Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique
Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique
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DOI：
10.1038/sj.cr.7290254
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发表时间：
2004-12-01
期刊：
影响因子：
44.1
通讯作者：
Koide, SS
中科院分区：
文献类型：
--
作者：
Liang, G;Zhang, XD;Koide, SS
采用激光捕获显微切割（LCM）结合抑制性消减杂交（SSH）技术，从人睾丸组织切片中分离特异性生殖细胞，并对生殖细胞分化发育过程中表达的基因进行鉴定。本研究成功地用LCM法分离了10，000多个初级精母细胞和圆形精子细胞。利用初级精母细胞和圆形精子细胞的cDNA，构建了初级精母细胞特异性的SSH cDNA文库。从75个随机挑选的白色克隆中分离的cDNA的平均插入片段大小为500 bp，范围为250 bp至1.7 kb。使用斑点印迹法，共421个克隆进行了检查，导致390个阳性克隆发射强信号的鉴定。测定了从每个克隆制备的cDNA的部分序列，总成功率为84.4%。编码细胞色素c氧化酶II和拯救因子humanin的基因在初级精母细胞中表达最频繁，表明它们参与减数分裂。
The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.