A mouse model of the zebrafish meltdown mutant

描述(由申请者提供)：在这份拨款提案中，我们概述了一项基于我们在斑马鱼上的最新发现来模拟人类癌细胞在老鼠身上侵袭的计划。我们已经证明，在斑马鱼熔融(MLT)突变幼体中，肌球蛋白重链(MYH11)基因突变会导致后肠囊性扩张。MLT(MYH11)蛋白在斑马鱼MLT幼体的肠道中非自主地激活人前侵袭基因同源物的上皮表达，仅限于平滑肌细胞。因此，MLT突变体可能被用来模拟人类癌细胞的侵袭。在这里，我们建议获得转基因小鼠，在其中MLT点突变被工程到小鼠MYH11基因座中。在哺乳动物模型系统中复制MLT肠道缺陷将使我们能够解决有关上皮结构和癌细胞侵袭的问题，这些问题在斑马鱼身上是不可行的。首先，MLT小鼠可以用来获得基质细胞系，用于基因芯片实验和器官培养试验。这些实验可能导致识别编码保守的间质信号的基因，这些信号指导人类癌细胞的侵袭。其次，MLT突变体可能与其他模拟人类息肉综合征的小鼠突变体交配。这些双突变将有助于确定MYH11的改变是否促进良性肠息肉的侵袭性转化。第三，MLT基因敲入小鼠可能受到化学诱变方案的影响，已知的突变方案会导致肠癌的形成。这些实验将使我们能够测试MYH11突变是否促进了肿瘤的生长和/或转移。因此，MLT突变小鼠的衍生将使可能有助于确定人类MYH11在癌细胞侵袭中的作用的实验成为可能。这项拨款提案中描述的工作将有助于确定癌细胞侵袭和转移的分子机制，并可能导致防止癌症进展的新疗法。

DESCRIPTION (provided by applicant): In this grant proposal, we outline a plan to model human cancer cell invasion in the mouse based on our recent discoveries in zebrafish. We have shown that mutation of the smooth muscle myosin heavy chain (myh11) gene leads to cystic expansion of the posterior intestine in zebrafish meltdown (mlt) mutant larvae. The Mlt (Myh11) protein, which is restricted to smooth muscle cells, non-autonomously activates epithelial expression of human proinvasion gene orthologs in the intestine of zebrafish mlt larvae. Thus, mlt mutants may be used to model human cancer cell invasion. Here, we propose to derive transgenic mice in which the mlt point mutation is engineered into the mouse Myh11 locus. Phenocopy of mlt intestinal defects in a mammalian model system will allow us to address questions concerning epithelial architecture and cancer cell invasion that are not feasible using zebrafish. First, the mlt mice may be used to obtain stromal cell lines for use in microarray experiments and organ culture assays. These experiments may lead to the identification of genes encoding conserved stromal signals that direct human cancer cell invasion. Second, the mlt mutants may be mated to other mouse mutants that model human polyposis syndromes. These double mutants will help determine if alteration of Myh11 promotes invasive transformation of benign intestinal polyps. Third, the mlt knock-in mice may be subjected to chemical mutagenesis protocols known to cause formation of intestinal cancers. These experiments will allow us to test whether the myh11 mutation enhances tumor growth and or metastasis. Thus, derivation of mlt mutant mice will enable experiments that may help define the role of human MYH11 in cancer cell invasion. The work described in this grant proposal will help define the molecular mechanisms of cancer cell invasion and metastasis and may lead to new treatments to prevent cancer progression.