Production And Study Of Mouse Reconstructed Oocytes With Somatic Cells

Mouse reconstructed oocytes means transfering the late stage nucleus into enucleated Metaphase II (M II ) oocytes in. order to investigate the developmental potential of the later developmental nucleus. As donor cells , cumulus cells from fresh oocytes complexes (COCs) in oviducts, foetal fibroblast cells from the foetal tissus in vitro cultured and passaged. M II oocytes were collected from oviducts of superovulated donor mouse and enucleated as recipient cytoplasts. Reconstructed oocytes by direct intracytoplasmic injecting ruptured donor cell into recipient cytoplasts were cultured for 3 to 4 hours before being activated with 10mM SrCl2. after activation for 6hours , reconstructed oocytes were directly transferred into oviducts of synchronized recipient mice .some reconstructed oocytes were cultured in vivo for 72h and recycled to observe the early development; some foster recipients were euthanazied at 9.5d p.c for determination of implantation sites; the others were caesareaed section at 21d.p.c to observe the development of the reconstructed embryo, study of enucleation, compared the effect of two different microoperation medium to the enucleated rate .Grouping 3489 MII oocytes into two randomly, group A, 2452 oocytes, microoperation medium without 2.5% sucrose; group B, 1037 oocytes, microoperation medium with 2.5% sucrose , the enucleated rate were 87.64% and 89.97%, respectively. There was no significant difference between two groups (p>0.05) .the study of injection ,the diameter of the injection pipettes proved to be an important factor for survival.There was no significant difference between 5-7μm and > 12μ m pipettes but the surviving rate of injected cytoplasts (lysis) rapidly increased when using 7-10 M m pipettes ( P< 0.05 ) Activation is the key to nuclear transfer, first using MⅡ oocytes after injection HCG 18h to acquired the optimum concentration and effective time of activation, then activated the reconstructed oocytes with the optimum concentration and effective time. From a total 475 reconstructed oocytes were submitted to the strontium treatment, 274 (57.05%.) formed pseudo-pronuclei were considered activated, the proportion of morula and blastocysts were no significant difference between cumulus cells (16.17%) and foetal fibroblast cells( 15.38%) (p>0.05) .now we already builded up a procedure of mouse nuclear transfer .