• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.43-52
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• 1996

This study was carried out to establish the in vitro short-term culture system of developing male germ cells by purifing germ cells of various stages. The decapulated testicular cells were incubated with collagenase (lmg/ml) and try psin (2.5mg/ml) in HBSS. After separating male germ cell, the separated germ cells were stained with heamatoxylin/eosin and determined developing stages under light microscopy. The purity of pachtene spermatocytes a and round spermatid were 85%, respectively. Yield of total male germ cells was highly variable between individuals, with a mean value of 3.5 to 4.5 ${\times}$ 10$^7$ cells/testis. Viability of the cell was over 97% after separation. In DMEM medium, the optimal cell number for culture is approximately 1 x 10$^5$ cells/dish, but low cell den-sities than 1 ${\times}$ 10$^5$ cell/dish showed a decreased cell viability. Furthermore, about :36.8% of pac-hytene cells was successfully cultured for 6 days and some of cells were developed to secondary spermatids and round spermatids. Therefore, our data suggested that this culture conditions will be utilize as a feasible tools to produce tran-sgenic livestock using techniques such as intrac-ytoplasmic injection and cell fusion.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.213-213
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• 2004

Exogeneous DNA can reproducibly be delivered by co-injected spermatozoa and this transgenesis method is very efficient protocol. But, mosaic patterns of transgenic embryos and offspring were shown frequently. Whole blastomere integration is important in transgenic animal economics. (omitted)

• Korean Journal of Animal Reproduction
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• v.25 no.4
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• pp.305-315
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• 2001

In this study, the production of transgenic embryo was attempted by microinjection or round spermatid cultured with foreign DNA. At first, the expression of haploid spermatids specific gene, mTP1 in mouse and hPrm2 in hamster spermatids were investigated by RT-PCR method in testes of young mice and hamster testis. The specific gene expression first appeared at 18 days post partum (dpp) in mice spermatid and 20 dpp in hamster spermatid. Therefore, the round spermatids isolated from 17 dpp mice and 19 dpp hamster were used for the introduction of foreign EGFP gene into haploid round spermatids. For the introduction of EGFP gene haploid round spermatids suspended in medium including EGFP gene were treated with a different electric field strength at 0.11, 0.18 and 0.44 ㎸/cm. After electrical stimulation, viability of testicular sperm cells and 67.6%, 66.4% and 49.9%, in mice and 62.6%, 57.9% and 27% in hamster, respectively. These values were significantly lower than those of non-treated control groups 80.5% in mouse and 69.1% in hamster After 72 hrs culture, the highest expression rate of EGFP gene, 28.5% in mice and 32.1% in hamster were obtained from tile spermatogenic cells electroporated by the field strength or 0.18 ㎸/cm. Then, the ability of fertilization and embryonic development of haploid spermatids transfected with foreign EGFP gene were estimated by the microinjection of spermatids into hamster oocytes. The Irate pronuclear formation rate (77.5%) was lower than non-treated control (80%), and the cleavage rate of the treated group (58.8%) was lower than control (65%). To prove the foreign EGFP integration in hamster embryos, 2-cell stage hamster embryos were subjected to the observation under the fluorescence microscope, and the PCR analysis. As a result, about 44% of 2-cell embryos were showed the integration of EFGP gene into their genome. Therefore, These results suggest the possibility to produce transgenic hamsters by microinjection of haploid spermatid transfected with foreign DNA.