Identification of bovine QTL for growth and carcass traits in Japanese Black cattle by replication and identical-by-descent mapping.

To map quantitative trait loci (QTL) for growth and carcass traits in a purebred Japanese Black cattle population, we conducted multiple QTL analyses using 15 paternal half-sib families comprising 7860 offspring. We identified 40 QTL with significant linkages at false discovery rates of less than 0.1, which included 12 for intramuscular fat deposition called marbling and 12 for cold carcass weight or body weight. The QTL each explained 2%-13% of the phenotypic variance. These QTL included many replications and shared hypothetical identical-by-descent (IBD) alleles. The QTL for CW on BTA14 was replicated in five families with significant linkages and in two families with a 1% chromosome-wise significance level. The seven sires shared a 1.1-Mb superior Q haplotype as a hypothetical IBD allele that corresponds to the critical region previously refined by linkage disequilibrium mapping. The QTL for marbling on BTA4 was replicated in two families with significant linkages. The QTL for marbling on BTA6, 7, 9, 10, 20, and 21 and the QTL for body weight on BTA6 were replicated with 1% and/or 5% chromosome-wise significance levels. There were shared IBD Q or q haplotypes in the marbling QTL on BTA4, 6, and 10. The allele substitution effect of these haplotypes ranged from 0.7 to 1.2, and an additive effect between the marbling QTL on BTA6 and 10 was observed in the family examined. The abundant and replicated QTL information will enhance the opportunities for positional cloning of causative genes for the quantitative traits and efficient breeding using marker-assisted selection.

Investigation of the fate of Sry-expressing cells using an in vivo Cre/loxP system.

Sry (sex-determining region on Y chromosome) is expressed in the undifferentiated, bipotential genital ridges of mammalian XY fetuses. The expression of Sry initiates testis development, but the lineage of Sry-expressing cells is unclear. In this study, double-transgenic mice were analyzed using the Cre/loxP system. Cre under the control of the Sry promoter was expressed in the fetal gonads of transgenic mice similarly to endogenous Sry. The Sry/Cre-transgenic mice were crossed with CAG(cytomegalovirus immediate-early enhancer, chicken beta-actin promoter and fusion intron of chicken beta-actin and rabbit beta-globin)/loxP/CAT/loxP/LacZ-transgenic mice, in which the transgene expressed beta-galactosidase after a Cre-mediated recombination event. Sertoli cells, germ cells of testes and granulosa cells of ovaries of double-transgenic mice stained positive with X-gal. Cre expression was detected in germ cells and peritubular/Sertoli cells in adult testes. It is not clear whether beta-galactosidase expression in the Sertoli cells of the testes occurred as a result of Cre expression in the adult or in the fetal gonads. These analyses indicate that cells expressing Sry-inducing factors in female fetal gonads become granulosa cells.

Detection of elements responsible for stage- and tissue-specific expression of mouse Sry using an in vitro Cre/loxP system.

We have successfully specified essential sequences of the 5' upstream region for the stage- and tissue-specific expression of mouse Sry by using an in vitro Cre/loxP system. Sry/Cre plasmids carrying Sry 5' sequences of various sizes were transfected into the primary cultured cells from different tissues of CAG/loxP/CAT/loxP/LacZ transgenic fetuses on 11.5-day post coitus (dpc) or 13.5-dpc. Stage- and tissue-specific regulation of Sry expression was disrupted by the deletion of positions 7549-7660 (from -0.4 to -0.5 kb region). In vitro transcription assay also suggested that the region contains element(s) responsible for stage- and tissue-specific expression of mouse Sry. SRY promoter of Shiba goat (Capra hircus var Shiba), a native Japanese miniature goat, showed the tissue-specific activity in the cells from urogenital ridges of the male mouse, but not in the cells from female mice, indicating a possibly different mechanism among species in the regulation of Sry expression.

Stage-specific regulatory element of mouse Sry gene.

Sry expression is essential for initiating male sex differentiation, and the expression occurs only during a restricted period in the developing gonad. It is thought that Sry is part of a pathway of genes that regulate sex determination. Although the interactions of several genes with Sry expression have been suggested, the exact cascade of gene expression regulating Sry transcription is entirely obscure because there is no available cell line expressing Sry and reflecting an in vivo condition. The present study was carried out to investigate the cis-acting element of the mouse Sry that responds stage specifically to its expression, in part, using transgenic mice expressing GFP on the Y chromosome. Ten DNA fragments were generated by digesting the 5' upstream region (positions 5491-8039; 2,549 bp) of mouse Sry with appropriate restriction enzymes. In an electrophoretic mobility assay with these fragments, the region from position 5491 to position 5799 (309 bp) was identified as forming specific protein-DNA complexes with nuclear extracts from 11.5 days post coitus (dpc) gonads, but not from 12.5 and 13.5-dpc gonads. This region also formed specific protein-DNA complexes with the nuclear extracts from adult testicular germ cells that generate only a circular form from Sry. This stage-specific responsive region was narrowed down to positions 5559-5616 by DNase I footprinting analysis. The assay of DNase I hypersensitive (HS) using the nuclear lysates from the 11.5-dpc urogenital ridges demonstrated that the novel HS site was located in the proximity of position 5600. This region DNase I HS was also detected at the same position when the lysates from adult testicular germ cells were applied. The results indicate that the present HS site may be involved in the transcriptional regulation of the linear and/or circular molecule transcripts from mouse Sry gene.

In vitro Cre/loxP system in cells from developing gonads: investigation of the Sry promoter.

There have been few studies on the regulatory elements of the Sry gene, mainly because no Sry-expressing cell lines have yet been established. This paper describes a useful tool for investigating the regulation and upstream region of Sry by means of the in vitro Cre/loxP system. Using plasmids containing the 9.9 kb mouse genomic Sry previously shown to induce testis development in XX transgenic mice, we constructed a Sry/Cre fusion gene plasmid in which Cre expression is controlled by the 5' and 3' untranslated regions of mouse Sry. To distinguish between male and female gonads of 11.5 days post-coitus (d.p.c.) fetuses, double transgenic fetuses carrying both the CAG (cytomegalovirus enhancer and beta-actin promoter)/loxP/lacZ transgene on the autosome and the green fluorescent protein transgene ubiquitously expressed on the Y chromosome were produced by crossing between two transgenic mouse lines. When Sry/Cre plasmids were transfected into the cells that had been prepared from the gonads, brains and livers of double transgenic fetuses, only a small number of X-gal-stained cells were detected among the primary cultured cells from male and female gonads, and none were detected among the cells from the other tissues. The X-gal-positive cells were negative for alkaline phosphatase, indicating that these cells were somatic cells expressing Sry. The Sry/Cre plasmids with a 0.4 kb upstream region of Sry yielded a large number of X-gal-positive cells in the cells from gonads, including various tissues of 11.5 d.p.c. fetuses, indicating the loss of the tissue-specific expression of Sry. The Sry/Cre with a 1.4 kb upstream region maintained tissue-specific activity of Sry. The results indicate that the present in vitro Cre/loxP system using transgenic mice is a simple and useful system for investigating the regulatory element of sex determination-related genes, including Sry.