小鼠转基因研究方法.pptxVIP

Transgene and gene knockout in studying gene functions;功能缺失(Loss of function)： KO, CKO, RNAi 获得功能(Gain of function)：Transgenic, virus-based 在体 in vivo与离体 in vitro 描述性报告 vs 机理研究报告 研究设计：假说(可能性)—验证—总结(论文) ;小鼠基因敲除和转基因 ; 基因敲除（knock out ）和基因敲入（ knock in） KO过程也称基因打靶(gene targeting)，利用DNA同源重组，在基因组中的某一特定部位进行定点的基因置换，用无功能基因替换目的基因或Delete目的基因。 根据需要（如验证A与B在功能上有某种关联），在敲除基因A时，将有功能的基因B放在A基因的位置，即A是KO，B是Knock in。 ;基因敲除的基本程序;positive selection neor ：新霉素抗性基因，neor基因存在于打靶载体内，当重组（同源或随机整合）后，ES细胞能在含新霉素G418的培养液中生长 negative selection 1. HSV-tk：单纯疱疹病毒??胸腺嘧啶激酶基因，该基因产物可分解单核苷酸类似物(ganciclovir)生成毒性产物，产生自杀效果 同源重组时：Tk-基因丢失，ES细胞存活 随机整合时：Tk-基因存在，ES细胞死亡 2. DTA (diphtheria fragment A) : 不用给药 ;2、将载体导入ES细胞 ;Homologous recombination;Recombinants with random insertion-containing both neo and tk;4 、将基因敲除ES细胞注射入囊胚，形成嵌合胚胎，得到嵌合体小鼠，而后筛选培育阳性子代小鼠。 ;Conventional Knock out;Mutation of Lmx1b in mice;Lmx1b is essential for development of 5-HT+ neurons ;Conditional knock out (CKO) (Tissue- or cell type-specific);Raphe nuclei-specific deletion of Lmx1b ;Mating ;CKO mice lose essentially all 5-HTergic neurons in the brain;Time-control CKO;Generation of Pet1-CreER mice ;Lmx1b is required for 5-HT expression in Adult Brain;Other methods in gene Knock out;The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR): RNA-Guided Endonuclease technology for genome engineering. Two components: (1) a guide RNA (2) an endonuclease, CRISPR associated (Cas) nuclease, Cas9. ;Transgene ;Transgene ; Founder mice: transgenic mice that develop from the injected eggs Transgenic mice: mice that are determined to presence of the injected gene Stable line: due to multiple insertion ;美国The Jackson Laboratory－世界最大的小鼠品系仓库;南京国家小鼠资源中心 ;资源库坐落于南京市浦口高新区，占地100亩，建筑面积7800平方米，小鼠笼位3万多个，各项指标已通过省实验动物环境与设施的监测。 引进和自主构建小鼠品系达150多种。;SPF饲养要求;Mouse Care: Barrier Accommodation with Individually Ventilated Cage Racks;子宫内胚胎电转技术in utero electroporation;功能缺失（LOF）： KO, CKO, RNAi 获得功能（GOF）：Transgenic,