金 珍會 韓国建国大学, 動物資源研究所, 研究員
李 勲澤 韓国建国大学, 動物資源研究所, 助教授
鄭 吉生 韓国建国大学, 動物資源研究所, 教授
TAKAHASHI Seiya National Inst.Anim.Industry, Resercher, 繁殖部, 研究員
TOKUNAGA Tomoyuki National Inst.Anim.Industry, Resercher, 繁殖部, 研究員
IMAI Hiroshi National Inst.Anim.Industry, Lab.Chief, 繁殖部細胞操作研究室, 室長
OKUDA Kiyoshi Okayama University, Associate Professor, 農学部, 助教授 (40177168)
KIM Jin-hoi Kon-Kuk University, Reserch Student
LEE Hoon-taek Kon-Kuk University, Associate Professor
CHUNG Kil-saeng Kon-Kuk University, Professor
Transgene for microinjection into mouse eggs was constructed by the use of the regulatory region of rabbit beta-casein gene (2.2 kb), fused with the genomic sequences of human luteinizing hormone (LH) beta-subunit gene.
In vitro fertilization and in vitro culture of mouse eggs were conducted for an efficient production of fertilized eggs for transgene injection. Ovulated eggs were inseminated by epididymal spermatozoa and cultured for 120 hours in M16 mediim. All eggs examined were fertilized and 95% (122/128) of the eggs developed to the blastocyst stage.
Total of 998 eggs were microinjected with the transgene (5 mug/ml) and 474 eggs developed to the blastocyst stage following in vitro culture. Among 65 pups produced after uterine transfer to the recipient mice, 5 transgenic mice was obtained. Three of five transgenic mice expressed the products of LHbeta gene in the milk at the level of 0.8-1.6 mIU/ml.
One of the powerful tool for producing transgenic animals is the use of pluripotencial cells such as ES cells and EG cells. The establishment of cell lines from mouse blastocysts and primordial germ cells (PGCs) was carried out and the effect of growth factors on the proliferation of the cells was examined. ES cells was established from the blastocysts, without the addition of growth factors, on mouse embryonic fibroblast cell feeder (MEF). In contrast to embryonic cells, PGCs did not proliferate in the absence of growth factors and supported their growth for only 5 days with the addition of LIF and bFGF in the culture medium on mouse born marrow derived stromal cell feeder, but not on MEF.