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Full term development of animals from enucleated oocytes reconstituted with adult somatic cell nuclei

A technology of oocytes and somatic cells, used in the preparation of hybrid cells, cells modified by introducing foreign genetic material, fermentation, etc.

Inactive Publication Date: 2001-08-01
UNIV OF HAWAII
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Similarly, Sendai virus-mediated fusion of adult mouse thymocytes with enucleated Met II oocytes, followed by activation with 7% ethanol for 30 to 60 minutes, resulted in 70% of 20 oocytes reaching the 2-cell stage, but none A development beyond the 4-cell stage

Method used

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  • Full term development of animals from enucleated oocytes reconstituted with adult somatic cell nuclei
  • Full term development of animals from enucleated oocytes reconstituted with adult somatic cell nuclei
  • Full term development of animals from enucleated oocytes reconstituted with adult somatic cell nuclei

Examples

Experimental program
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Effect test

Embodiment 1

[0085] Preparation of Somatic Cells

[0086] In this example, cumulus cells were isolated from mouse oviducts as a source of adult somatic nuclei for injection into enucleated mouse oocytes. The method for obtaining the cloned mice produced in Table 2 and in Series A-D described below is also described in Wakayama, et al. 1998, Nature 394, 369-374.

[0087] Female mice B6D2F1 (C57BL / 6×DBA / 2, series A and B), B6C3F1 (C57BL / 6×C3H / He, adopted in series C) or B6C3FI clone mice generated in series D were superovulated. Thirteen hours after hCG injection, cumulus-oocyte complexes were collected from the oviduct (see figure 1 A), cumulus cells were dispersed by treatment in Hepes-CZB medium supplemented with bovine testicular hyaluronidase (0.1% [w / v], 300 units / ml, ICN Biochemicals, Costa Mesa, CA). Medium-sized cumulus cells (10-12 microns in diameter) were the most common (>70%) and were selected for injection. After dispersion, the cells were transferred to Hepes-CZB containi...

Embodiment 2

[0089] Preparation of Somatic Cells

[0090] In this example, podocytes and brain cells (neurons) were isolated from adult mice. These cells are characterized by nondividing in adult animals and are permanently arrested in the G0 phase of the cell cycle.

[0091] Separate the seminiferous tubules from the testes and contact with 1 mg / ml collagenase in Hepes-CZB solution at 30°C for 20 minutes. Vials were then minced with a razor blade and placed in Hepes-CZB containing 1 mg / ml trypsin with occasional agitation. The resulting suspension was then allowed to stand. The podocyte-rich fraction settles first. Aspirate the suspended cells and resuspend the remainder with fresh medium. Podocytes with characteristic morphological features are easily identified under a low power microscope. Manipulation of single podocytes is performed with large injection pipettes (approximately 10 µm inner diameter).

[0092] Neurons were isolated from the cerebral cortex of adult B6D2F1 female ...

Embodiment 3

[0093] The preparation of embodiment 3 somatic cells

[0094] Fibroblasts were prepared from the tail of adult B6C3F1 mice. The tails of the mice were isolated, the skin removed, cut into small pieces, and then placed in 5 ml of Dulbecco's Modified Eagle's Medium (DMEM, Sigma). at 5% CO 2 After incubation in air at 37.5°C for 5-7 days, many fibroblasts were seen scattered along the inner surface of the dish. In some experiments, the medium in the plates was replaced with DMEM without FCS and incubated for an additional 3 to 5 days. To detach fibroblasts from plates, use Ca-free media containing 0.25% trypsin and 0.75 mmol ethylenediaminetetraacetic acid (EDTA, Specialty Media, Lavallette, NJ). 2+ , does not contain Mg 2+ Phosphate buffered saline (PBS) was used instead of medium. After 10 minutes, the medium was agitated by pipetting for several minutes to release the cells from the plate surface. The medium was collected and centrifuged (150 xg, 10 minutes) to pellet t...

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Abstract

Animals are produced following injection of adult somatic cell nuclei into enucleated oocytes. The invention provides a method for cloning an animal by directly inserting at least a portion of the adult somatic nucleus into a recipient enucleated oocyte. Preferably, the nucleus is inserted by microinjection and, more preferably, by piezo electrically-actuated microinjection. The oocyte is activated prior to, during, or up to about 6 hours after insertion of the nucleus, by electroactivation or exposure to a chemical activating agent, such as Sr2+. The activated renucleated oocyte is allowed to develop into an embryo and is transplanted to a host surrogate mother to develop into a live offspring.

Description

[0001] This application is a continuation-in-part of U.S. Patent Application No. 09 / 132,104, filed August 10, 1998, which claims U.S. Provisional Patent Application No. 60 / 072,002, filed January 21, 1998, and the 1998 Priority of No. 60 / 072,002 filed June 19. [0002] The United States Government has a paid license in this invention and has the right under certain circumstances to require the patent owner to license on reasonable terms under the terms of the Department of Public Health Service National Institutes of Health grant contract No. R01-HD-03402 others. Background of the invention [0003] The present invention relates to a method for cloning animals. The method is to insert the nuclei of adult somatic cells into enucleated oocytes, so that the host oocytes can form embryos and develop into live animals. In one embodiment of the invention, insertion of the nucleus is achieved by piezo-actuated microinjection. [0004] Rapid production of large numbers of nearly iden...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/02C12N5/10C12N15/877
CPCC12N15/8775A01K67/027C12N15/02
Inventor T·瓦卡雅玛R·杨吉麦希
Owner UNIV OF HAWAII
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