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
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[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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