Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus

Genomic imprinting causes parental origin-specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.     

Detection of the EGFP sequence in breast muscle of 3‐year‐old chicken after transfection using sonoporation

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG‐EGFPac) and microbubbles were injected into the central disc of stage‐X blastoderm or the germinal crescent of stage‐4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP‐positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3‐year‐old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.     

Effect of the strain combination of the donor and recipient on the production efficiency of W‐bearing sperm in mixed‐sex germline chimeric chickens

Effect of the strain combination of the donor and recipient on production efficiency of W‐bearing sperm in mixed‐sex chimeric testes was analyzed. The combinations of the donors and recipients were White Leghorn (WL) and Rhode Island Red (RIR), and vice versa. Generated mixed‐sex chimeras that had the male phenotype at sexual maturity were classified into four groups: (1) a female WL donor and a male RIR recipient; (2) a male WL donor and a female RIR recipient; (3) a female RIR donor and a male WL recipient; (4) a male RIR donor and a female WL recipient. The mean number of W‐bearing sperm detected by in situ hybridization among 10 000 sperm observed were 147, 165, 30 and 45 in groups 1, 2, 3 and 4, respectively. The numbers in groups 1 and 2 were both significantly higher than those of groups 3 and 4 (P < 0.05). The combination of a WL donor and a RIR recipient produced W‐bearing sperm more efficiently than the reverse combination.     

Fixation to the canine bone of artificial implant with new surface structure

Screw and laser (SL) column by making screw threads and forming small holes using laser irradiation on the base metal and conventional beads coating (BC) columns were embedded into the shaft of canine femurs, and compared the implant fixation to the host bone. The interfacial strength in SL columns was almost equivalent as BC columns, and bone-column contact rate was higher than BC columns significantly at twelve weeks after implantation. The newly devised SL surface had almost equivalent bone fixation strength comparable to the conventional BC surface. Also, this surface should provide a useful porous surface for use in artificial joints since there is no risk of surface structure detachment.     

Identification of bovine dendritic cell phenotype from bovine peripheral blood

Dendritic cells (DCs) are professional antigen presenting cells, which initiate primary immune responses and also play an important role in the generation of peripheral tolerance. There is no reliable method established for the isolation of bovine peripheral blood DCs, and furthermore, the phenotypes and the functions of bovine DCs are still not fully clear. In the present study, we have attempted to identify bovine peripheral blood DCs by negative-selection. In bovine peripheral blood mononuclear cells (PBMC), we have newly characterized the phenotype of DCs, which is CD11c+/CD172a+. These cells display features of myeloid type DCs. In the thymic medulla, CD11c+/CD172a+ cells were also present and CD1+/CD172a+ cells were additionally detected as a population of DCs. The data suggest that one of the bovine DCs phenotypes from PBMC is derived from myeloid lineages lacking a CD1 molecule, which then drift to several tissues, and that they then may express a CD1 molecule upon their functional differentiation.     

Variation in heading date conceals quantitative trait loci for other traits of importance in breeding selection of rice

To identify quantitative trait loci (QTLs) associated with the primary target traits for selection in practical rice breeding programs, backcross inbred lines (BILs) derived from crosses between temperate japonica rice cultivars Nipponbare and Koshihikari were evaluated for 50 agronomic traits at six experimental fields located throughout Japan. Thirty-three of the 50 traits were significantly correlated with heading date. Using a linkage map including 647 single-nucleotide polymorphisms (SNPs), a total of 122 QTLs for 38 traits were mapped on all rice chromosomes except chromosomes 5 and 9. Fifty-eight of the 122 QTLs were detected near the heading date QTLs Hd16 and Hd17 and the remaining 64 QTLs were found in other chromosome regions. QTL analysis of 51 BILs having homozygous for the Koshihikari chromosome segments around Hd16 and Hd17 allowed us to detect 40 QTLs associated with 27 traits; 23 of these QTLs had not been detected in the original analysis. Among the 97 QTLs for the 30 traits measured in multiple environments, the genotype-by-environment interaction was significant for 44 QTLs and not significant for 53 QTLs. These results led us to propose a new selection strategy to improve agronomic performance in temperate japonica rice cultivars.     

Traits of shattering resistant buckwheat ‘W/SK86GF’

Seed shattering is a significant problem with buckwheat, especially at harvesting time. Several reports have shown that a green-flower mutant of buckwheat, such as W/SK86GF, has a strong pedicel. Although a strong pedicel may provide some resistance to shattering in the field, no study has thoroughly examined this issue. In this paper, we demonstrate that a W/SK86GF has shattering resistance by comparing the degrees of shattering of W/SK86GF and Kitawasesoba (leading variety of Hokkaido with non-green-flower traits) through a test for four years, including a typhoon hit year in the field. In a non-typhoon year, the shattering seed ratio (shattering seed weight/(yield + shattering seed weight) × 100) of W/SK86GF at maturing time +15 days (+15D) was lower than that of Kitawasesoba. In a typhoon hit year, the shattering seed ratios of Kitawasesoba at maturing time and +15D were surprisingly high, 14.4 and 21.1%, respectively. On the other hand, those of W/SK86GF were only 3.08% and 2.57%, respectively; indicating W/SK86GF is promising as a shattering resistant line even in a typhoon hit year. From these results, shattering resistance of W/SK86GF can be evaluated after maturing time such as +15D and pedicel strength would confer W/SK86GF a shattering resistant trait.     

Peripartum back fat thickness of multiparous Holstein-Friesian cows with displacement of the abomasum or ketosis

To establish a method to predict postpartum diseases using prepartum back fat thickness (BFT), the peripartum BFTs of 54 healthy multiparous cows before calving, which were diagnosed with postpartum displacement of the abomasum (DA), clinical ketosis or subclinical ketosis were compared with those of healthy cows from 8 weeks before the expected calving date to 8 weeks after calving. The peripartum BFTs of the cows with DA or clinical ketosis were significantly higher than those of healthy cows. The peripartum BFTs of the cows with subclinical ketosis were not significantly higher than those of the healthy cows.