Cultivation Of Primary Mice Fully Derived From Embryonic Stem Cell(F0-ESC Mouse) And Its Methodology

Objective: To produce primary ESC mice (F0-ESC mice) which fully derived fromembryonic stem cell(ESC) with the velocimouse technic and analyze the technicaldetails in order to promote application of this technology for the identification ofESC/Ips and the preparation of gene modified mice to provides a more convenienttechnology platform for life-science.Methods:10～15G1-ESCs were micro-injected into8-cell stage embryos ofwild-type ICR mouse utilizing laser-assisted micro-manipulation system. Injectedembryos were cultured in KSOMAAat37°C in an atmosphere of5%CO2for48hours.The rates of chimeric/homozygous blastocysts were calculated under invertedfluorescence microscope. Some of injected embryos were cultured in KSOMAAat37°C in an atmosphere of5%CO2for2hours or overnight and then were transferredinto the oviducts of0.5dpc or the uterine horns of2.5dpc pseudo-pregnant ICRfemales. The supposed ESC mouse was recognized by black coat color and greenfluorescence all over skin from the newborns. The supposed ESC mouse was matedwith wild-type ICR mouse and the phenotype of their offspring(F1) was analyzed tojudge whether the mouse exhibited germline transmission. Finally, the mouse wasdissected and all of organs examined for green fluorescence in order to confirmed theF0-ESC mouse which fully derived from G1-ESC.Results:（1）After ESC micro-injection and embryos cultured in vitro, the embryosdeveloped into blastocysts and18.54%blastocysts owned the fully-green inner cellmass that is fully derived from G1-ESC;（2）3F0newborns carrying G1-ESC wereobtained and one of them was F0-ESC mouse（male），two of them were chimera;（3）The F0-ESC mouse was mated with wild-type ICR females and exhibited its ability of germline transmission from their offspring;（4）All of organs of F0-ESC mouseexhibited green fluorescence confirming the mouse is fully derived from G1-ESCs.Conclusion: Velocimouse technic is a novel method to efficiently produce F0-ESCmouse. It is a relative simple and powerful technic for the identification of ESC/iPSand the preparation of gene modified mice to provide a more convenient technologyplatform for life-science. The home-made G1-ESC line is pluripotency and anexcellent embryonic stem cell line.