[Cloning of compaction-related gene Crg1 of mouse preimplantation embryos].
A total of 181 8-cell embryos and 241 8-cell compacted embryos were collected respectively from C57BL/6 mouse and their cDNA was synthesized by SMART-PCR. Then PCR productions after SSH were 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 ESTs (GenBank accession No.: BQ740263 and BQ740251), which were reverse complementary to each other, assemble into a cDNA fragment, which include a whole open read frame (ORF) and submitted to GenBank 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, mouse embryo stem cell and mouse embryo fibroblast. We analyzed the expression of Crg1 in 8-cell embryos and 8-cell compacted embryos using semi-quantity RT-PCR. Northern-blot analysis of Crg1 expression in some adult mouse tissues was done too. The results showed that the length of Crg1 is 810 bp. It include only one exon and code a 150 amino acid 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-cell embryos, 4-cell embryos, 8-cell embryos, compacted embryos and blastocyst), and expressed little higher in compacted embryos. Crg1 also expressed in mouse embryo stem cell but not expressed in mouse embryo fibroblast. The gene only expressed weakly in adult mouse ovary, but not expressed in other adult mouse tissues (brain, spleen, heart, skeletal muscle, kidney, testis, epididymis, liver and lung). So, Crg1 may related to compaction in compacted embryos and maintaining cell's pluripotentiality.
Li
Wen
W
Institude of Reproduction and Stem Cell Engineering, Central South University, Changsha 410078, China. wenshui14@sina.com
Lu
Guang-Xiu
GX
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism*; D003001:Cloning, Molecular; D020224:Expressed Sequence Tags; D005796:Genes; D051379:Mice; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction