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

在项目III中(可以预测表达的CDH候选基因，然后从功能上 在动物模型和IPS细胞中得到验证)，我们正在解决基因组和遗传学社区面临的最令人生畏的困境。除了对每个基因组平台产生的大量数据集进行优先排序的挑战外，还需要获得资源和专门知识，以确认检测到具有计算意义的变异的候选者的功能和因果关系。在这个项目中，每个变体都将按照严格的标准进行评估，这些标准要求序列变体不能在数据库SNP和1000基因组计划中报告，并且它们是非同义的，会导致蛋白质结构变化或引入终止密码子(中间变体应该影响物种)。然后，我们将在多纳霍实验室或我们与杰克逊实验室的合作下，在多纳霍实验室或纽约罗切斯特大学共同研究员凯特·阿克曼的实验室的指导下，对这些变体进行评估，以便在多种动物模型系统中进行测试。如果有小鼠基因敲除，并且存活超过EL 5.5，我们将在杰克逊实验室饲养这些动物，并检查横隔膜是否存在相当于人类横隔膜的缺陷。如果动物不能存活超过EL 5.5(图2的右臂)，将使用NKX3.2等启动子在Ackerman博士的指导下将表达引导到横隔膜，从而创建条件基因敲除。这些后代的胚胎将在E15.5接受横隔膜缺陷的检查。发现缺陷将证实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，从而在高通量筛选中挽救表型。 如果没有小鼠基因敲除(图2的中栏)，我们在实验室里配备了通过在子宫内直接注射到RCAS受体转基因胚胎(包含该基因变体的RCAS-TVA载体)来敲除目标基因的表达。这项工作将在由Maria Loscertales博士指导的Expression Core中完成，她还将使用禽类模型来确定在卵子中是否存在肺表型的改变。然后，可以在器官培养中培养受影响的肺，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横隔膜上正确完成。她在微操作技术方面也有相当多的经验，能够在E10的小鼠胚胎和卵子中的雏鸟中应用这项技术数据。Loscertales博士的专业知识与项目III的合作研究员Kate Ackerman博士的专业知识相辅相成，她在识别小鼠横隔膜结构和评估其畸形方面拥有六年多的经验。

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.