转基因与基因敲除knockoutmice.ppt

Conventional Knockout, knockin Transgenic Vectors E1 E2 Genomic Targeted locus Targeting vector E1 E2 MC1-TK Neo 8bp deletion E1 E2 Neo 8bp deletion POU4F3 Knockin Scheme ppp2ca flox小鼠 小鼠交配方案 表型分析 生育力检测 ♂ ♀ 平均子代数目 基因型 数目 基因型 数目 HETZ 9 WT 18 5.8±3.2 HOMO 7 WT 14 0 胚胎时期，生殖嵴结构 胚胎时期，PGCs数目 不同时期，小鼠睾丸外形、重量 不同时期，小鼠睾丸切片 小鼠附睾切片 睾丸细胞DNA含量流式分析 睾丸细胞增殖情况 睾丸细胞凋亡情况 对睾丸管腔中细胞性质的鉴定 CONTACT ME PLUSGENE 南京汉中路140号 南京医科大学 江苏省医药实验动物基地 转基因中心  T0 XIAWEIWEIXIA@NJMU.EDU.CN PHONE025WEB 1 of 10 转基因 ? 基因敲除? 表型分析 ?影像分析? 慢病毒? 基因资源 PLUSGENE PROFESSIONAL TRANSGENIC CENTER THANK YOU! PlusGENE 1 of 10 转基因 ? 基因敲除? 表型分析 ?影像分析? 慢病毒? 基因资源 PLUSGENE PROFESSIONAL TRANSGENIC CENTER * * fluorescent zebrafish * * * DNA recognition and cleavage by zinc-finger nucleases (ZFNs). (a) Sketch of a ZFN dimer bound to a typical, nonpalindromic DNA target. Each ZFN consists of the cleavage domain of FokI fused to a zinc-finger protein (ZFP) that has been customized to specifically recognize either a ‘left’ or ‘right’ half-site (indicated by blue and red boxes), which are separated by a spacer of either 5 or 6 bp. Simultaneous binding by both ZFNs enables dimerization of the FokI nuclease domain and DNA cleavage. Note that endogenously active ZFNs have been created using four fingers (indicated here) that each bind 12-bp sites5, as well as three fingers that each bind 9-bp sites4,6,20,21. (b) Three-dimensional model corresponding to the arrangement in a. DNA is shown in gray, ZFNs are colored blue (left ZFN) or red (right ZFN), and reflective spheres denote zinc ions. Zinc-finger helices that mediate sequence recognition in the major groove are represented by cylinders, whereas the more centrally located FokI cleavage domains are represented entirely as ribbon diagrams. The cleavage domain interface that has been reengineered in these studies is located at the center of the image. (c) Sketch of the problem motivating this work. Coexpression of Lwt (left ZFP fused to the wild-type FokI cleavage dom