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Y-chromosome modification method and application thereof

A Y chromosome, in vitro technology, applied in the field of Y chromosome modification, can solve the problem of no research on sex control of large animals, and achieve the effect of improving breeding efficiency

Active Publication Date: 2015-03-25
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although transgenic technology continues to advance, there is no research on the use of this technology for sex control of large animals

Method used

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  • Y-chromosome modification method and application thereof
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  • Y-chromosome modification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0081] The preparation method of the maturation solution is as follows: mix M199 medium and fetal bovine serum (FBS) according to the volume ratio of 9:1 to obtain a mixed solution, add 0.01U / mL bFSH (follicle-stimulating hormone), 0.01U / mL bLH (luteinizing hormone) and 1 μg / mL estradiol.

[0082] The preparation method of the operating solution is as follows: M199 medium and fetal bovine serum (FBS) were mixed at a volume ratio of 9:1 to obtain a mixed solution, and 7.5 μg / mL cytochalasin B was added to the mixed solution.

[0083] Zimmerman's solution was prepared as follows: containing 0.3M mannitol, 0.1M MgSO 4 , 0.05M CaCl2 , 0.5mM HEPES, 0.05g / 100mL BSA aqueous solution, pH7.2, filter with 0.22μm filter membrane

[0084] A23187 solution was purchased from sigma, the product number is C9275.

[0085] The CR1aa culture solution was prepared as follows: an aqueous solution of 114mM sodium chloride, 3.1mM potassium chloride, 26.2mM sodium bicarbonate, and 20.4mM sodium py...

Embodiment 1

[0101] Example 1. Preparation of Somatic Cells of Bull Breeding Bulls Precisely Modified by "Self-killing Sperm Containing Y Chromosome" Element

[0102] 1. Establishment of Bull Fibroblast Cell Line

[0103] Take the ear skin tissue of the Holstein bull, remove the hair on the back side of the lower edge of the ear, clean it with 70% ethanol water solution by volume, and then use a razor to pick an area of ​​1 cm from the back side of the lower edge of the ear 2 The left and right skins were placed in DMEM / F12 medium at 0°C and transported back to the laboratory as soon as possible, washed several times with PBS and 70% ethanol water solution by volume, and cut into 1mm pieces 3 For the left and right small pieces, wash them twice with DMEM / F12 and then plant them in batches in a 25cm medium containing 1mL DMEM / F12+10%FBS. 2 In the culture flask, add DMEM / F12+10% FBS to 6mL after the tissue block is firmly adhered to the wall, and store at 37°C, 5% CO 2 Culture in an incuba...

Embodiment 2

[0140] Example 2. Somatic cell breeding of bulls using the "self-killing sperm containing Y chromosome" element to precisely modify the Y chromosome sex control gene SRY somatic cell cloned bulls

[0141] 1. Maturation and culture of oocytes

[0142] The ovaries of adult cattle were collected from the slaughterhouse, placed in 30°C normal saline, and sent to the laboratory within 4 hours. After the ovaries were washed three times in 37°C PBS solution, a needle with a diameter of 0.7mm was used to extract a diameter of 2- 8mm follicles, recovery of cumulus-oocyte complexes (COCs) with uniform shape and compact structure, washed twice with maturation solution, and then 50-60 cumulus-oocyte complexes / well were placed in the 4-well plate of maturation solution at 38.5 °C, 5% CO 2 After maturing and culturing in the incubator for 18-20 hours, mature cells are obtained. Put the mature cells into a tube containing an aqueous solution of 0.1% hyaluronidase by volume and vibrate for 2...

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Abstract

The invention discloses a Y-chromosome modification method and an application thereof. The invention discloses an animal Y-chromosome modification method which comprises performing specific suicide component modification on the Y-chromosome of an isolated animal body cell by use of a TALEN method. The Y-chromosome modification method lays a foundation for selectively breeding animals of specific gender and improving the breeding efficiency.

Description

technical field [0001] The invention relates to a Y chromosome modification method and application thereof, belonging to the field of biotechnology. Background technique [0002] Animal sex control (sex control) technology is a biological technology that makes adult female animals produce offspring of the desired sex by artificially intervening in the normal reproductive process of animals. Sex control techniques are of great significance in livestock production. First, by controlling the sex ratio of the offspring, the maximum economic benefits of sex-restricted production traits (such as lactation) and gender-affected production traits (such as growth speed, meat quality, etc.) can be fully utilized. Secondly, controlling the sex ratio of offspring can increase the intensity of selection and speed up the breeding process. By controlling the sex of embryos, the phenomenon of heterosexual twin infertility in bovine embryo transfer can also be overcome, and the harm of sex-...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/11C12N15/877A01K67/027
CPCA01K67/027C12N15/09C12N15/11C12N15/877
Inventor 戴蕴平孙照霖丁方荣王海萍李京李玲
Owner CHINA AGRI UNIV
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