Morimoto Mitsuru, Kopan Raphael
Department of Developmental Biology, Division of Dermatology, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Dev Biol. 2009 Jan 1;325(1):171-8. doi: 10.1016/j.ydbio.2008.10.013. Epub 2008 Nov 1.
The doxycycline (DOX)-inducible gene expression systems allow tight temporal and spatial control of transgene expression, invaluable in studies of organ development and disease pathogenesis. Transgenic mice using the human Surfactant Protein C promoter to drive the expression of the reverse tetracycline transactivator (SP-C-rtTA) enabled functional analysis of essential gene function during lung development. Here we report that DOX-fed SP-C-rtTA mice during the period in which Type II cells differentiate results in cellular toxicity that may have confounded the interpretation of previous reports using this line. These effects included impaired alveologenesis, loss/reduction in expression of surfactant-associated proteins, and death. Severity was dependent on genetic background: outbred mice or those on a CD1 background are highly susceptible, whereas the C57BL/6 background appeared resistant by morphological criteria. However, quantitative analysis reveled that DOX-fed, SP-C-rtTA C57BL/6 pups had reduced surfactant mRNA accumulation that could contribute to synthetic lethality when combined with other genetic alterations. We conclude that the combination of genetic backgrounds, length of DOX exposure and the presence of the SP-C-rtTA transgene contributed more than previously appreciated to the similarities seen in the phenotypes reported by investigators using the SP-C-rtTA, (tetO)(7)-Cre. These studies demonstrate the importance of using appropriate SP-C-rtTA only controls in all experiments.
强力霉素（DOX）诱导的基因表达系统能够对转基因表达进行严格的时空控制，这在器官发育和疾病发病机制研究中具有极高价值。利用人类表面活性蛋白C启动子驱动反向四环素反式激活因子（SP-C-rtTA）表达的转基因小鼠，可用于肺发育过程中关键基因功能的功能分析。在此我们报告，在II型细胞分化期间用DOX喂养SP-C-rtTA小鼠会导致细胞毒性，这可能使此前使用该品系的报告的解读产生混淆。这些影响包括肺泡形成受损、表面活性剂相关蛋白表达缺失/减少以及死亡。严重程度取决于遗传背景：远交系小鼠或CD1背景的小鼠高度易感，而C57BL/6背景的小鼠从形态学标准来看似乎具有抗性。然而，定量分析显示，用DOX喂养的SP-C-rtTA C57BL/6幼崽的表面活性剂mRNA积累减少，当与其他基因改变相结合时可能导致合成致死性。我们得出结论，遗传背景、DOX暴露时间长度以及SP-C-rtTA转基因的存在，对使用SP-C-rtTA、(tetO)(7)-Cre的研究人员报告的表型相似性的影响比之前认为的更大。这些研究证明了在所有实验中使用合适的SP-C-rtTA对照的重要性。
