Identification of ESTs of genes and a new gene related to compaction in preimplantation embryos of mouse

Abstract

Objective: To investigate genes expressed differently between early 8-cell embryos and 8-cell compacted embryos and identified new compaction-related genes. Design: Collecting early 8-cell embryos and 8-cell compacted embryos and synthesizing them directly using SMART PCR. Genes, which expressed differently between, were investigated by using the method of suppression subtractive hybridization (SSH). Then identify new genes related to compaction. Materials and Methods: One hundred eighty-one 8-cell embryos and 241 8-cell compacted embryos were collected respectively from C57BL/6 mouse and their cDNA was synthesized. Then PCR production after SSH was cloned into pUCm-T vector according to the size after isolated and purified. Choice the positive clones for sequencing after being confirmed with PCR. All fragment cloned were blastered matching in GenBank for homology analysis. On the base of this work, we select two of the 17 ESTs( BQ740263 and BQ740251), which were reverse complementary to each other. the two ESTs, assemble into a a cDNA fragment, which include a whole open read frame (ORF) and submissed to GeneBank with an accession number, AY134859. The target fragment were amplified from cDNA of mouse compacted embryos using primers designed according to the ORF and. cloned into pUCm-T vector and sequenced. It’s confirmed that the gene, Crg1, was the same as AY134859. Crg1 were also blastered matching in GenBank for homology analysis and mapped by database analyses. RT-PCR analysis of Crg1 were done in series mouse embryos, some adult mouse tissues, mouse embryo stem cell and mouse embryo fibroblast. Results: 27 ESTs (expressed sequence tags ) of genes expressed differently between early 8-cell embryos and 8-cell compacted embryos have been isolated and cloned. 17 of those were novel ESTs and they were banked into GenBank with accession numbers: BQ740249,BQ740250,BQ740251,BQ740252,BQ740253,BQ740254,BQ740255,BQ740256,BQ740257,BQ740258,BQ740259,BQ740260,BQ740261,BQ740262,BQ740263,BQ740264,BQ740265. Length of Crg1 is 810bp. It include only one exon and code a 150aa protein with a theoretical molecular weight of 17670.34 Dalton. The protein is similar to a protein encoded by a known gene, Stella. Crg1 was mapped to chromosome 14 by database analyses. RT-PCR analysis shows that Crg1 expressed in series mouse embryos (2-c embryos, 4-c embryos, 8-c embryos, compacted embryos and blastocyst), and expressed little higher in compacted embryos. It also expressed in mouse embryo stem cell but not expressed in mouse embryo fibroblast. It expressed in some adult mouse tissues (kidney, testis, epididymis, ovary, liver and lung) too but not expressed in other adult mouse tissues (brain, spleen, heart, skeletal muscle). Conclusion: All 17 ESTs might be for novel genes related to compaction in compacted embryos. And longer ESTs may obtained by cloning according to the size. Crg1 may related to compaction in compacted embryos and maintaining cell’s pluripotentiality.