Mitrani E, Coffin J, Boedtker H, Doty P
Proc Natl Acad Sci U S A. 1987 May;84(9):2781-4. doi: 10.1073/pnas.84.9.2781.
We have developed a protocol that allows us to infect chicken early embryonic (CEE) cells with high efficiency. This was achieved by exposing the CEE cells to a semicontinuous dose of Rous sarcoma virus (RSV) for a period of 20 hr. Southern blot analysis indicated that an average of one proviral copy is integrated per embryonic cell. However, there was no production of infectious viral particles by the cells containing the proviral genome, although low levels of full-length genomic RNA could be detected by RNA transfer blot analysis. These low RNA levels contrast with the 100- to 1000-fold higher levels found in RSV-infected chicken embryo fibroblasts. We conclude that in cells derived from pregastrulating chicken embryos, RSV DNA is integrated into the cell genome but fails to be expressed in an efficient manner. These primary cells can therefore be used to identify factors involved in regulation of retroviral gene expression in normal cells. Such factors may also be instrumental in elucidating basic mechanisms involved in gene regulation during early development in higher vertebrates.
我们已经开发出一种方案，可使我们高效感染鸡早期胚胎（CEE）细胞。这是通过让CEE细胞在20小时内接触半连续剂量的劳氏肉瘤病毒（RSV）实现的。Southern印迹分析表明，每个胚胎细胞平均整合有一个前病毒拷贝。然而，含有前病毒基因组的细胞并未产生传染性病毒颗粒，尽管通过RNA转移印迹分析可检测到低水平的全长基因组RNA。这些低RNA水平与RSV感染的鸡胚成纤维细胞中高出100至1000倍的水平形成对比。我们得出结论，在原肠胚形成前的鸡胚胎来源的细胞中，RSV DNA整合到细胞基因组中，但未能有效表达。因此，这些原代细胞可用于鉴定正常细胞中参与逆转录病毒基因表达调控的因子。这些因子也可能有助于阐明高等脊椎动物早期发育过程中基因调控所涉及的基本机制。
