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首页> 美国卫生研究院文献>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引入转录调节子 进入哺乳动物细胞进行肿瘤维持分析

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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 β-actin TVA transgenic mice with this vector permits efficient regulation of tet-responsive transgenes. Sarcomas arise when p53-deficient murine embryonic fibroblasts carrying β-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with RCAS-rtTA-IRES-GFP and introduced into nude mice treated with the tet analog, doxycycline (dox); when dox is withdrawn, K-ras4bG12D levels fall, cells undergo apoptosis, and tumors regress.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末端剪接受体的长末端重复序列（RCAS）]载体将逆向tet转录反式激活因子（rtTA）基因传递给体细胞哺乳动物细胞来研究肿瘤维持的系统。 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应答转基因。当携带β-肌动蛋白TVA和tet-o-K-ras4b G12D 转基因的p53缺陷型鼠胚胎成纤维细胞被RCAS-rtTA-IRES-GFP感染并引入经tet处理的裸鼠中时，会产生肉瘤 类似物，强力霉素（dox）;当dox撤回时， K-ras4b G12D 水平下降，细胞发生凋亡，并且 肿瘤消退。可以通过遗传防止回归 补体试验，在停药后肿瘤被过度感染 与其他RCAS病毒一起感染，例如带有 K-ras。许多TVA和tet调节的转基因小鼠已经 产生;因此，该方法用于体细胞特定的和暂时的 受控的基因表达可能在研究 肿瘤发生和肿瘤维持以及其他细胞功能和 发展。

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