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Method for efficiently preparing cloned embryos of sheep

A technology for cloning embryos and sheep, applied in the field of embryo transfer, can solve the problems of waste of embryos, low embryo fusion rate, high cost, etc., and achieve the effects of improving efficiency, improving maturation rate, and improving success rate.

Inactive Publication Date: 2011-11-02
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Moreover, the embryo fusion rate is low, resulting in a large amount of embryo waste and high cost. Therefore, it is urgent to develop a set of efficient methods for preparing sheep cloned embryos, including the preparation of high-quality nuclear donor cells, and the initial screening of sheep oocytes by stirring method to improve the quality of sheep. Maturation efficiency of oocytes, increase efficiency of nuclear transfer operation, increase fusion efficiency

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0015] The maturation of embodiment 1 sheep oocyte

[0016] Place freshly collected sheep ovaries in sterilized physiological saline at 30°C-35°C, and transport them back to the laboratory within 1h-2h. Wash the ovary with pre-warmed saline, and use a 10ml syringe with an 8# needle, which contains egg suction solution (containing penicillin (100IU / ml), streptomycin (100IU / ml), 0.5% FBS, 25mg / ml heparin TCM199), absorb follicles with a size of 2mm-6mm on the surface of the ovary, collect the follicular fluid in a beaker, let the follicular fluid coagulate into flocs after standing for 5 minutes, gently stir the agglomerates with a thin rod, and pull the agglomerates after the agglomerates are aggregated Crumble and continue stirring. Place the stirred follicle fluid under a stereo microscope to collect sheep oocytes wrapped with more than two layers of cumulus cells for maturation, wash the collected COCs in the maturation solution for 3 times, and place them in 100 μl of matu...

Embodiment 2

[0018] Example 2 Preparation of Donor Cells

[0019] The skin of the head of the 40-day-embryonic sheep fetus was taken by laparotomy and shredded as much as possible. The tissue pieces were inoculated at the bottom of a 50 ml Karl Fischer flask at intervals of about 5 mm. About 25 pieces were inoculated in each bottle. Stick the cut surface of the block on the bottom of the bottle to make the tissue block adhere to the bottom of the bottle, put it into the incubator, let it stand for 3h-4h, add 5ml of DMEM / F12 culture solution containing 15%FBS, put it in 37℃, 5%CO2 1. After standing in a saturated humidity incubator for 3 days, replace 5ml of DMEM / F12 fresh culture medium containing 15% FBS. After that, change the culture medium every 3 days. From 3 days to 4 days, it is obvious that there are cells growing around the tissue block, and the cells expand to the surrounding area. From 5 days to 8 days, the cells grown from the tissue block are confluent and can be digested and ...

Embodiment 3

[0020] Example 3 Production of cloned embryos

[0021] 1. Place the mature sheep oocytes after 22h-24h in serum-free H-M199, blow and suck the mature sheep oocytes repeatedly with a pipette, until the cumulus cells around the mature sheep oocytes partly fall off , and then transfer the mature sheep oocytes into H-M199 containing 0.25% hyaluronidase for 5 minutes, and then wash the sheep oocytes with H-M199 containing 10% FBS until there are no cumulus cells. Take the discharge of the first polar body as the maturity standard, and check the maturity under a microscope.

[0022] 2. Select naturally growing confluent fibroblasts within 5 passages as nuclear donors and digest with 0.05% TE at 37°C for 1 min. After the digestion is terminated, collect the cells by centrifugation, wash 3 times with operating solution and store in a 4°C refrigerator for later use.

[0023]3. Select sheep oocytes that discharge the first polar body and have normal morphology, place them in 7.5 μg / ml ...

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Abstract

The invention discloses a method for efficiently preparing high-quality cloned embryos, belonging to the field of embryo transplanting methods. The method provided by the invention comprises the following steps of: agitating and screening to obtain sheep oocytes in the same cell period and transplanting a supplier cell into the sheep oocytes through utilizing microscopy injection; then treating by utilizing a fusion solution and putting the treated embryo into a mature solution to be cultured to form a fused embryo; and chemically activating and then starting and reconstructing embryo cleavage. According to the method provided by the invention, the sheep oocytes which are in the same cell period and have the uniform mature time through an agitating method are acquired, a new method for picking the good sheep oocytes is provided and the mature rate of the sheep oocytes is improved. According to the method provided by the invention, the operation efficiency of nuclear transfer is improved by utilizing a method of simultaneously denucleating through extrusion and carrying out nucleus injection by utilizing the same injection needle. The fusion efficiency is improved to more than 95% by using the fusion solution which is developed by the method provided by the invention, the successful rate of the cloned embryo is greatly improved and the subsequent embryo development is not influenced. The method provided by the invention has the advantages of reducing the loss of the embryos in the process of preparing the cloned embryo and improving the efficiency of preparing the sheep cloned embryos; and the method provided by the invention is a method for efficiently preparing the sheep cloned embryos with good quality and has a wide application prospect.

Description

technical field [0001] The invention relates to a method for efficiently preparing sheep cloned embryos, belonging to the field of embryo transplantation methods. Background technique [0002] Currently, there are two main methods for producing mammalian clones: embryo segmentation and nuclear transfer. The cloned sheep "Dolly", as well as various cloned animals bred by scientists from various countries, all adopted the technology of cell nucleus transfer. The so-called nuclear transfer refers to the process of transplanting the nuclei of embryos or adult animals at different developmental stages into oocytes through microinjection and cell fusion methods, reconstituting embryos and making them mature. Different from embryo segmentation technology, nuclear transfer technology, especially serial nuclear transfer technology can produce unlimited genetically identical individuals. Because nuclear transfer is an effective method to produce cloned animals, it is often called an...

Claims

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Application Information

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IPC IPC(8): C12N15/877
Inventor 周欢敏刘羿羿张东
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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