| 主办单位: |
| 中国科学院遗传与发育生物学研究所 |
| 中国遗传学会 |
| 编辑单位: |
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《遗传学报》 编辑部
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| 主 编: 薛勇彪 |
| 主 任: 张颖 |
编委阵容 |
| 审稿专家 |
| 国内刊号： |
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CN 11-5450/R
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| 国际刊号： |
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ISSN 1673-8527
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| 邮发代号： 2-819
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遗传学报 2004.3 Vol 31, No.3 246-251
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| 小鼠植入前胚胎紧密化相关基因Crg1的克隆 |
| Clonging of Compaction-Related Gene Crg1 of Mouse Preimplantation Embryos |
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| 李汶，卢光xiu |
| 中南大学生殖与干细胞工程研究所，长沙 410078 |
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| Key Words： 基因克隆；植入前胚胎；紧密化；RT-PCR |
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Abstract
从已获得的运用抑制消减杂交技术（Suppression Subtractive Hybridization,SSH）分离、克隆和筛选代表8-细胞早期胚胎和紧密化8-细胞胚胎差别表达基因的ESTs片段(GenBank登录号：BQ740263、BQ740251)入手，经比较二者的同源性发现这两个EST末端反向互补，拼接成一个cDNA片段，经分析此序列包含一个完整的阅读框，提交给GenBank，登录号为AY134859 。根据此序列设计引物从小鼠8-细胞紧密化胚胎cDNA中经PCR扩增出目的片段，克隆入pUCm-T载体后测序而获得全长cDNA，为小鼠植入前胚胎紧密化相关基因Crg1，分析比较证明Crg1基因与AY134859基本吻合。Crg1基因的cDNA全长为810 bp，只有一个外显子，编码由150个氨基酸组成，分子量理论值为17.67 kD的蛋白质。与最新的小鼠基因组工作草图进行电子杂交，该基因被定位在小鼠的14号染色体上。RT-PCR实验证明在小鼠植入前各个时期的胚胎、小鼠胚胎干细胞中均有表达，在小鼠胚胎成纤维细胞中没有表达。半定量RT-PCR实验证明Crg1基因 在紧密化胚胎中表达较8-细胞胚胎高。采用Northern-blot手段分析Crg1基因在成年小鼠的8种组织中的表达情况，结果表明该基因只在小鼠卵巢中有微弱的表达，转录本大小为1.2 kb，而在成年小鼠的脑、心脏、肾、睾丸、肝脏、肺、脾等中没有表达。研究表明，Crg1基因可能与小鼠胚胎紧密化及保持细胞的全能性相关。 |
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Abstract
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. |
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中国科学院遗传与发育生物学研究所
