Pcdh18基因抑制对小鼠胚胎中枢神经系统发育的影响

Studies had shown that down regulation of protocadherin18 (Pcdh18), a kind of cadherin, induced abnormal embryonic neurogenesis and caused fetal death in a dose-dependent fashion in zebrafish, which provided clues to determine the gene function of human Pcdh18. The homology of Pcdh18 nucleotide sequence in zebrafish and human is only 66%, and their protein structures are not exactly the same. These differences made it necessary to study Pcdh18 in mammalian models to get more reliable information for determining the gene function. .As the data suggested, Pcdh18 was essential for embryonic survival and loss of Pcdh18 resulted in fetal death mainly because of neurogenesis defects. Thus, it was not optimistic to obtain the Pcdh18 knockout mice by traditional knockout technology for further research..Combination of RNA interference (RNAi) theory and transgenic mice technology, a sound method was to construct Pcdh18 RNAi mice in which Pcdh18 was properly knockdown but not knockout by controlling the rate of gene inhibition. The advantage of the RNAi mice model was twofold: (1) to avoid fetal death; and (2) to produce defective phenotype for researching the gene function..Generation of Pcdh18 RNAi mice as a model, the effects of Pcdh18 down regulation on the embryonic central nervous system would be identified by morphological observation, detection of apoptosis, and by in situ hybridization which is for tracing the expression and alteration of neural markers. The results of our research would provide reliable information for determining the gene function of human Pcdh18.

Pcdh18是一种钙黏附蛋白。Pcdh18基因抑制可致斑马鱼神经系统发育异常并呈剂量依赖性胚胎死亡，该结果可为人类Pcdh18基因功能确定提供线索。但是，斑马鱼与人类Pcdh18仅有66%的序列同源性，且结构也不尽相同，若要获得更为可靠的参考，须以哺乳动物为对象做进一步研究。研究显示，Pcdh18极有可能是必须基因，基因敲除可因神经系统发育缺陷导致胚胎死亡，因此，获得Pcdh18基因敲除小鼠的前景不容乐观。将RNA干扰理论与转基因小鼠技术相结合，通过控制基因抑制效率获得Pcdh18恰当"下调"而非完全"敲除"的RNAi小鼠，既可规避胚胎死亡，又可获得缺陷表型进而倒推基因功能。以所建立的RNAi小鼠为模型，通过原位杂交技术追踪神经发育过程中结构标志基因的表达变化，并结合形态学观察和凋亡检测的手段，研究Pcdh18基因抑制对小鼠胚胎中枢神经系统发育的影响，其结果可为该基因功能确定提供可靠参考。

研究背景: Pcdh18是一种钙粘附蛋白。研究表明，Pcdh18基因敲除可因神经系统发育缺陷导致斑马鱼胚胎死亡，因此，获得Pcdh18基因敲除小鼠的前景不容乐观。将RNA干扰理论与转基因小鼠技术相结合，通过控制基因抑制效率获得Pcdh18恰当“下调”而非完全“敲除”的RNAi小鼠，既可规避胚胎死亡，又可获得缺陷表型，为进一步研究该基因功能奠定基础。.研究方法/主要发现：利用RT-PCR技术自14.5d小鼠胚胎中扩增mPcdh18基因片段（缺终止密码子）并进行测序鉴定；将扩增片段定向克隆入载体pEGFP-N1，使其同框融合于EGFP报告基因N-端，构建并鉴定mPcdh18、EGFP融合表达载体pEGFP-mPcdh18。通过pEGFP-mPcdh18和候选siRNA共转染体外培养的293T细胞，筛选出高效抑制mPcdh18基因表达的siRNA序列。将siRNA相对应的反向互补DNA序列插入pSingle-tTS-shRNA载体构建siRNA诱导表达载体pSingle-tTS-shRNA。将pEGFP-mPcdh18与siRNA诱导表达载体瞬时共转染293T细胞，在适当浓度DOX诱导下，观察诱导表达载体所表达的siRNA对外源性Pcdh18的抑制效果。将已初步鉴定的pSingle-tTS-shRNA重组质粒载体转入LLC细胞，经G418筛选，得到稳定细胞株。通过qRT-PCR和Western-Blotting手段，测定在不同剂量的DOX诱导下，稳定细胞株内mPcdh18的表达水平，同时观察所筛选细胞株的增值活性和细胞形态变化，测定其生长曲线和迁移能力，观察诱导表达载体所表达的siRNA对内源性Pcdh18的抑制效果以及Pcdh18表达下调对细胞生物学特性的影响。线性化pSingle-tTS-shRNA，以传统小鼠受精卵原核注射的方法争取获得转基因小鼠做进一步研究。同时熟练小鼠整体原位杂交技术和小鼠切片原位杂交技术，为相关功能研究奠定基础。.结论/本研究的意义：本研究基于Pcdh18基因特性，希望将RNAi理论与转基因技术相结合，以构建RNAi诱导表达细胞系为基础进一步建立RNAi诱导表达小鼠来研究功能未明基因Pcdh18对小鼠胚胎中枢神经系统发育的影响。研究结果表明：稳定转染细胞株在DOX 的诱导下能有效表达siRNA， 所表达的siRNA下调Pcdh18后导致细胞的生