Fertilization and Early Embryogenesis
(W354) EFFECT OF THE KINDS OF GENES USING FOR TRANSFECTION ON IN VITRO DEVELOPMENT OF TRANSFECTED SOMATIC CELL NUCLEAR TRANSFERRED EMBRYOS IN CATTLE.
Yang, ByoungChul1, Kim, Dong Hoon1, Kim, Se Woong1, Lee, Poong Yeon1, Lee, Yen Keun1, Park, Hyo Suck1, Yang, Boh Suk 1, Chang, Won Kyong 1, 1 National Livestock Research Institute, RDA, Suwon, Republic of Korea
ABSTRACT- Nuclear transfer technology is being used to clone desirable animals as well as transgenic animals. However, production of transgenic animals using nuclear transfer technique has a much lower success rate than production of normal adult clones. The objective of this study was to compare with the kinds and clones of genes for transfection to bovine fetal fibroblast cells (BFF) on in vitro development of nuclear transferred embryos. BFF were established from day 45 of pregnant Korean native cattle and were transfected with a human tissue plasminogen activator gene (tPA) including puromycin-resistant genes and a plasmid containing the enhanced green fluorescence protein (EGFP) with neomycin-resistant genes by electroporation. The 4×106 cells at passage 2 were transfected with 10 g of linearized tPA or EGFP. The selection of transfected colonies was done by culture in 10% FBS contained DMEM with puromycin for tPA and neomycin for EGFP. Two different tPA-positive cell clones (tPA1, tPA2), one EGFP-positive cell clone (EGFP), and non-transfected cells (control) were used for subsequent nuclear transfer. The enucleated oocytes were injected with transfected clones. We compared the fusion, cleavage and development rates of NT derived embryos from three transfected or one non-transfected cells. The fusion rate of tPA1 (73.02%) and tPA2 (72.59%) was lower than that of the GFP (96.29%), but the cleavage rate of the tPA1 (92.97%) was higher than that of the tPA2 (48.95%), and control (85.39%) and GFP (82.22%) were no significant differences (p<0.05). The blastocysts rate of tPA2 (10.49%) was lower than that of the any other groups (control, 35.22%; GFP, 29.80% and tPA1, 32.66%, p<0.05). The morulae and blastocysts from tPA1 and tPA2 clones were examined by PCR. The transfected rates of tPA1 (n=33, 51.86%) and tPA2 (n=29, 80.68%) clones were different (p<0.05). All blastocysts from EGFP clone were expressed with GFP under UV light, no mosaic (n=62, 100%). GFP transfected cells were without negatively affecting their development into blastocyst, while tPA transfected cells was negatively affecting their embryo development. Therefore, an exogenous gene on embryo development should be considered in future transgenic animal studies.
KEY WORDS: nuclear transfer, fetal fibroblasts, tissue plasminogen activator, green fluorescence protein