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. Copyright 2003 Wiley-Liss, Inc.