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Unactivated oocytes as cytoplast recipients for nuclear transfer

Inactive Publication Date: 2010-06-10
CAMPBELL KEITH HENRY STOCKMAN +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If any of the DNA either fails to replicate or is replicated more than once then the ploidy of that nucleus at the time of mitosis will be incorrect.
These improvements in the frequency of development of reconstructed embryos have as yet not addressed the question of nuclear reprogramming.

Method used

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  • Unactivated oocytes as cytoplast recipients for nuclear transfer

Examples

Experimental program
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Effect test

example 1

“MAGIC” Procedure using Bovine Oocytes

[0074]Recipient oocytes the subject of this experimental procedure are designated MAGIC (Metaphase Arrested GI / GO Accepting Cytoplast) Recipients.

[0075]The nuclear and cytoplasmic events during in vitro oocyte maturation were studied. In addition the roles of fusion and activation in embryos reconstructed at different ages were also investigated. The studies have shown that oocyte maturation is asynchronous; however, a population of matured oocytes can be morphologically selected at 18 hours (FIG. 1)

Morphological Selection of Oocytes

[0076]In FIG. 1 ovaries were obtained from a local abattoir and maintained at 28-32° C. during transport to the laboratory. Cumulus oocyte complexes (COC's) were aspirated from follicles 3-10 mm in diameter using a hypodermic needle (1.2 mm internal diameter) and placed into sterile plastic universal containers. The universal containers were placed into a warmed chamber (35° C.) and the follicular material allowed to...

example 2

“MAGIC” Procedure Using Ovine Oocytes

[0093]Similar observations to those in Example 1 have also been made in ovine oocytes which have been matured in vivo. Freshly ovulated oocytes can be retrieved by flushing from the oviducts of superstimulated ewes 24 hours after prostaglandin treatment. The use of calcium magnesium free PBS / 1.0% FCS as a flushing medium prevents oocyte activation. Oocytes can be enucleated in calcium free medium and donor cells introduced as above in the absence of activation. No organised spindle is observed, multiple nuclei are formed upon subsequent activation and this may be suppressed by nocodazole treatment

Results

[0094]In preliminary experiments in sheep, a single pregnancy has resulted in the birth of a single live lamb. The results are summarised in Tables 4 and 5.

[0095]Table 4 shows development of ovine embryos reconstructed by transfer of an embryo derived established cell line to unactivated enucleated in vivo matured ovine oocytes. Oocytes were obtai...

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Abstract

A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the GO or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This is a continuation of U.S. application Ser. No. 09 / 658,862, filed Sep. 8, 2000, which is a division of U.S. application Ser. No. 08 / 803,165, filed Feb. 19, 1997 (now U.S. Pat. No. 6,252,133), which is a continuation of International Application No. PCT / GB96 / 02098, filed Aug. 30, 1996, which claims the benefit of GB 9517779.6, filed Aug. 31, 1995, in Great Britain, all of which are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]This invention relates to the generation of animals including but not being limited to genetically selected and / or modified animals, and to cells useful in their generation.[0003]The reconstruction of mammalian embryos by the transfer of a donor nucleus to an enucleated oocyte or one cell zygote allows the production of genetically identical individuals. This has clear advantages for both research (i.e. as biological controls) and also in commercial applications (i.e. multiplication of genetic...

Claims

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Application Information

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IPC IPC(8): A01K67/027A01K67/02A61K35/54C12N5/10C12N15/02C12N15/873C12N15/877
CPCA61K35/54C12N15/8773C12N15/8771C12N15/873A01K67/027
Inventor CAMPBELL, KEITH HENRY STOCKMANWILMUT, IAN
Owner CAMPBELL KEITH HENRY STOCKMAN
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