Construction Of A Mouse Noggin Site-Specific Intergration Targeting Vector And Its Transfection Into Murine Embryonic Fibroblasts And Myoblasts

Objective:In order to study the function of Noggin,we clone Noggin gene in mouse,and constructe a hair follicle specific-expression vector pDs-MSKTLNE of Noggin gene. Then use linearization pDs-MSKTLNE transfer mouse embryonic fibroblast (MEF) cells and mouse myoblast (C2C12) to obtain a transgenic cell clones,which stable expresses Noggin gene in hair follicle cells specifically, and verify the rationality of the vector’s construction.The results can lay a foundation for noggin gene function research.Another purpose of this test is to verify that whether this interval can be used as a target site,the mouse chromosome11spacing between Keratin16and keratin14.1. Construct of mouse noggin gene site-specific integrated targeting vectorBuild a set of targeting vector,which noggin gene site-specific integrated,named pDs-MSKTLNE.pDs-MSKTLNE is routine homologous recombination targeting vector, contains5.0kb homologous long arm and4.0kb homologous short arm human keratin14promotor, Noggin gene,and contained Neor gene as a positive marker, HSV-TK as negative selection marker.First,we cloned these fragments,and then in this order connected these fragment to the vector frame,homologous short arm, negative selection marker HSV-TK,homologous long arm,human keratin14promotor,Noggin gene.2. Prepare mouse embryonic fibroblast (MEF) cells and myoblast (C2C12) used for site-specific integrating mouse Noggin geneFirst,we use fastdigest enzyme Agel to linearization targeting vector of pDs-MSKTLNE,and according to the liposomes kit instructions,linearization vector700ug plus liposome2ul, incubation30min,then transfect mouse embryonic fibroblast (MEF) cells,that these cells deal with hunger in advance.Added G418and GANC to select for15days and received a total of103resistance clones, the PCR identification, no homologous recombinant positive clones. Among them, the fibroblasts random integration efficiency is23.3%(24/103). First,we use fastdigest enzyme Agel to linearization targeting vector of pDs-MSKTLNE,and according to the liposomes kit instructions,linearization vector1000ug plus liposome2ul, incubation30min,then transfect mouse myoblast (C2C12), that these cells deal with hunger in advance.Added G418and GANC to select for15days and received a total of124clones, the PCR identification, no homologous recombinant positive clones. Among them, the fibroblasts random integration efficiency is34.6%(43/124).