| Diploid-tetraploid (2N-4N) embryo aggregate usually has a developmental property with 4N cells restrictively distributed into PrE and its derivatives, including parital yolk sac, placental trophoblast, as was confirmed by many previous results. Most of current investigations on its origin were focused on the peculiarities of 4N blastomere such as doubled ploidy and the comparatively enlarged cell size.Though uncertainty on the detailed developmental mechanism of 2N-4N aggregates may present now, we are still able to apply 2N-4N aggregation to studies on gene functions in mammalian early development. In addition, postimplanted reconstructed embryo by nuclear transfer (NT) saw massive loss due to incomplete development in extraembryonic tissues, especially, the placenta. In inter-species NT, the implantaion seems impossible at all because of mismatched genotypes for implantaton recognition between maternal uterus and the transfered NT embryos. If provided with a normally developed extraembryonic tissues by tetraploid embryos aggregating with NT embryos or its derived ES cell toe, then the implantation or the developmental potentials of NT embryos might be well improved, hence increasing the birth rate of cloned animals. Besides its biological implications, 2N-4N aggregation can be performed as an important transgenic technical system following microinjection and NT for transgenesis. Thus, establishment of 2N-4N aggregation may be of theoretical and practical significance as well.The objectives of this research are just to ascertain the basic conditions for aggregation, obtain viable mice by 2N-4N aggregation, and finally characterize the development of 2N-4N aggregates by tracing the dynamic changes of GFP fluorescence markered to 4N blastomeres. On such basis, futher application of 2N-4N aggregation to NT related research may be feasible in the future.1 Establishment of 2N-2N aggregation systemThe aggregation system to be established includes: means for removing zona pellucida, carrier selection for aggregates culture, chemical reagent for promoting aggregation, and recipient mice for ET as well.The research results were as follows: (1)0.5% protease is comparable to tyrode's in developmental capacities of the zona-removed embryos. (2) There was no significant difference between culture plate with 96 microwells and petri dish with microdrops in the effiency of aggregation, still, the latter was an ideal culture carrier for its convenience in operation. (3)M16 cultrue medium added with PHA-P at 180 g/ml may be effective for aggregation. (4)Some aggregates produced from two diploid embyos are capable of developing into blastocysts in M16 culture medium andgiving birth to viable chimaeric mice in coat color after transferred to recipient mice.2 Production of 2N-4N aggregates and derivation of viable miceProcedures for 2N-4N aggregation basicly accorded that of 2N-2N aggregation except for: (l)Induction of 4N embryos by electrofusing 2N blastomeres at 2-cell stage. (2)Embryo combination by 2N embryo at 4 or 8-cell stage with 4N embryo at 4-cell stage. (3)Agggregation by routine l:l(one 2N embryo paired with one 4n embryo) and sandwiched 1:2 (one 2N embryo put between two 4N embryos)modes.The results were that; (1) 2N-4N chimaeric blastocysts could be developed from aggregates in Ml 6 culture medium. (2) There was no significant difference in the ratio of aggregates formed between compacted and non-compacted 8-cell 2N embryos adopted for aggregation,however, the former saw higher ratio of chimaeric blastocysts developed. (3)Timing for completion of aggregation and the ratio of aggregates formed were comparable between routine 1:1 and sandwiched aggregation modes,(4)2N-4N chimaericblastocysts transferred to recipient mice were capable of implanting and developing to mid-late gestated conceptus with fetus(designated to fetus) as well as viable mice to term. (5)Fetus displayed somewhat developmentally delayed, while the accompanying placentas remaining unaffected, as was similar to 2N-2N con...
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