Molecular biology method of early embryo sex determination of transgenic dairy cattle

A technology of molecular biology and genetic modification, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high semen price, slow separation speed, and semen intolerance to freezing

Inactive Publication Date: 2012-05-30
TIANJIN MENGDE GROUP
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  • Abstract
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Problems solved by technology

[0003] So far, sex control methods are mainly divided into two aspects: one is the separation technology of X and Y sperm, combined with artificial insemination, to control the sex of offspring, but the current se

Method used

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  • Molecular biology method of early embryo sex determination of transgenic dairy cattle

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Embodiment Construction

[0022] 1. Sample preparation and storage

[0023] Isolate 4-6 cells from bovine embryos to be tested, wash them with sterile ultrapure water for 3 times, put them into a 0.5mL PCR tube, add 10uL of sterile ultrapure water, boil at 100°C for 10min-15min, and immediately place After cooling in crushed ice, centrifuge at 4°C for 5 min, and take the supernatant and store it in a refrigerator at 4°C for later use.

[0024] 2. Primer design

[0025] Y-chromosome-specific primer Forward: 5′-TGATAGTTGATCCCCACAGAAGG-3′

[0026] Reverse: 5′-TGAACTTACAAGCAGCTGAGG-3′

[0027] Autosome-specific primer Forward: 5′-TGAGGCATGGAACTCCGCTTG-3′

[0028] Reverse: 5′-GGTGGTTCCACATTCCGTAGG-3′

[0029] 3. PCR reaction

[0030] Amplify with Y-chromosome-specific primers and autosome internal standard primers. The total volume of the reaction system for PCR amplification is 20uL, of which 10×PCRBuffer 2uL, the concentration of 4dDNTP is 200pmol / L, and th...

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Abstract

The invention belongs to the technical field of animal reproduction, and in particular relates to the field of early embryo sex determination of transgenic dairy cattle. The invention discloses a molecular biology method of early embryo sex determination of transgenic dairy cattle, which comprises the following steps of: incising 4-6 cells from an embryo to be detected by applying a micromanipulation technology, washing the incised cells by using sterile ultra-pure water and putting the cells in a PCR (Polymerase Chain Reaction) tube; adding a proper amount of the sterile ultra-pure water, boiling and placing in trash ice; cooling, centrifuging at a low temperature, and taking a supernatant liquor to perform PCR reaction; detecting PCR amplification products by using agarose gel electrophoresis so as to obtain an electrophoretogram; observing the electrophoretogram, determining the embryo as a bull when a 131bp stripe and a 250bp stripe are simultaneously existent in the electrophoretogram, and determining the embryo as a cow when only a 250bp stripe is existent in the electrophoretogram.

Description

technical field [0001] The invention belongs to the technical field of animal reproduction, in particular to the field of sex identification of bovine early embryos. Background technique [0002] In order to give full play to the potential of transgenic cows and expand the scale of transgenic cows, the reproductive speed of transgenic cows can be improved through embryo transfer technology, and the cost of producing transgenic cows can be greatly reduced through embryo transfer technology. Gender identification of early embryos of transgenic dairy cows, and then transplantation, can effectively inherit foreign genes to the next generation, which has important scientific research value and commercial value in production. [0003] So far, sex control methods are mainly divided into two aspects: one is the separation technology of X and Y sperm, combined with artificial insemination, to control the sex of offspring, but the current separation speed is too slow (1.8×107 / h), the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王洋马毅张俊瑜王巍许静董钧
Owner TIANJIN MENGDE GROUP
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