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Ouiescent cell populations for nuclear transfer

A nuclear transfer, cell technology, applied in fusion cells, hybrid cell preparation, cells modified by introducing foreign genetic material, etc., can solve problems such as uncontrollable development and cell changes

Inactive Publication Date: 2006-05-10
ROSLIN INST (EDINBURGH) ET AL +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the long-term culture of established cell lines, when these cells are used as nucleus donors to transfer nuclei into what the above paper refers to as "universal recipients", these cells change and thus become unable to control development

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Induction of quiescence in donor cells

[0078] Various methods have been shown to induce quiescence in cultured cell lines including: contact inhibition or serum starvation (reviewed by Whitfield et al., Contril of Animal Cell Proliferation, 1 331-365 (1985)). We consider the method of inducing quiescence to be unimportant, the important step is that the cell exits the cell cycle and arrests in the G0 state while having a diploid DNA content and remaining viable. In Examples 3 and 4, bovine cell lines established from 7-day-old inner cell mass clumps generated in vivo from embryo sacs were induced to arrest cell cycle arrest using serum starvation of bovine primary fibroblasts G0 phase. Likewise, serum starvation was used to induce quiescence of the donors as described in Example 5.

Embodiment 2

[0079] Example 2: Isolation of oocytes and nuclear transfer

[0080] Oocytes may be obtained by (i) in vitro maturation of abattoir material, or transvaginally by follicle puncture; (ii) in vivo maturation and surgical recovery; or (iii) any other suitable method. All in vivo matured oocytes should be harvested by flushing the oviducts in calcium-free magnesium phosphate buffered saline (PBS) containing 1.0% fetal calf serum (FCS) and transferred to calcium-free M2 (Whittingham and Wales, Aust. J. Biol. Sci. 22 1065-1068 (1969)). The oocytes of the cumulus cells were peeled off and enucleated according to the aforementioned method (Campbell et al., Biol. Calcium medium. The fusion step modifies those previously reported (Campbell et al., 1993, 1994 loc cit) and follows the method described in the following sections; alternatively, the nucleus can be introduced by injecting donor cells into the enucleated oocyte (Ritchie and Campbell, J. Reprod. Fertil. Abstract Series (5)...

Embodiment 3

[0081] Example 3: Sheep cell nuclear transfer

[0082] 3.1. Hyperstimulation of donor ewes and recovery of oocytes

[0083] Scottish blackface ewes were synchronized with progestogen sponges for 14 days (Veramix TM , Upjohn, UK), by a single injection of 3.0 mg / day (total 6.0 mg) of sheep follicle-stimulating hormone (FSH) (Ovagen TM , Immuno-chemical Products Ltd, New Zealand), to induce superovulation. Twenty-four hours after the second injection of FSH, a single dose of 8 mg of gonadotropin-releasing hormone analog (GnRH Receptal TM , Hoechst, UK) to induce ovulation.

[0084] At 24-29 hours after GnRH injection, oviducts were flushed with Dulbecco's phosphate buffered saline (kept at 37°C until use) containing 1.0% fetal calf serum (FCS), and unfertilized metaphase II oocytes were recovered.

[0085] 3.2. Oocyte manipulation

[0086]The recovered oocytes were kept at 37°C, washed in PBS containing 1.0% FCS, and transferred to calcium-free M2 medium containing 10...

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Abstract

Methods for reconstituting animal embryos involve transferring the nuclei of quiescent donor cells into appropriate recipient cells. The donor cells are quiescent in that they exit the growth and division cycle in the G1 phase and cease in the G0 state. Nuclear transfer can be performed by cell fusion. The recombinant embryo can then produce one or more animals. The present invention can be used for the production of transgenic as well as non-transgenic animals with high genetic value.

Description

[0001] This application is a divisional application, the filing date of the original application is August 30, 1996, the application number is 96197891.0, and the invention name is "quiescent cell population for cell nuclear transfer". technical field [0002] The present invention relates to the production of animals including, but not limited to, genetically selected and / or genetically modified animals. technical background [0003] Mammalian embryos are reconstituted by transferring the nucleus of a donor embryo into an enucleated oocyte or a zygote, resulting in the production of genetically identical individuals. This is clearly beneficial both for research (ie as a biological control) and for commercial applications (ie breeding of genetically valuable livestock, uniformity of meat products, animal management). One problem with using early embryos as nucleus donors is that the number of offspring that can be produced from a single embryo is limited by both the number o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/00A01K67/02C12N5/16A01K67/027A61K35/00A61K35/54C12N5/10C12N15/02C12N15/06C12N15/877
CPCA61K35/54C12N15/8771C12N15/8773A01K67/027
Inventor K·H·S·坎贝尔I·维慕特
Owner ROSLIN INST (EDINBURGH) ET AL
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