神经科学导论 III 101713.pptVIP

Genomic Information Included in Conventional versus BAC Transgenic Constructs: Bigger is Better Gene X Gene X An Average Mammalian Transcription Unit: 25kb Conventional construct 20 Kb BAC construct ~180 Kb Gene W Gene Y Regulatory Elements ( ): Promoter, Enhancer, Silencer, LCR, Splicing Enhancer, Imprinting Signal, etc. BAC Modification by Homologous Recombination in E. coli RecA A mHtEx1 B Vector A B Chlor(+) Co-integration Vector A B Chlor(+) A B RecA Shuttle Vector (pLD53.SCAB) Resolution Widtype BAC A B BAC Ab Ab mHtEx1 mHtEx1 mHtEx1 SacB R6kg R6kg SacB BAC Transgenic Expression of EGFP in Cerebellar Cell Types Monyer Lab Heintz Lab Heintz Lab Calbindin: Purkinje cells BLBP: Bergmann Glia Parvalbumin: Stellate, Basket cells Zipro 1: Granule Cells (Monyer lab) Calbindin Partial List of BAC Modification/Transgenic Mice in Literature(up to June, 2001) Gene Marker Expression Patterns References a9AchR IRES.EGFP Inner hair cells Zuo et al, PNAS, 1999 NPY Cre Hypothal/Cortex/Medulla/Olfa Defalco et al, Science, 2001 Myf5 LacZ/AP Dermomyotome/myotome,etc Carvajal et al, Develop., 2001 Neuronatin LacZ/AP Correct/Imprinted Expression John et al, Dev. Biol., 2001 P57/Kip2 LacZ/AP Skeletal Muscle/Cartilage John et al, HMG, 2001 Rag1/Rag2 EGFP/EYFP T cells/B cells Wong et al, Science, 1999; Wong et al, Nature, 1999 Renin-1 IRES.bgeo Juxtaglomerular cells Mullins et al, JBC, 2000 PU.1 Thy1.1 Peritoneal Macrophage/BM/Spleen Li et al, Blood, 2001 Gensat BAC Transgenic Strategy: Select genes for analysis Prepare marked Bacterial Artificial Chromosomes (BACs) Generate transgenic mice Analyze anatomically: gene expression, possible anatomic phenotypes