PROJECT llI; EXPRESSED CDH CANDIDATE GENES CAN BE PREDICTED THEN FUNCTIONALLY

在项目III中（表达的CDH候选基因可以预测，然后在功能上 在动物模型和IPS细胞中验证），我们正在解决基因组和遗传学界面临的最令人生畏的困境。除了对来自每个基因组平台的大型数据集进行优先级排序的挑战之外，还需要获得资源和专业知识，以确认其中检测到计算上显著的变体的候选物的功能性和因果关系。在这个项目中，每个变体将通过严格的标准进行评估，这些标准要求序列变体在dbSNP和1000个基因组项目中未报告，并且它们是非同义的，会导致蛋白质的结构变化或引入终止密码子（Intertronic变体应该影响物种）。然后将在多纳霍实验室或通过我们与杰克逊实验室的合作，在Carol Bult博士的指导下，在多个动物模型系统中对此类变体进行评估以进行测试，项目III的共同主要研究者，或共同研究者Kate阿克曼，医学博士的实验室，在纽约的罗切斯特大学。如果敲除小鼠可用并且存活超过E15.5，我们将在杰克逊实验室饲养这些动物，并检查横膈膜是否存在等同于人类膈疝的缺陷。如果动物不能存活超过E15.5（图2的右臂），则在阿克曼博士的指导下，使用启动子如NKX3.2产生条件性敲除以将表达引导至隔膜。这些后代的胚胎将在E15.5时检查隔膜缺陷。发现缺陷将确认表达基因的功能重要性，并使其成为寻找下游基因的候选基因，这些基因可以在高通量筛选中拯救表型。 如果小鼠敲除不可用（图2中的中间列），我们在实验室中配备了通过直接子宫内注射到RCAS受体转基因胚胎中来敲低感兴趣基因的表达的设备，RCAS-TVA载体含有基因变体。这项工作将在由Maria Loscertales博士指导的表达核心中完成，其还将使用禽类模型在卵内确定是否存在肺表型的修饰。受影响的肺可以在器官培养中培养，并进行抢救测试。Loscertales博士还将通过免疫组织化学和原位杂交来确认表达，这需要相当多的专业知识才能在微小的胚胎隔膜中正确完成。她也有相当丰富的显微操作技术经验，能够在E10小鼠胚胎和鸡卵中使用该技术数据。Loscertales博士的专业知识得到了项目III联合研究员Kate阿克曼博士的补充，他在识别小老鼠横膈膜结构和评估其畸形方面拥有超过六年的经验。

In PROJECT III (Expressed CDH Candidate Genes Can Be Predicted, Then Functionally Validated in Animal Models and IPS Cells) we are addressing a most daunting loggerjam facing the genomic and genetics community. In addition to the challenge of priortizating the large data sets emanating from each of the genomic platforms, is to garner the resources and expertise to confirm the functionality and causality of the candidates in which a computationally significant variant is detected. In this project, each variant will be evaluated by stringent criteria that requires that the sequence variants be unreported in dbSNP and the 1000 Genomes Project, and that they are nonsynonymous and cause structural changes in the protein or introduce stop codons (Intertronic variants should affect species). Such variants will then be assessed for testing in the multiple animal model systems uniquely available In the Donahoe laboratory or through our collaborations with The Jackson Laboratory, under the direction of Carol Bult, Ph.D., the Co-PI of Project III, or the laboratory of Co-investigator Kate Ackerman, M.D., at University of Rochester, New York. If a mouse knockout is available and survives beyond El 5.5, we will breed these animals at The Jackson Laboratory and examine the diaphragms for defects equivalent to human diaphragmatic hernia. If the animals do not survive beyond El 5.5 (Right arm of Figure 2), a conditional knockout will be created using a promoter such as NKX3.2 to direct expression to the diaphragm under the direction of Dr. Ackerman. Embryos of these offspring will be examined at E15.5, for defects in the diaphragm. Finding a defect will confirm the functional importance of the expressed gene and make it a candidate in the search for downstream genes which can rescue the phenotype in high throughput screens. If a mouse knockout is not available (Middle column of Figure 2), we are equipped in the laboratory to knock down the expression of a gene of interest by direct in utero injection into RCAS receptor transgenic embryos, the RCAS-TVA vector containing the gene variant. This work will be done in the Expression Core directed by Maria Loscertales, Ph.D., who will also use the avian model to determine in ovo if there is a modification of the lung phenotype. Affected lungs can be then be cultured in organ culture and tested for rescue. Dr. Loscertales will also confirm expression by immunohistochemistry and in situ hybridization, which requires considerable expertise to be done properly in the tiny embryonic diaphragm. She also has considerable experience with micromanipulation techniques to be able to employ this technique data in the mouse embryo at E10 and in the chick in ovo. Dr. Loscertales' expertise is complemented by that of Dr. Kate Ackerman, co-investigator of Project III, who has over six years of experience in identifying the small mouse diaphragm structures and evaluating their dysmorphology.