The mouse Rad5l gene is a mammalian homologue of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad5l null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad5Sl/-) pups among 148 neonates examined but a few Rad5-/-embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cell. The homozygous Rad5l null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability.Genetic recombination leads to new associations of genetic elements. In meiosis, recombination between closely paired homologous chromosomes results in extensive reshuffling of paternal and maternal genes, and the progeny can be better fitted to cope with the environment. Recombination occurring in somatic cells is manifested as sister chromatid exchange and the outcome, by itself, does not alter the cellular genotype.Molecular mechanisms of recombination have been studied extensively in bacteria and lower eukaryotes. The recA gene of Escherichia coli plays an essential role in recombination as well as in DNA repair and induction of SOS functions (1-3). The RecA protein has the potential to promote homologous pairing and strand exchange of DNA in the presence of adenosine 5'-triphosphate (ATP) (2-6). In yeast Saccharomyces cerevisiae, RAD51, RAD52, and RAD54 genes, belonging to the RAD52 epistasis group, were initially identified as those involved in the repair of DNA damage induced by ionizing radiation (7,8), and subsequently were shown to be responsible for mitotic recombination (9-12). Among them the RAD51 gene is a homologue of the E. coli recA gene and plays crucial roles in both mitotic and meiotic recombination as well as in repair of double-strand breaks of Isolation of Targeted ES Cell Clones. The ES cell line CCE was cultured on a feeder cell layer and electroporated, using 5 X 107 cells and 50 ,tg of the linearized targeting vector DNA, as described (25,26). Colonies doubly resistant to G418 (250 ,ug/ml) and ganciclovir (5 ,uM) were selected and expanded on feeder layers in 24-well plates. Homologous recombinants were identified by Southern blot analysis of restriction enzymedigested DNA. DNAs were prepared from cells cultured in the absence of feeder cells and subjected to Southern blot analysis.The DNA (8 jig) was cleaved with BamHI, subjected to agarose gel electrophoresis, blotted onto Hybond N+ membrane (Amersham), and hybridized to probe A. To ensure targeted disruption of the Rad5l gene, the DNA was digested with EcoRV or HindIll, followed by hybridization with ...

We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I-III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of M r 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G 1 , resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately onehalf of wild-type Haspin was able to bind to a DNAcellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells.

Non-invasive selection of transgenic mice was performed at the stage of preimplantation embryos. The morulae collected from wild female mated with hemizygous transgenic male expressing Aequorea victoria green fluorescent protein (GFP) under chicken ~-actin promoter could be classified as green or non-green under a fluorescent microscope. All the green embryos were shown to carry the transgene by PCR analysis. Taking advantage of the detection of GFP expression can be done non-invasively, the selected embryos were demonstrated to be able to developed to term with 100% of accuracy of the selection.

We have cloned cDNAs involved in germ cell-specific expression. For this, a subtracted cDNA library was generated by subtracting cDNAs derived from supporting cells of mutant testis from wild-type testis cDNAs. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in testes and were developmentally controlled.Key words: Testis; Mouse; Protamin; cDNA library; Subtraction; WnV mutant; Actin capping protein IntillctlonMouse spermatogenesis is an excellent model system to study regulation of gene expression during differentiation. Postnatal development of the mouse seminiferous epithelium is a complicated process which finally generates a tissue able to produce functional spermatozoa. The whole process can be subdivided into three parts: (i) a premeiotic phase characterized by an increase in cell number due to mitotic divisions of diploid spermatogonia; (ii) a meiotic prophase, which leads to the formation of haploid round spermatids; and (iii) a post-meiotic phase, which includes the morphogenetic events required for spermatozoa formation (spermiogenesis) [ 1,2]. Specific cells derived from the stem cells in seminiferous epithelium of the adult testis undergo these processes and continuously provide the mature sperms. The precise regulation of such germ cell differentiation requires a strict program of stage-and cell-specific gene expression in germ cells as well as in surrounding somatic cell types [3]. To understand the mechanism of testicular germ cell differentiation, it is of great interest to isolate specific genes and characterize their functions as well as their regulation. Materials and methods Preparation of cDNA libraries carrying directional insertsTotal RNA followed by purification of poly (A)+ RNA was extracted by the guanidme thiocyanate/CsTFA method from the testes of adult wild-type B6 mice and 4-month-old W/W" mutant mice [4]. Their cDNA libraries were prepared as described by Gubler and Hoffmann with some modifications (Kobori et al., manuscript in preparation) [5]. Briefly, cDNA was synthesized in a reaction mixture including 'MedCTP with reverse. transcriptase (Superscript II) from 2-5 pg of mouse testis poly(A)+ RNA and 1.6 pug of oligo (dT) primer carrying a Not1 site. The reaction mixture was treated with RNase H, followed by reaction with DNA polymerase I. Each end was blunt-ended with T4 DNA polymerase and ligated to an unphosphorylated BglII-SmaI adaptor. After digestion with NotI, small DNA fragments of less than 300 bp were removed by a CROMA spin-400 column (Clontech, USA). The cDNA fragments were directionally inserted between the Not1 (dephosphorylated) and BgZII sites of vector pAP3neo (H. Nojima, unpublished). The ligation mixture was electroporated into MC1061A cells as described [q. The complexities of the cDNA libraries used here *Corresponding author. Fax: (81) (6) 879-8339.were 6.0 x lo6 colony forming units (cfu) for the B6 wild-type mouse and 2.8 x lo6 cfu for the Wm mutant mouse. Preparation of a s...

No abstract