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Clone embryo transplantation method

A technology for cloning embryos and subspecies, applied in the field of bioengineering, can solve the problems of no special selection of subspecies and low conception rate, and achieve the effect of increasing compatibility, improving conception rate and facilitating conception.

Inactive Publication Date: 2009-03-11
SHANGHAI JIAO TONG UNIV AFFILIATED CHILDRENS HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventor thus thinks that the current conventional embryo transfer method does not have a special choice for the subspecies, as long as the surrogate is in synchronous estrus, it will be transplanted, so due to the difference in the internal environment between the cloned embryo cytoplasm and the subspecies of the surrogate, May lead to reduced conception rates

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 , Preparation of recipient oocytes

[0019] Cattle and dairy cows are two subspecies of the genus BOS primigenius in the family Bovidae of the order Artiodactyla Mammalia. Egg extraction is carried out from the living body of heifers of yellow cattle or dairy cows. For the specific method, see the Chinese invention patent application with application number 200510023510.1.

[0020] Pour the collected solution into the egg sorting cup, and use calcium-magnesium-free Duchenne's phosphate buffer solution (DPBS, containing 8gNaCl, 0.2g KCl, 1.44g NaCl in 1 liter of deionized water 2 HPO 4 , 0.24g KH 2 PO 4 , 30g BSA and 2000u heparin), sorted out oocyte complexes (cumulus oocyte complexes, COCs) and moved them into the culture dish, graded the COCs under a stereo microscope, selected uniform cytoplasm, tightly arranged surrounding cumulus cells, and contained oocytes. Surround the oocytes with more than three layers, put into the maturation medium (TCM-199 (G...

Embodiment 2

[0021] Example 2 , Preparation of donor cells

[0022] 1. Take young bovine fibroblasts and cumulus cells respectively, and culture them with DMEM / F12 (Gibco Company, product number 11039-021) containing 10% FBS.

[0023] ① Primary culture of fibroblasts

[0024] Take newborn bovine ear tissue, put it in 0.9% normal saline containing double antibody (2% penicillin streptomycin), soak and disinfect the ear tissue with tincture of iodine and 75% alcohol, wash 3 to 5 times with sterile normal saline, cut into pieces, Add 0.25% trypsin, digest at 37°C for 2h, add 10% FBS-containing DMEM / F12 medium, 37°C, 5% CO 2 1. Primary culture in a saturated humidity incubator, observe the cell attachment under an inverted microscope, and then replace the medium to continue the culture.

[0025] ②Primary culture of cumulus cells

[0026] Put COCs matured for 20 hours (see Example 1) into calcium and magnesium-free DPBS containing 0.5% hyaluronidase, digest for 1 to 2 minutes, and at the s...

Embodiment 3

[0031] Example 3 , nuclear transfer

[0032] 1. Preparation of ACM culture medium required for nuclear transfer

[0033] The formula of ACM culture medium is as follows:

[0034] NaCl (Sigma S-5886) 0.580g

[0035] KCl (Sigma P-5405) 0.022g

[0036] NaHCO 3 (Sigma S-5761) 0.209g

[0037] L-Glutamine (Sigma G-8540) 0.015g

[0038] CaCl 2 2H 2 O (Sigma C-7902) 0.004g

[0039] Pyruvate (2.2mg / ml stored in PBS) 2ml

[0040] BME amino acid (Sigma B-6766) 2ml

[0041] MEM amino acid (Sigma M-7145) 1ml

[0042] Penicillin streptomycin (GIBCO-BRL company) 1ml

[0043]Lactic acid (Sigma L-7900) 14μl

[0044] Phenolsulfonphthalein (Sigma P-0290) 100μl

[0045] BSA (fatty acid free) (Sigma A-6003) 0.300g

[0046] Dissolve all dry powder ingredients except BSA in a beaker filled with 70ml of embryo water. To prevent BME / MEM from being contaminated, add all liquid ingredients and cover the beaker with a cover for stirring. Then add BSA dry powder and continue stirring until...

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Abstract

The invention discloses a clone embryos transplanting method. In the method, a surrogate motherhood which is the same with the cytoplasm subspecies of the receptor of the clone embryos is selected for carrying out clone embryos transplanting. The method increases the compatibility between the clone embryos and the surrogate motherhood as well as can remarkably improve the conception rate of the clone embryos after being transplanted.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a cloned embryo transplantation method. Background technique [0002] Embryo transplantation in the usual sense refers to taking out the early embryos of a fine-bred female animal after mating, and transplanting it into another female animal that is in synchronous estrus, so that it can continue to develop into a new individual, also known as gestation by abdomen. The method can greatly improve the utilization rate and reproduction rate of excellent female animals, and fully tap the genetic and reproductive ability of female animals. [0003] Animal cloning technology is to transplant donor somatic cell nuclei into the cytoplasm of enucleated oocytes through micromanipulation and / or cell fusion to reconstitute embryos (reconstructed embryos). The reconstituted embryo is cultivated and developed into a blastocyst, and then the blastocyst is transplanted into th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61D19/04C12N15/06C12N5/06C12N15/877
Inventor 曾溢滔赵磊文黄淑帧
Owner SHANGHAI JIAO TONG UNIV AFFILIATED CHILDRENS HOSPITAL
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