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Nucleus transplantation method

A cell nucleus and cell technology, applied in the field of cell nuclear transfer, can solve the problems affecting the developmental ability of reconstructed embryos, and achieve the effect of increasing the blastocyst rate and the fusion rate.

Active Publication Date: 2008-02-06
SHANGHAI INST OF MEDICAL GENETICS SHANGHAI CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventor thinks that the embryos currently obtained by conventional nuclear transfer methods (allogeneic nuclear transfer) contain different recipient and donor mtDNA haplotypes, and these haplotypes may affect the developmental ability of reconstituted embryos

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 , Amplification of full-length mitochondrial DNA

[0019] Extract peripheral blood samples from different young cattle, anticoagulate with heparin, separate and extract DNA with phenol-chloroform, use the extracted DNA as a template, and use the following four fragments of H1, H2, H3, and H4 as primers to amplify the DNA of different cattle Mitochondrial DNA:

[0020] H1:S:5'-CTGCAGTCTCACCATCAACC-3';

[0021] A: 5'-GTGTAGATGCTTGCATGTAAGT-3';

[0022] H2:S:5'-TTATCCGTTGGTCTTAGGAA-3';

[0023] A: 5'GCGGCATGGTAATTAAGCTC-3';

[0024] H3:S:5'-TTATCACAATCCAGAACTGAC-3';

[0025] A: 5'-CTAGTGAGAGTGAGGAGATATG-3';

[0026] H4:S:5'-TGTGCATGTGACACGTATCC-3';

[0027] A: 5'-AAGCGATTGCTTACTAGTCGG-3';

[0028] The specific amplification conditions are as follows:

[0029] H1: Pre-denaturation at 94°C for 5 minutes, then enter the cycle; the cycle conditions are: 94°C for 30s, 58°C for 30s, 72°C for 1min, 35 cycles; the final extension at 72°C for 10min.

[0030] H2:...

Embodiment 2

[0064] Example 2 , analysis of enzyme-digested fragments and classification of mitochondrial DNA

[0065] The fragments with different lengths in the RFLP analysis of various enzymes were summarized, and the results are shown in Table 1 below.

[0066] Table 1. Summary of fragments with length differences in endonuclease RFLP analysis

[0067] Endonuclease

(Cut Fragment Name)

Digestion type

fragment length

AvaII (H3)

+

0.17+1.75kb

-

1.92kb

BamHI(H3)

+

1.38+1.44kb

-

2.82kb

BglII(H4)

+

2.12+2.3kb

-

4.42kb

HpaII(H1)

+

0.25+0.13kb

-

0.38kb

HpaII(H2)

+

2.12+0.34kb

-

2.46kb

NlaIIIa(H1)

+

149+309bp

-

458bp

NlaIIIb(H1)

+

149+310bp

-

459bp

PstI(H2)...

Embodiment 3

[0073] Example 3 , Preparation of recipient oocytes

[0074] Live oocyte retrieval is carried out on haplotype A young cattle. For specific methods, see the Chinese invention patent application with application number 200510023510.1.

[0075] Pour the collected solution into the egg sorting cup, and use calcium-magnesium-free Duchenne's phosphate buffer solution (DPBS, containing 8g NaCl, 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 (Gibco, Grand Island, NY) plus 10% fetal bovine serum (FBS), 10 μg / ml luteinizing hormone, 1 μg / ml estradiol and 1 μg / ml follicle-stimulating hormone) for 15 ...

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Abstract

The present invention discloses a method for the nuclear transplantation. The method is characterized in that donor cells and recipent oocytes of non-human mammals of the same mitochondria haplotype are nuclear transferred. The method in the present invention can remarkably improve fusion, cleavage and blastocyst rates of the reconstructed embryo, thus remarkably improving the nuclear transplantation efficiency.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a cell nucleus transplantation method. Background technique [0002] Somatic cell nuclear transfer (SCNT) technology has been widely used in the production of cloned animals and transgenic animals, and cloned animals of many different species have come out one after another (Chesne P., Adenot PG., Viglietta C., et al.Nat Biotechnol. 2002, 20: 366-369; Woods GL., White KL., Vanderwall DK., et al. Science 2003, 301: 1063). Compared with conventional transgenic animal preparation methods, cloning technology has unique advantages, because cloning technology advances the screening work to the cell level, thus greatly shortening the breeding cycle of elite animals. Currently, inefficiency is the bottleneck limiting the technology. [0003] Mitochondria (mitochondrion, mt) is not only the organelle with the most content in the cytoplasm, but more importantly, due to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/06C12N5/16C12N15/877
CPCC12N15/8771
Inventor 曾溢滔焦飞曾凡一黄淑帧
Owner SHANGHAI INST OF MEDICAL GENETICS SHANGHAI CHILDRENS HOSPITAL
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