Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitro-transfected somatic cells
Transgene expression of green fluorescent protein and germ line transmission in cloned calves derived from in vitro-transfected somatic cells
复制标题
DOI：
10.1095/biolreprod.102.010066
复制
发表时间：
2003-06-01
影响因子：
3.6
通讯作者：
Smith, LC
中科院分区：
文献类型：
--
作者：
Bordignon, V;Keyston, R;Smith, LC
体外转染培养细胞结合核移植是目前生产转基因家畜最有效的方法。在本研究中，牛原代胎儿成纤维细胞转染绿色荧光蛋白（GFP）报告基因，并作为核供体细胞在卵母细胞重建。由于细胞同步化方案在转染后不太有效，激活的卵母细胞可能更适合作为核转移的宿主。为了研究宿主细胞质对转基因表达和发育结果的作用，将表达GFP的成纤维细胞融合到激活前（中期）或激活后（末期）重建的卵母细胞中。GFP的表达在早期胚胎发生过程中，在克隆小牛的组织中，并再次在胚胎发生过程中，通过生殖系使用精液从转基因克隆后代。无论使用何种宿主细胞质，GFP在重建后约80小时的8- 16细胞阶段变得可检测，并且在所有后期阶段保持阳性。出生后，虽然通过这两种程序获得的克隆小牛在所有组织中表达GFP，但表达水平在组织之间和同一组织内的细胞之间都不同，表明在细胞分化期间GFP表达部分关闭。此外，来自转基因后代的非表达成纤维细胞在核转移和发育到胚泡阶段后不能直接表达GFP，这表明转基因的不可逆沉默。尽管如此，GFP表达在约一半的囊胚获得精子从转基因克隆，确认通过生殖系的转基因传输。
In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP)-reporter transgene and used as nuclear donor cells in oocyte reconstructions. Because cell synchronization protocols are less effective after transfection, activated oocytes may be more suitable as hosts for nuclear transfer. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either before (metaphase) or after (telophase) activation. Expression of GFP was examined during early embryogenesis, in tissues of cloned calves, and again during embryogenesis, after passage through germ line using semen from the transgenic cloned offspring. Regardless of the kind of host cytoplasm used, GFP became detectable at the 8- to 16-cell stage, approximately 80 h after reconstruction, and remained positive at all later stages. After birth, although cloned calves obtained through both procedures expressed GFP in all tissues examined, expression levels varied both between tissues and between cells within the same tissue, indicating a partial shutdown of GFP expression during cellular differentiation. Moreover, nonexpressing fibroblasts derived from transgenic offspring were unable to direct GFP expression after nuclear transfer and development to the blastocyst stage, suggesting an irreversible silencing of transgenes. Nonetheless, GFP was expressed in approximately half the blastocysts obtained with sperm from a transgenic clone, confirming transmission of the transgene through the germ line.