Mehta Parul, Kaushik Ramakant, Singh Karn Pratap, Sharma Ankur, Singh Manoj Kumar, Chauhan Manmohan Singh, Palta Prabhat, Singla Suresh Kumar, Manik Radhey Sham
Embryo Biotechnology Laboratory, Animal Biotechnology Center, ICAR-National Dairy Research Institute , Karnal, India .
Cell Reprogram. 2018 Apr;20(2):135-143. doi: 10.1089/cell.2017.0013. Epub 2018 Feb 15.
The aim of the present study was to compare transgenic cells, containing human insulin gene kept under the control of mammary gland-specific buffalo beta-lactoglobulin promoter, and their counterparts, that is, nontransgenic cells, for examining their potential for the production of embryos following somatic cell nuclear transfer (SCNT). The gene construct was delivered into buffalo fetal fibroblasts (BFF) by nucleofection following which, the transfected cells were selected by culture in the presence of G418 for 3 weeks. Transgene integration into BFF genome was confirmed by polymerase chain reaction (PCR) and reverse transcriptase PCR. At passage 8-10, the growth rate, cell proliferation rate, and quantitative expression of certain genes were compared between transgenic and nontransgenic cells. The growth rate and cell proliferation rate was significantly lower (p < 0.05) for transgenic than for nontransgenic cells. Using quantitative real-time PCR it was found that the expression level of CASPASE 3, CASPASE 9, BAX, and P53 was significantly higher (p < 0.05) and that of HDAC1 and IGF-1R was significantly lower (p < 0.05) in transgenic compared with nontransgenic cells. The differences in the relative expression level of BCL-XL, MCL-1, DNMT1, DNMT3a, GDF9, FGF2, and G6PD between the two groups were not significant. Furthermore, when the two cell types were used as donor cells for production of embryos by handmade cloning, the blastocyst rate was significantly lower (p < 0.05) with transgenic (35.69% ± 1.78%) than with nontransgenic cells (48.75% ± 2.38%). In conclusion, these results indicate that differences were present between transgenic and nontransgenic cells, which may affect the efficiency of SCNT when used as donor cells.
本研究的目的是比较含有在乳腺特异性水牛β-乳球蛋白启动子控制下的人胰岛素基因的转基因细胞及其对应物，即非转基因细胞，以检测它们在体细胞核移植（SCNT）后产生胚胎的潜力。通过核转染将基因构建体导入水牛胎儿成纤维细胞（BFF），之后，在含有G418的培养基中培养3周以选择转染的细胞。通过聚合酶链反应（PCR）和逆转录PCR确认转基因整合到BFF基因组中。在第8 - 10代时，比较转基因细胞和非转基因细胞之间的生长速率、细胞增殖速率以及某些基因的定量表达。转基因细胞的生长速率和细胞增殖速率显著低于非转基因细胞（p < 0.05）。使用定量实时PCR发现，与非转基因细胞相比，转基因细胞中CASPASE 3、CASPASE 9、BAX和P53的表达水平显著更高（p < 0.05），而HDAC1和IGF - 1R的表达水平显著更低（p < 0.05）。两组之间BCL - XL、MCL - 1、DNMT1、DNMT3a、GDF9、FGF2和G6PD的相对表达水平差异不显著。此外，当将这两种细胞类型用作手工克隆生产胚胎的供体细胞时，转基因细胞（35.69% ± 1.78%）的囊胚率显著低于非转基因细胞（48.75% ± 2.38%）（p < 0.05）。总之，这些结果表明转基因细胞和非转基因细胞之间存在差异，这可能会影响它们作为供体细胞时体细胞核移植的效率。
