor into maturation to cultivate acceptor oocyte, enucleating under spindle image system, cultivating and processing donor cells with DNA synthesized depressant Aphidicolin, carrying out hungry cultivation, activating the recombinant embryo with ionomycin and 6-dimethoxy aminopurine, and proceeding embryo extracorporal cultivation with modified TCM 199 and estrus cow's serum.

Description

technical field [0001] The invention belongs to the animal biotechnology of bioengineering, in particular to the field of buffalo breeding and directional genetic improvement. Background technique [0002] Animal somatic cell cloning is an epoch-making breakthrough in the field of biotechnology at the end of the 20th century. It not only proves that the differentiated adult animal somatic cells can return to the initial state of development under suitable conditions and develop into a complete individual. Moreover, it has very broad application prospects for accelerating the genetic improvement of animals and improving the efficiency of transgenic animals. Although somatic cell cloning has been successfully performed in various animals such as cattle, mice, rats, pigs, goats, horses, and mules, buffalo clones of this species ( Buffaloes, including embryonic cell cloning) have so far not been internationally successful. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N15/877
Inventor 石德顺陆凤花杨素芳韦英明
Owner GUANGXI UNIV
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