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Generation of Embryonic Stem Cell-derived Transgenic Mice by Using Tetraploid Complementation |
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S. M. Park, S. J. Song, S. J. Uhm, S. G. Cho, S. P. Park, J. H. Lim, H. T. Lee |
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| Abstract |
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The objective of this study was to generate transgenic mice expressing human resistin gene by using the tetraploid- embryonic stem (ES) cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR, cloned into pCR짰2.1 TOPO짰 vector and constructed in pCMV-Tag4C vector. Mammalian expression plasmid containing human resistin was transfected into D3-GL ES cells by Lipofectamine 2,000, and then after 10-12 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 sec (fusion rate: 2,114/2,256, 93.5%) and cultured up to the blastocyst stage (development rate: 1,862/2,114, 94.6%). The selected 15-20 ES cells were injected into tetraploid blastocysts, and then transferred into the uteri of E 2.5 d pseudopregnant recipient mice. To investigate the gestation progress, two E 19.5 mused fetuses were recovered by Cesarean section of which one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, our findings demonstrate that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mice for the rapid analysis of gene function in vivo. |
| Keywords:
Human Resistin; Tetraploid; Embryonic Stem Cell; Mouse |
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