Hou Ling, Loftus Stacie K, Incao Arturo, Chen Amy, Pavan William J
Mouse Embryology Section, Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA.
Dev Dyn. 2004 Jan;229(1):54-62. doi: 10.1002/dvdy.10468.
An in vitro gene complementation approach has been developed to dissect gene function and regulation in neural crest (NC) development and disease. The approach uses the avian RCAS virus to express genes in NC cells derived from transgenic mice expressing the RCAS receptor TVA, under the control of defined promoter elements. Constructs for creating TVA transgenic mice were developed using site-specific recombination GATEWAY (GW), compatible vectors that can also be used to facilitate analysis of genomic fragments for transcriptional regulatory elements. By using these GW vectors to facilitate cloning, transgenic mouse lines were generated that express TVA in SOX10-expressing NC stem cells under the control of the Pax3 promoter. The Pax3-tv-a transgene was bred onto a Sox10-deficient background, and the feasibility of complementing genetic NC defects was demonstrated by infecting the Pax3-tv-a cells with an RCAS-Sox10 expression virus, thereby rescuing melanocyte development of Sox10-deficient NC cells. This system will be useful for assessing genetic hierarchies in NC development. Developmental Dynamics 229:54-62, 2004.
已开发出一种体外基因互补方法，用于剖析神经嵴（NC）发育和疾病中的基因功能与调控。该方法利用禽RCAS病毒，在特定启动子元件的控制下，在源自表达RCAS受体TVA的转基因小鼠的NC细胞中表达基因。使用位点特异性重组GATEWAY（GW）构建了用于创建TVA转基因小鼠的构建体，这些兼容载体还可用于促进对转录调控元件的基因组片段的分析。通过使用这些GW载体来促进克隆，生成了在Pax3启动子控制下在表达SOX10的NC干细胞中表达TVA的转基因小鼠品系。将Pax3-tv-a转基因培育到Sox10缺陷背景上，通过用RCAS-Sox10表达病毒感染Pax3-tv-a细胞，从而挽救Sox10缺陷NC细胞的黑素细胞发育，证明了补充遗传性NC缺陷的可行性。该系统将有助于评估NC发育中的遗传层次。《发育动力学》229:54 - 62，2004年。
