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Method for breeding transgenic buffalos by using somatic cell nuclear transfer technology

A somatic cell nuclear transfer and transgene technology, which is applied in the production of transgenic cloned buffaloes and in the field of animal biology, can solve the problems of restricting the development of the buffalo milk industry, low milk production and reproduction rate, etc.

Inactive Publication Date: 2011-11-23
GUANGXI UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the milk yield and reproduction rate of buffalo breeds in my country are low, which restricts the development of the buffalo milk industry, and it is urgent to use embryo engineering technology to improve its breed

Method used

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  • Method for breeding transgenic buffalos by using somatic cell nuclear transfer technology
  • Method for breeding transgenic buffalos by using somatic cell nuclear transfer technology
  • Method for breeding transgenic buffalos by using somatic cell nuclear transfer technology

Examples

Experimental program
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Effect test

preparation example Construction

[0020] 2. Preparation of transgenic cloned buffalo:

[0021] Buffalo ovaries were collected from the slaughterhouse and placed in a + , Ca 2+ ,, Na + , Mg 2+ Put it in a thermos bottle of 35-37°C normal saline, send it to the laboratory within 4 hours, wash it with 37°C normal saline, cut off the suspensory ligament around the ovary, and use a 10ml syringe with a 12-gauge needle to extract Oocytes in 6 mm follicles. Under a solid microscope, the cumulus oocyte complex with uniform cytoplasm and complete compact cumulus cells was selected, washed twice with egg washing liquid, and then put into 1.5ml buffalo in vitro maturation solution (TCM-199+5% OCS+0.1μg / ml FSH) in a 30×10mm glass dish at 38.5°C, 5% CO 2 In vitro maturation in an incubator with maximum saturated humidity for 22 hours.

[0022] Select the buffalo oocytes with the first polar body, and place 10-15 oocytes in a group of 5 μg / ml cytochalasin B operating solution droplets, and cover with paraffin oil. Fin...

Embodiment

[0028] In 2010, we transplanted 72 fresh or frozen / thawed buffalo transgenic cloned blastocysts expressing the EGFP gene into the uterine horns of 36 recipient buffaloes, and each recipient cow transplanted 2 blastocysts, of which 2010 Three recipient buffaloes transplanted on January 28, February 5 and April 3 were pregnant, and after more than 300 days of full-term pregnancy, they gave birth to two bull calves on December 19, 2010. One of them survived and the other died. One bull calf was born on January 4, 2011 and March 19, 2011. Under the irradiation of ultraviolet light, the heads and limbs of these transgenic cloned buffaloes obviously expressed the EGFP marker gene. This example proves that the method of the present invention for producing transgenic cloned buffalo by somatic cell nuclear transfer technology is feasible.

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Abstract

The invention discloses a method for breeding transgenic buffalos by using somatic cell nuclear transfer technology, which comprises: obtaining buffalo fetal fibroblasts (BFF) by separation from buffalo fetuses and culture, implanting a transgenic vector (pCE-EGFP-IRES-neo), which carries a neomycin resistant gene and an enhanced green fluorescent protein (EGFP) gene, into the BFF by an electroporation process, and selecting BFF into which the EGFP gene is transferred by using G418; transplanting the BFF into which the EGFP gene is transferred into denucleated oocytes of the buffalo to construct cloned embryos, treating the cloned embryos for 5 minutes with ionomycin at a concentration of 5 mu mol / L, culturing cloned embryos in 6-dimethyl-aminopurine at a concentration of 2mmol / L for 3 hours, performing chemical activation treatment, co-culturing the cloned embryos in granulose cell monolayer liquid drops for 6 to 7 days, and obtaining the transgenic cloned blastaea; selecting transgenic cloned blastaea in which the EGFP is expressed under a reversed fluorescence microscope, and transplanting the transgenic cloned blastaea into receptor buffalos; and obtaining the transgenic buffalos after full-term pregnancy. After the irradiation by an ultraviolet lamp, the EGFP marker genes are obviously expressed in the heads and limbs of the transgenic buffalo calves, and this proves the method disclosed by the invention can successfully breed transgenic cloned buffalos.

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. Specifically, it is a method for producing transgenic cloned buffalo by using somatic cell nuclear transfer technology. Background technique [0002] Transgenic animal technology is a technology in which exogenous genes are introduced into the genome of recipient animals by means of genetic engineering technology, and are stably expressed in vivo, and the exogenously introduced genes can be stably passed on to offspring. Its main feature is that humans change the genetic structure of animals purposefully, in a planned, based, and predictable way, and endow transgenic animals with new phenotypic characteristics. [0003] Buffalo is an important livestock with great development prospects in southern my country, because of its strong adaptability, high temperature and high humidity resistance, strong dis...

Claims

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Application Information

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IPC IPC(8): C12N15/873C12N15/85A01K67/027
Inventor 石德顺陆凤花罗婵李楠韦英明刘庆友李湘萍蒋建荣
Owner GUANGXI UNIV
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