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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Use of avian retroviral vectors to introduce transcriptional regulators into mammalian cells for analyses of tumor maintenance.
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Use of avian retroviral vectors to introduce transcriptional regulators into mammalian cells for analyses of tumor maintenance.

机译：使用禽逆转录病毒载体将转录调节因子引入哺乳动物细胞，以分析肿瘤的维持情况。

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A key issue in cancer biology is whether genetic lesions involved in tumor initiation or progression are required for tumor maintenance. This question can be addressed with mouse models that conditionally express oncogenic transgenes, i.e., under the control of tetracycline (tet)-dependent transcriptional regulators. We have developed a system for studying tumor maintenance by using avian retroviral [i.e., replication-competent avian leukosis virus long terminal repeat with splice acceptor (RCAS)] vectors to deliver the reverse tet transcriptional transactivator (rtTA) gene to somatic mammalian cells. rtTA can regulate any transgene in which the protein coding sequence is preceded by a tet-operator (tet-o); RCAS viruses infect only cells engineered to express ectopically the avian retroviral receptor, TVA. One vector, RCAS-rtTA-IRES-GFP, also encodes GFP to identify infected cells. Infection of cells from beta-actin TVA transgenic mice with this vector permits efficient regulation of tet-responsive transgenes. Sarcomas arise when p53-deficient murine embryonic fibroblasts carrying beta-actin TVA and tet-o-K-ras4bG12D transgenes are infected with RCAS-rtTA-IRES-GFP and introduced into nude mice treated with the tet analog, doxycycline (dox); when dox is withdrawn, K-ras4bG12D levels fall, cells undergo apoptosis, and tumors regress. Regression can be prevented by means of a genetic complementation assay in which tumors are superinfected before dox withdrawal with other RCAS viruses, such as those carrying an active allele of K-ras. Many TVA and tet-regulated transgenic mice have been generated; thus, this method for somatic cell-specific and temporally controlled gene expression may have broad applications for the study of oncogenesis and tumor maintenance, as well as other cell functions and development.
机译：癌症生物学中的关键问题是维持肿瘤是否需要涉及肿瘤起始或进展的遗传性病变。这个问题可以用有条件地表达致癌转基因的小鼠模型来解决，即在四环素（tet）依赖性转录调节子的控制下。我们已经开发了一种通过使用禽逆转录病毒[即具有复制能力的禽白血病病毒带有剪接受体（RCAS）的长末端重复序列]载体研究逆转录的tet转录反式激活因子（rtTA）基因传递给体细胞哺乳动物的肿瘤维持系统。 rtTA可以调节任何蛋白质编码序列后接tet-operator（tet-o）的转基因； RCAS病毒仅感染经工程改造以异位表达禽逆转录病毒受体TVA的细胞。一种载体，RCAS-rtTA-IRES-GFP，也编码GFP以鉴定感染的细胞。用该载体感染β-肌动蛋白TVA转基因小鼠的细胞后，可有效调节tet应答性转基因。当携带β-肌动蛋白TVA和tet-o-K-ras4bG12D转基因的p53缺陷型鼠胚胎成纤维细胞被RCAS-rtTA-IRES-GFP感染并引入到用tet类似物强力霉素（doxcycline）处理的裸鼠中时，就会出现肉瘤。撤消dox后，K-ras4bG12D水平下降，细胞发生凋亡，肿瘤消退。可以通过遗传互补测定法来预防退化，在遗传互补测定法中，在用其他RCAS病毒（例如携带K-ras活性等位基因的那些病毒）进行Dox撤离之前，将肿瘤重复感染。已经产生了许多TVA和tet调控的转基因小鼠。因此，这种用于体细胞特异性和时间控制的基因表达的方法可能在肿瘤发生和肿瘤维持以及其他细胞功能和发育的研究中具有广泛的应用。

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