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Method of nuclear transfer

A technology for nuclear transfer, purpose, applied in the field of nuclear transfer

Inactive Publication Date: 2004-08-25
MONASH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To avoid unplanned aggregation of embryos, either singly or in "aggregates" of two or three reconstituted nuclear transfer embryos, reconstituted zona-free embryos are cultured in special systems, as conventional systems are not suitable for this purpose of

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  • Method of nuclear transfer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Embodiment 1 uses granulosa cell as the nuclear transfer method of donor

[0133] All chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA) unless otherwise stated.

[0134] In vitro maturation of bovine oocytes (150 total per day) was performed with minimal modifications as described in detail previously (Vajta et al., 1996). Briefly, oocytes were aspirated from abattoir-derived ovaries, matured in 4-well culture dishes (Nunc, Roskilde, Denmark) for 24 hours, with 15% cow serum supplemented with 10 IU / ml pregnant horse Serum gonadotropin and 5 IU / ml human chorionic gonadotropin (Suigonan R Vet, Intervet, Australia) in bicarbonate-buffered TCM-199 medium (GibcoBRL, Paisley, UK) and at 39°C, containing 5% CO 2 Insulate with mineral oil in moist air.

[0135] Cumulus cells were removed by vortexing 19 hours after the initiation of the maturation process. From this point forward (unless otherwise stated) all manipulations were performed on a heat stage ...

Embodiment 2

[0145] Example 2 Results of Nuclear Transfer Using Granulosa Cells as Donors

[0146] The average efficiencies and approximate times for the main steps of the nuclear transfer experiments in seven replicates using a total of 1016 immature oocytes are summarized in Table 1:

[0147] Table 1

[0148] Average efficiency and approximate time required for the zona-free somatic cell nuclear transfer step

[0149] Method Individual Efficiency Cumulative Efficiency Time Required

[0150] PB rate determination - - 30 minutes

[0151] Zona pellucida removal 99% 99% 10 minutes

[0152] Bisection 89% 88% 20 minutes

[0153] UV observation 91% 80% 30 minutes

[0154] First fusion 94% 75% 40 minutes

[0155] Second fusion 91% 69% 15 minutes

[0156] (related work) - - 35 minutes

[0157] Total 180 minutes

[0158] Oocytes without clearly visible polar bodies (28% of the total) were discarded. However, this loss does not affect the nuclear transfe...

Embodiment 3

[0171] Example 3 simplified zone-free somatic cell cloning technique

[0172] The method used in this example was the same as that described in Example 1 except for the following.

[0173] Recombinant nuclear transfer embryos were cultured either singly, or 2 recombinant nuclear transfer embryos as aggregates, and cultured either in glass capillary tubes or in WOWs in 4-well Nunclone dishes. After activation embryos were aspirated into glass tubes or placed in WOW pits, either individually or 2 "recombined" nuclear transfer embryos were cultured in each glass capillary or in WOW pits, and post-activation as aggregates nourish.

[0174] Tables 3, 4 and 5 show the blastocyst development and conception rates for the culture and transfer of nuclear transfer embryos produced using the technique described in Example 1. The recombinant nuclear transfer embryos were cultured individually (Table 3) or in aggregates of two recombinant nuclear transfer embryos (Table 4 and 5).

[0175...

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Abstract

The present invention relates to nuclear transfer methods and embryos developed therefrom. In particular, the invention relates to methods of nuclear transfer comprising the transfer of somatic cells or somatic cell nuclei into zona-free, enucleated oocytes.

Description

field of invention [0001] The present invention relates to methods of nuclear transfer and embryos developed therefrom. Also included are methods of culturing embryos and recombining embryonic animals produced from the nuclear transfer methods of the present invention. Background of the invention [0002] A number of recent publications address the potential benefits of nuclear transfer (Galli et al., 1999; Colman, 1999; Wells & Powell, 2000; Lewis et al., 2001; Trounson, 2001). Methods of nuclear transfer have been actively explored and developed over the past two decades and are described in numerous references (see, e.g., Campbell et al., Theriogenology, 43:181 (1995); Collas et al., Mol. ReportDev ., 38: 264-267 (1994); Keefer et al., Biol. Reprod., 50: 935-939 (1994); Sims et al., Proc. Natl. Acad. Sci., USA, 90: 6143-6147 (1993 ); WO97 / 07668; WO97 / 07669; WO94 / 26884; WO94 / 24274; and US Patent Application Nos. 4,944,384 and 5,057,420 (describing bovine nuclear transfer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/09C12N15/873A01K67/027
CPCC12N15/873
Inventor 伊恩·刘易斯加博尔·瓦杰塔泰弗·特西里奥格鲁
Owner MONASH UNIV
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