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Transferring nucleus of long-period cultured female or male cell including one changed by artificial induction gene into denucleated receptor cell

一种体细胞、细胞核的技术，应用在活体全能胞质杂合体领域，能够解决低克隆效率、限制经济应用前景、不允许靶向(定向)基因改造等问题

Inactive Publication Date: 2007-01-03
UNIV OF CONNECTICUT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Obviously such a low cloning efficiency greatly limits the economic application prospects of this technology
[0024] The success of somatic cell cloning is largely limited by the use of donor cells, which must be fresh (Wakayama et al., Nature (London) 394:369-374 (1998)) or in (Wilmut et al., Nature 385: 810-813 (1997); Kato et al., Science 282: 2095-2098 (1998); Wells et al., Biol. Reprod.60: 996-1005 (1997); Schnieke et al., Science 278: 2130-2133 (1997); Cibelli et al., Science 280: 1256-1258)), this method does not allow targeted (directed) genetic modification, make current technology limited

Method used

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  • Transferring nucleus of long-period cultured female or male cell including one changed by artificial induction gene into denucleated receptor cell
  • Transferring nucleus of long-period cultured female or male cell including one changed by artificial induction gene into denucleated receptor cell
  • Transferring nucleus of long-period cultured female or male cell including one changed by artificial induction gene into denucleated receptor cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1: Cloning of cattle with long-term cultured mature male fibroblasts

[0107] Materials and methods

[0108] Collection and culture of mature somatic cells:

[0109] A skin biopsy was obtained from the ear of a genetically superior 17-year-old Japanese black bull. The tissue slices were cut into small pieces (3mm 2 ) and put the small pieces as explants into DMEM (GIBCO, Cat. No. 12100-061) (in which 10% FBS and antibody (GIBCO, Cat. No. 15240-013) were added) for culture. CO 2 And a humid environment with 95% air. After one week in culture, a monolayer of fibroblasts formed around the tissue explants. The explants were removed and the fibroblasts were grown to confluency. Cell lines were kept on culture dishes until passage 17, and then stored frozen as follows. For each generation (estimated cell proliferation 2-fold per generation), cells were cultured to confluence and treated with 0.1% (wt / vol) insulin (Difco) and EDTA (Nacalai Tesque, Kyoto) solutio...

Embodiment 2

[0156] Example 2: Preparation of cloned embryos from long-term cultured mature rabbit fibroblasts

[0157] We have successfully cloned rabbit embryos using long-term cultured fibroblasts using the following method.

[0158] Raw Materials and Methods

[0159] Source of oocytes and fertilized eggs:

[0160] Mature Dutch striped female rabbits were subcutaneously injected twice with 0.3 mg and four times with 0.4 mg of follicle stimulating hormone (FSH) (FSH-P, Schering-Plough Animal Health, Kenilworth, NJ), each interval 12h, to stimulate It hyperovulates (Yang et al., Mol. Reprod. Dev. 27: 110-117 (1990)). Twelve hours after the last FSH injection, ovulation was induced by intravenous injection of 100 IU human chorionic gonadotropin (hCG) (Sigma, St Louis, MO). 13.5 hours after hCG injection, animals were laparotomed and expelled oocytes were flushed out with Dulbecco-amended PBS containing 3 mg / ml BSA (Fraction V, Sigma A-9418). Cumulus cells were removed by a short treatm...

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Abstract

An improved method of nuclear transfer employing long-term cultured somatic cells as the donor cells and enucleated oocytes as the recipient cells to produce dividing cybrids. Such cybrids are useful for developing viable animals clones when nurtured in a suitable host environment.

Description

[0001] related technology [0002] This application claims priority to US Patent Applications 60 / 174,383 and 60 / 174,424, filed January 4, 2000, the disclosures of which are hereby incorporated by reference in their entirety. technical field [0003] The present invention relates to an improved method of nuclear transfer which enables the efficient development of cytoplasmic hybrids obtained using male or female cells as donors. The present invention provides a way to transfer the nucleus of long-term cultured, non-embryonic somatic cells to an enucleated oocyte to obtain living totipotent cytoplasmic hybrids capable of producing embryos, fetuses and / or animals. Background technique [0004] Recent discoveries in animal cloning research have sparked a new revolution in the scientific world. There is no longer any doubt about the application potential of cloning technology in the fields of agriculture, medicine and basic biological research. Cloning technology provides a mor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/00A01K67/00C12N15/00A01K67/027C12N5/10C12N15/09C12N15/873
CPCA01K2217/05A01K67/0275C12N15/873
Inventor 杨向中洼田力
Owner UNIV OF CONNECTICUT