Isolation And Identification Of Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs), derived from preimplantation embryos, areundifferentiated, immortal cells capable of differentiating into derivatives of all threeembryonic germ layers. Pluripotential ES cells have been used extensively in studies ofembryogenesis, gene expression and regulation, seek the mechanism of cell differentiation,the cell repair and the transplantation of cells, tissues and organs. The production oftetraploid embryos has become a common experimental manipulation in the mouse.Although development of tetraploid mice has generally not been observed beyondmidgestation, tetraploid:diploid chimeras are widely used as a method for rescuingextraembryonic defects and elucidating gene function in vivo. For utilization of the mouseembryonic stem (ES) cells for various purposes, it is desirable that the cell lines areestablished from various sources such as inbred and outbred mouse strains. The majorobject of this paper is to generate mouse embryonic stem cell lines from(C57BL/6J×129/Sv)F1 and (C57BL/6J×DBA/2)F1×129/Sv mouse and tested their abilityto produce completely ES cell-derived mice at early passage numbers by tetraploid embryoaggregation.Three days after ovulation, 58 blastocysts from (C57BL/6J×129/Sv) F1 mice and 63blastocysts from (C57BL/6J×DBA/2)F1×129/Sv mice were recovered by a surgical uterineflush technique and played on passage 2 to passage 5 mouse embryonic fibroblasts feederlayer mitotically inactivated with mitomycin C (1mg/100ml for 3 hours). After 4-6 days ofgrowth, the intact inner cell mass (ICM) was separated with 0.25% trypsin-EDTA andagain seeded on feeder in Dulbecco's modified Eagle medium (4500mg of glucose per liter)supplemented with 15% newborn bovine serum, 0.1mM nonessential amino acids, 0.1mM2-mercaptoethanol, 2mM glutamine, 50 units/ml of penicillin, and 50 units/ml ofstreptomycin,1000IU/ml LIF(Leukemia Inhibitory Factor). Cultures were grown in 5%CO₂, 95% humidity and 37℃in incubator. After 6 to 7 days of culture, Eleven ES celllines were selected and explanted.ESCs were injected to different position: Subcutaneous implant, under kidney andcapsule testes membrance. We gain derivatives of all three embryonic germ layers. We alsoobserved that implant the ESCs under the testes membrance was a simple, efficientprocedure for testing the pluripotency of ESCs.The oviducts of superovulated (C57BL/6J×DBA/2)F2 black females were flushed 44-46 hours after treatment with human chorionic gonadotropin to collect 1979 latetwo-cell-stage embryos. The embryos were placed twenty at a time between two platinumelectrodes laid 1mm apart in 0.3M mannitol supplemented with 0.1mM CaCl₂ and 0.1mMMgCl₂ in the electrode chamber. The blastomeres were fused by two short electric pulse(100V for 50μsec) applied by a pulse generator. Fusion of blastomeres should becompleted in 10-30 minutes.The developmental ability of duple embryos and the tetraploid embryos were assessedby explanted in the mice oviduct. We conclude that there was no significant difference ofthe developmental ability between the duple embryos and the tetraploid embryos. Thetetraploid embryos could develop 12-13 days, several could develop 14-15days. Ultimately, we did not gain the alive tetraploid foetus.After 24 hours of culture, most of the fused embryos developed to the four-cell stage.Zonae pellucidae of the 1444 four-cell-stage fused embryos were removed by treatmentwith 0.5% Pronase solution. The embryos were aggregated with five ES cell lines. After 24hours of aggregation coculture, 1122 embryos aggregated with ES cells were collected andtransferred into the uteri of 51 pseudopregnant recipients. Five recipients were pregnant.These pregnant recipients were routinely subject to a Caesarean section or spontaneousdelivery and got 1 fetus derived from five ES cell lines, designated B1, B2, B3, B4, B5. Inresult, the only one ES cell-tetraploid neonates derived from B4 ES cell lines survived onlyone day. ES cell-tetraploid pups obtained from hybrid ES cell displayed increased placentaland birth weights.The present results prove that ES cell-tetraploid mice (ES mice) are completelyderived from embryonic stem cells and can be obtained upon aggregation of hybrid EScells and tetraploid embryo. Genetic heterozygosity is a crucial parameter for postnatalsurvival of mice that are entirely derived from ES cells by tetraploid embryocomplementation. This method represents a simple, efficient procedure for immediategeneration of targeted mouse mutants from genetically modified ES cell clones, in contrastto the standard protocol, which involves the production of chimeras and several breedingsteps.