Influence Of Mouse Genetic Background On The Developmental Potential Of Tetraploid Embryo

Polyploidy is generally incompatible with the normal development of most mammalian tissues, although there is a low rate (appreximatily 0.1%) of spontaneous polyploidy occurred on the development of organism which would induce miscarriage , aberrant development of the bones,vertebral column and facial malformation on the fetus. Tetraploid embryo can be used as a power tool on life science such as on the research of mechanism in the inchoate embryo development which regulatory the cell volume, mumber and cleavage rate. Tetraploid embryo can also be used to change the ratio of genome of parents on the progeny; More importantly we can obtain completely ES cell-derived mice through tetraploid embryos complementation and the ES mice is significance in analysis of gene function and built disease mouse modle. For fully utilization of the tetraploid embryos complementation, it is desirable to understand the different developmental potential of tetraploid embryos affect by genetic backreound. The major object of this experiment is to compare the tetraploid embryos among different genetic backgrounds on electrofusion rate, preimplantation and postimplantation developmental potential and capability to compose ES:tetraploid chimera.In this study, 2-cell stage embryos with different genetic backgrounds were fused by two short electric pulse(100V for 50usec)applied by a pulse genetator. The electrofusion rate of 2-cell stage embryos with hybrid genetic background (B6D2F2 and B6C3D2F2) were higher than that of with inbred (C57,ICR and BALB/c) genetic (p<0.05).The ectogenesis developmental potential of tetraploid embryos is affected by the genetic background. There are significant deviation in murula rate, blastula rate and cells number of tetraploid blastula between high hybridization tetraploid embryos and low hybridization tetraploid embryos on the preimplantation stage(P<0.05 or P<0.01). The post-implantation rates of tetraploid blastocysts with different genetic background were not significant (p>0.05), but we obtained 5 fetues develop tol3.5dpc from those tetraploid embryos with high heterozygosity and 0 fetue from inbred tetraploid embryos.It is unsensible to buy ESCs from other labrotaries, because of the pluripotency of ESCs would be affected by passage number and freeze thawing. So we need to built steadily mouse embryonic stem cells (ESCs)-derived and tested system. In this study, three days after ovulation, 43 blastocysts from C57BL/6J×129/Sv mice,38 blastocysts from (C57BL/6J×DBA/2)F₁×129/Sv mice,49 blastocysts from (C57BL/6J×C3H/HeJ) F₁×129/Sv mice and 91 blastocysts from B6C3F₁×B6D2F₁mice were recovered by a surgical uterine flush technique and played on passage 3 to passage 5 mouse(ICR) embryonic fibroblasts(MEF) feeder layer .After 5-6 days of growth, the intact inner cell mass (ICM) was separated with 0.25% trypsin-EDTA and again seeded on feeder in Dulbecco's modified Eagle medium (4500mg of glucose per liter) supplemented with 15% newborn bovine serum, 0.1 mM nonessential amino acids, 0.1 mM 2-mercaptoethanol, 2mM glutamine, 50 units/ml of penicillin, and 50 units/ml of streptomycin, 2000IU/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 cell lines were selected and explanted. At last we obtained 4,3,4 and 3 ES cell lines separately. One of the B6C3F₁×B6D2F₁ ES cell lines was selected to future study its competent for germline transmission.We founded the ES cell line has normal karyotype on the chromosome analyse.In teratoma experiment by injected ESCs into C57 mice's inguen, we gained teratoma contained cells come from all three embryonic germ layers. Then we obtained ES:2n chimeras with germ-line transmission (indentified by coat-colour).In the study,we obtained 3 newborns by aggregation the germline-competent ESCs with B6C3D2F₂(1 newborn)and B6D2F₂(2 newborns) tetraploid embryos,none of the pregnancies those transplanted chimeric embryos that made by ESCs aggregation with tetraploid embryos with ICRxICR,C57×C57 and BALB/c×BALB/c genetic backgrounds was gestation.All of above confirm that developmental potential of the tetraploid embryos with high heterozygosity is better then that tetraploid embryos with low heterozygosity.