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Mammal body-cell neucleus transplanting method

A somatic cell nuclear transfer, mammalian technology, applied in the fields of transgenic animal technology, embryo engineering and developmental biology, can solve problems such as oocytes that have not yet been seen, and achieve the effects of improving reprogramming ability, quality and development.

Inactive Publication Date: 2006-08-09
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

But so far, there has been no report of mammalian cloning using hybrid F1 oocytes as recipient cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the preparation of donor nucleus cell

[0028] The cumulus cells or fibroblasts from the 1st to 5th generations of Holstein dairy cows were cultured with D-MEM / F-12 (Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS), and the cells grew to the bottom of the bottle When it reaches 80-90%, replace the culture medium with the culture medium containing 0.5% FBS and serum starve for 2-3 days. Then use 0.25% trypsin to digest the adherent cells for about 3 minutes, wash the digested cells twice, add an appropriate amount of TCM199+10% FBS and repeatedly pipette into a suspension of single cells to obtain donor nuclear cells. use.

Embodiment 2

[0029] Embodiment 2, the preparation of recipient oocyte

[0030] The bovines of each experimental group are carried out to get oocytes in vivo to obtain oocytes, and the specific method of living ovum extraction is mainly such as Petyim et al. (Petyim SR, et al.J.Vet.Med.A.Physio1.Pathol.Clin.Med.2000, 47:627-640), as follows:

[0031] Make the cow stand upright, disinfect and anesthetize. The whole process was carried out under B-mode ultrasound monitoring. Gently insert the probe with the egg collection needle into the vaginal vault, the operator pulls the ovary to the end of the B-ultrasound probe through the rectum through the rectal wall, pushes the egg collection needle, and at the same time, uses the foot switch to control the vacuum pump to suck the follicle fluid , the pressure is about 95mmHg, after that, rinse the ovum collection needle and catheter with DPBS (containing 3% BSA and 2u / ml heparin), and put the washing solution into the collection tube together. T...

Embodiment 3

[0033] Embodiment 3, nuclear transfer and fusion

[0034]The donor nuclear cells obtained in Example 1 and the oocytes obtained in Example 2 were moved into the operating solution (TCM199+10% FBS+5 μg / ml cytochalasin B), according to Park et al. (Park SH, et al.Mol Reprod Dev 2004; 68:25-34), the oocyte nucleus was removed under a microscope, and then the donor nucleus cells were transferred into the perivitelline space.

[0035] The specific operations of enucleation and nucleation are as follows: after the oocytes are moved into the operating solution for 20 minutes, the nucleus-donating cells and oocytes are placed on a glass slide together, and the oocytes are fixed with an egg-holding needle under a microscope. The polar body discharged by the mother cell is at the 3 o'clock position of the clock, and the enucleation needle is facing or slightly away from the polar body to remove the polar body and a part of the cytoplasm. Immediately after enucleation, move one donor ce...

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PUM

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Abstract

This invention provides a nucleus transplantation method for mammal somatic cells characterizing in taking the oocyte of cross-fertilized F1 generation of the mammal as the recipient somatic cell for nucleus transplantation, which can increase the re-programming ability of recipient cytoplast of nucleus transplantation of mammals obviously, improve the growth of re-structured embryo to increase the clone efficiency of somatic cells.

Description

technical field [0001] The invention relates to the fields of transgenic animal technology, embryo engineering and developmental biology. In particular, it relates to a method for mammalian somatic cell nuclear transfer. Background technique [0002] Animal cloning technology is to transplant donor somatic cell nuclei into the cytoplasm of enucleated oocytes through microsurgery and / or cell fusion to reconstitute embryos (reconstructed embryos). The reconstituted embryos are cultured and developed into blastocysts, and then the blastocysts are transplanted into the uterus of the host mother who is in estrus synchronously, and the animals are delivered after they mature. Cell cloning technology" or "somatic cell nuclear transfer technology" (Nuclear transfer, NT). [0003] Mammalian somatic cell cloning technology has made great progress after several years of development, and cloned animals such as sheep, goats, cows, pigs, mice, cats, and rabbits have been born one after ...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N15/873
Inventor 曾溢滔杨晓煜黄淑帧
Owner SHANGHAI JIAO TONG UNIV AFFILIATED CHILDRENS HOSPITAL
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