摘要
哺乳动物合子基因组激活和植入前胚胎阶段特异性表达基因的研究一直是发育生物学研究的重点。发现和克隆植入前胚胎阶段特异性表达的基因需要有效的方法 ,阶段特异性cDNA文库的构建和筛选是一种较好的方法。用小鼠单个成熟卵细胞、受精卵、2 细胞、4 细胞和 8 细胞胚胎分别构建了cDNA文库。滴度分别为 :6 2× 1 0 5、8 3× 1 0 5、1 0 4× 1 0 6、1 5 1× 1 0 6和 1 62×1 0 6。随机挑选了 2 9个克隆并进行了序列分析 ,结果表明 ,和已知表达序列标签 (ESTs)同源的序列为 65 5 2 % ( 1 9 2 9) ,未知序列为 1 3 79% ( 4 2 9) ,并发现了 2个重复序列。用特异性引物 ,分别对小鼠持家基因 ( β actin)和发育特异基因 (OCT 4)进行PCR扩增 ,结果表明 ,所建立的cDNA文库可以反映小鼠胚胎在不同发育阶段整个基因群体的转录活性 。
Molecular analyses of early mammalian embryos are important and necessary for identifying the genes involved in normal development.Analysis of the genetic and molecular mechanisms of embryogenesis requires effective methods to find and isolate genes that are differentially expressed in normal and altered conditions.Construction of cDNA libraries from single mouse embryo and screen can lead to the discovery of known or novel transcripts that change in association with critical events.We report on the construction of cDNA libraries from single unfertilized oocyte,fertilized oocyte,two cell,four cell and eight cell stage embryos respectively.The complexities of these libraries are 6 2×10 5 、8 3×10 5、1 04×10 6、1 51×10 6和1 62×10 6 respectively.A total of 29 clones were picked and sequenced,65.52%(19/29)to expressed sequence tags(ESTs) of mouse origin.Novel sequence were detected at a frequency of 13.79%(4/29) and found two repeat sequences in cDNA library from single four cell embryo.PCR analyses of the embryonic libraries for specific genes revealed transcripts for genes including housekeeping genes( β actin ),developmental gene (OCT4) .These results demonstrated that these cDNA libraries may represent the entire active gene population and are a valuable resource for the isolation of clones representing genes active at these early stages of mouse development.
出处
《中国生物工程杂志》
CAS
CSCD
2003年第6期50-54,共5页
China Biotechnology
基金
国家重大基础研究发展规划 (973 )资助项目 (2 0 0 0 1610 7)
