Effect of Vector Construction Strategies of the Target Genes on the Transfection Efficiency of Bovine Nuclear Transferred Embryos

• Main Authors: Hsing-Yi Lin, 林欣怡
• Other Authors: Perng-Chih Shen
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/40228988746924400021

碩士 === 國立屏東科技大學 === 畜產系 === 94 === The purposes of this study were to investigate the effects of different selection protocols on GFP (green fluorescent protein) gene transfection efficiency of bovine ear fibroblast cells, and to evaluate the feasibility of a vector containing two selectable markers, the RFP-(red fluorescent protein) and GFP- report genes, to improve the transfection efficiency of donor cells and NT embryos. In experiment 1 was to investigate the effect of screening pattern on the GFP gene transfection efficiency of bovine ear fibroblast cells. The result showed the GFP expression rate of transfected cells passaged at 50~60% cells proliferated (63.6%) after cultured with 0.8 mg/ml neomycin for a selection period of one month were higher than those cells passaged at 100% cells proliferated (11.5%) and those cells without being passaged (10.5%). Moreover, the GFP expression rate of transfected cells was dramatically raised with increased passage number in the selection protocol of passaged at 50~60% cells proliferation group. However, GFP expression rate could not be improved when transfected cells were culture in medium contained 0.3 μM 5-Azad and (or) 0.3 μM TSA for 3 days (40.3~45.1%, P>0.05). In experiment 2 was to evaluate the gene transfection efficiency of donor cells and NT embryos after the two selectable markers of the RFP- and GFP- report genes screening. The result showed the RFP expression rate of Tet-On-RFP gene transfected bovine ear fibroblast cells cultured for 2 days was higher than those cells cultured for others periods in culture medium contained 1ug/ml doxycycline (Dox)(17.7 vs. 3.0~8.6, P<0.05). Moreover, the RFP expression rate of Tet-On-RFP gene transfected bovine ear fibroblast cells cultured with 0.5~4.0 ug/ml doxycycline (Dox) (12.1~14.8%) was higher than those cells cultured with 0.1 μg/ml and without Dox for 2 day, respectively (5.7% and 10.3%) (P<0.05). The percentages of Tet-On-RFP- GFP gene transfected bovine ear fibroblast cells expressing the RFP and GFP both, RFP alone and GFP alone were 5.3% (52/975), 0.1% (1/975) and 5.9% (58/975), respectively. On the other hand, the hRLX examination rate of RFP and GFP both expressed cells (40%) transfected with Tet-On-RFP-hRLX-GFP gene was higher than those cells expressed GFP alone (0%) and unexpressed (0%), respectively, after single cell analysis by PCR method. The hRLX examination rate of NT embryos produced from RFP and GFP both expressed donor cells after analysis by PCR with 8 blastomeres was slight higher than those samples analysis from single donor cell (100 vs. 58.3%). In conclusion, the gene transfection efficiency of bovine ear fibroblast cells might be increased when the cells growth density was reduced in the screening period. Moreover, medium supplemented with 0.5 μg/ml Dox for 2 days culture period appeared helpless with the target gene expression of transfected cells, when the target gene regulated by Tet-On element. In addition, the two selectable markers used for donor cell screening could be raised the gene transfection efficiency of NT embryos.