Investigaton for molecular cloning of a gene on the Y chromosome crucial to spermatogenesis and its disorder

1)无精子因子（Azospermyfactor，AZF）基因的克隆我们找到了含有AZF基因的YAC，并将YAC上的粘粒重叠群亚克隆到质粒载体上。AZF包含在30 Kb的DNA中，然后，我们用外显子捕获法从每个粘粒中提取了几个功能性外显子。通过基因库中的DNA数据库对所有序列进行分析，发现不少外显子存在未知序列。2）人睾丸生殖细胞分化诱导基因的克隆为了克隆人睾丸生殖细胞分化诱导基因，我们以小鼠cDNA为探针，筛选人睾丸cDNA文库。我们分离了减数分裂前出现的SCP-1，并测定了其DNA和蛋白质的序列。3）人生殖细胞体外培养体系的建立我们建立了人生殖细胞体外培养体系。利用该系统，我们将能够进行体外分化人类生殖细胞的实验。

1) Cloning of Azoospermy factor gene (AZF)We found YAC in which AZF should be included and subcloned the cosmid contig mede from the YAC to a plasmid vector. AZF was included within 30 Kb DNA.Then, using Exon trapping we picked up severalfunctional exons from each cosmid. After all of sequences were analyzed using the DNA data base in the Gene Bank, not a few exons revealed unknown sequences. Screening using the testis cDNA libraryis is now on in progree.2) Cloning of genes which induce differentiation of human testis germ cellsFor molecular cloning of genes which induce differentiation of human testis germ cells we screened human testis cDNA library using mouse cDNAs as probes. We isolated human SCP-1 which appears in the phase before the miosis and determined sequences of the DNA and the protein.3) Establishment of in vitro culture of human germ cellsWe established in vitro culture system of human germ cells which has been very difficult. Using this system we will be able to do experiments in vitro differentiation human germ cells.