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  Vertebrate Reproductive Science & Technology
 
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RFD is the official journal of the International Embryo Transfer Society and the Society for Reproductive Biology.



Article << Previous     |     Next >>   Contents Vol 18(2)

70 NON-PLATED GRANULOSA AND CUMULUS CELLS AND FIRST PASSAGE FIBROBLASTS AS NUCLEUS DONOR FOR GOAT CLONING

D. Salamone A, M. Catala A, A. Gibbons B, F. Pereyra Bonnet B and M. Cueto B

A Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Argentina
B Instituto Nacional de Tecnología Agropecuaria, Estación Experimental Bariloche, CC 277 (8400) Bariloche, Argentina
   

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Abstract

Different types of somatic cells have been used as nucleus donors for cloning. Most of them were previously cultured in vitro as a monolayer through several plate passages. The experiment reported here was conducted to study the potential usages of granulosa and cumulus cells for cloning without previous culture as a monolayer. A first-plate-passage fibroblast was also used. Oocytes were aspirated by laparoscopy from Criolla goats and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg/mL bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg/mL) under UV light (<6 s). Granulosa and cumulus cells were also recovered by laparoscopy and maintained in maturation medium in cryotube for 20 h at room temperature or 39°C, respectively. Goat adult ear fibroblasts were cultured for 1 or 2 weeks and used 2 days after confluence. All types of donor cells were transferred to the perivitlline space of enucleated oocytes and fused by an electrical pulse. After 2 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium and atmosphere of 5% CO2 + 5% O2 + 90% N2. Cleavage (Day 2) and development to blastocysts (Day 6) were recorded and analyzed by chi-square test. The cleavage rate for non-plated granulosa cells was higher than for the other treatment goups; cumulus cells had a lower rate of development to blastocysts (Table 1). These results suggest that granulosa cells collected and maintained for 24 h at room temperature could be used to produce cloned blastocysts.

Reproduction, Fertility and Development 18(1343) 143–143   http://dx.doi.org/10.1071/RDv18n2Ab70
Submitted:     Accepted:     Published online: 14 December 2005




 
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