Cloned Mice: MATERIALS AND METHODS(4)

METHODS(4)Parthenogenetic Activation

As a positive control for oocyte quality and the method of activation used with the IE protocol, a sample of oocytes in each nuclear transfer experiment were activated with 7% ethanol in hCZB for 7 min, washed in hCZB, and cultured in the presence of 5 |xg/ml of CB to inhibit extrusion of the PB. After 5 h of incubation in the presence of CB, oocytes were washed in hCZB and transferred to CZB medium for culture at 37.5°C in 5% CO2 in air. canadian health & care mall

Embryo Culture and Transfer

Cloned and parthenogenetic embryos were assessed after 72 h of culture in CZB. At this point, nuclear transfer morulae/blastocysts were transferred into uteri of pseudopregnant, surrogate B6CBAF1 mothers that had been mated with vasectomized male mice 2.5 days earlier. Embryos (n = 5-10) were transferred into each uterine horn. Pups were recovered by cesarean section from recipients sacrificed at 19.5 days postcoitum.

Statistical Analysis

The frequency of oocytes at different stages of meiosis at successive times after activation and nuclear transfer development were analyzed by one-way ANOVA. Logistic regressions were fitted to the proportions of oocytes exhibiting IE and compartmentalization of chromatin in PBs to evaluate the efficacy of treatments with demecolcine.

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