南开大学 基因操作原理第十三章PPT.ppt

Chapter 13 Gene manipulation of animal ;1. Introduction 2. Genetic manipulation of animals;1.Introduction Characteristic：easy--gene transfer to cell hard--get the transgenic animals Basic research: essential for understanding gene functions, differentiation and development Genetic Products: authentic post-translational modifications Histroy: 1962, Szybalska and Szybalaki, HGPRT+ DNA+HGPRT- cell HAT HGPRT+ cell ;2. Genetic manipulation of animals 1) Methods for gene transfer into cells 2) Vector 3) Selectable markers 4) Reporter genes 5) Plasmids rescue 6) Transgenic animals;1) Method for gene transfer into animal cells A. Tansfection —DNA/calcium phosphate —DEAE-dextran —Lipofectmine B. Direct transfer — Electroporation — microinjection C. Viral transduction;Method A-2 DEAE-dextran;Method A-3 Lipofectmine Advantages： mild，high capacity, can be used to introduce YAC low toxicity, used in gene therapy.;Method B-1: Electroporation Advantage： adapted to many cell type. disadvantage： need specialized apparatus, must be determined empirically for different cell types Key ：optimal pulse length and amplitude depend on cell types;Viral vector ;Method C1: packaging cell line ;helper plasmid ;Method C3: wild type virus ; 2) Vector (1) pSV and pRSV plasmid (2) SV40 vector ;Plasmid: shuttle vector: Prokaryotic elements: Eukaryotic element： Promoter : from virus Intron Polyadenylation signal 5’UTR and 3’UTR Kozak’s sequence Signal peptid (selectable marker) ;; 3) Selectable markers;;①; 4) Reporter Genes;;;; 5.Plasmid rescue;; Ⅵ. Expression of exogenous genes in mice ;; left: giant mouse (44g) right: wild type (29g) Both are 10 weeks old. Growth speed of giant mouse is 2-3