兔植入前移核胚中发育相关基因的差异表达分析

Differential Expression Analyses of Development-related Gene in Rabbit Preimplantation NT Embryos

与早期胚胎发育相关的一些重要基因异常表达致使克隆胚细胞核的再程序化过程受阻,是导致动物克隆失败的重要原因.为了分离鉴定再程序化相关基因,我们改进了mRNA差异显示技术,成功地建立了单胚差示技术体系.以不同发育时期的兔克隆胚(MⅡ卵、2细胞、4细胞、8～ 16细胞克隆胚胎)为材料进行单胚差示,分离了 80个差异片段.经反向RNA印迹验证、亚克隆、序列分析及NCBIGenBank数据库检索,结果表明:A0 2 8片段与CstF3基因有 93%的同源性,在早期胚胎发育过程中的表达有阶段特异性,该基因在兔克隆胚的早期发育过程中起重要作用.RNA印迹显示:该基因在所检测的组织中,只在卵巢中有表达.

Abnormal expression of developmentally important genes leads to inadequate reprogramming development of NT embryo which subsequently causes failure in animal cloning. To identify reprogramming-associated genes in rabbit NT embryo, mRNA differential display has been developed and SPEDDRT-PCR method was set up successfully to overcome the paucity of the biological materials. Using this modified technique, the mRNA content of rabbit NT embryos at different developmental stages (from oocyte to 8 similar to 16-cell embryo) were compared, eighty differential displayed bands at different stage in rabbit NT embryo were isolated. A028 amplicon was confirmed by reverse Northern blot. Nucleotide sequences were determined for these cDNAs and database searches identified using NCBI BLAST program. The data suggested that A028 displayed high homology (93%) to CstF3 gene for cleavage stimulation factor, which involved in pre-mRNA 3'-end processing and is required for progression through mitosis. This gene was probably related with preimplantation embryo development, and might play important roles in development of rabbit NT embryo. Of seven organ tissues examined by Northern blot analysis, A028 was only found in ovary. This work paves the way of cloning of the full length of A028 cDNA and further study on gene function.