Establishment And Optimization Of Mouse Tetraploid Complementation

Tetraploid complementation is also known as the two-step cloning.Tetraploid embryos(4n embryos)can aggregation with diploid embryos(2n embryos)or embryonic stem cells(ES cells)to form chimeras.During development,4n cells mainly participate in extraembryonic tissues(such as trophoblast,yolk sac membrane,allantoic membrane,placenta,etc.)and hardly participate in the formation of embryoid bodies.Thus,almost completely 2n embryos or ES cell-derived mice can be obtained,which means tetraploid complementation.Currently,the efficiency of cloned mice obtained by somatic cell nuclear transfer is low,which is 1% to 4%.Most of the cloned embryos die in the early stages of development after implantation.And a small number of embryos that can grow to term also produce a large placenta and fetus,resulting in dystocia.Even if the baby is lucky enough to survive,most of them are difficult to survive due to problems in the respiratory system and blood circulation system.The tetraploid complementation can effectively increase cloning efficiency and become an effective method for producing transgenic animals.At present,tetraploid complementation has been widely used in testing cell totipotency,studying gene function,studying embryonic development mechanism and producting cloned animals.In this experiment,4n embryos were made by electrofusion method.And the optimal electrofusion parameters were initially explored.It was found that when the voltage is 100v/mm,the P.Length is 50 ?s,and the number of pulses is 2,fusion rate reaches 95.27%.To improvet the collection and culture methods of mouse embryo collection and culture methods,greatly improving embryo blastocyst rate.The blastocyst rate of 4n embryos was 93.42%,which was not significantly different from 2n embryos(94.41%)(P>0.05).Immunofluorescence staining showed that compared with 2n embryos,4n embryos had larger nuclei,fewer total cell numbers and low levels of pluripotency transcription factor Oct-4.The m RNA levels of Oct-4,Nanog,Bax,and Bcl-2 in 2n embryos and 4n embryos were detected by qRT-PCR.The results showed that the mRNA levels of Oct-4 and Nanog in 4n embryos were higher than those in 2n embryos,but the difference was not significant(P>0.05).The Bax mRNA level of 4n embryos was higher than that of 2n embryos(P<0.05).There was no significant difference in Bcl-2 m RNA levels between 4n embryos and 2n embryos.When 4n embryos developed to 2-8 cells embryos,ES cells were injected into the embryos by microinjection.Each embryo was injected with 6 to 8 ES cells.Or about 15 ES cells were injected into per blastocysts.And then,the embryos were transplanted into pseudopregnant mice.A total of 18 pseudopregnant mice were transplanted.There are two female mice with enlarged uterus and spherical protrusions.A full-term chimeric mouse,3.5cm in length and 2.06 g in body weight,was obtained by caesarean section.The chimeric mouse tissues were detected and the ES cells were tagged in his heart,liver,brain and testis,confirming that ES cells actually participate in fetal development.