A y-chromosome marking method and its application
A Y-chromosome and marker technology, which is applied in biochemical equipment and methods, DNA/RNA fragments, microbial determination/inspection, etc., can solve problems such as inability to meet production applications, and achieve the effect of improving breeding efficiency and accurate early identification.
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[0084] 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.
[0085] 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.
[0086] Zimmerman's solution was prepared as follows: containing 0.3M mannitol, 0.1M MgSO 4 , 0.05M CaCl 2 , 0.5mM HEPES, 0.05g / 100mL BSA aqueous solution, pH 7.2, filtered with a 0.22μm filter membrane.
[0087] A23187 solution was purchased from sigma, the product number is C9275.
[0088]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 so...
Embodiment 1
[0104] Example 1, the preparation of somatic cells of a breeding bull that precisely modifies the sex control gene SRY of the Y chromosome with the enhanced green fluorescent protein gene
[0105] 1. Establishment of Bull Fibroblast Cell Line
[0106] 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 s...
Embodiment 2
[0154] Example 2, Utilizing Enhanced Green Fluorescent Protein Gene to Precisely Modify the Y Chromosome Sex Control Gene SRY Somatic Cell Breeding of Somatic Bull Breeding Bull
[0155] 1. Maturation and culture of oocytes
[0156] 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 in the incubator for 18-20 hours, mature cells are obtained, and the mature cells are placed in a tube containing 0.1% hyaluronidase by volume and shaken for 2-3 minutes, and then gently blown with a glass ...
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