Application of restriction fragment length polymorphism analysis to simple and rapid genotyping of bovine viral diarrhea virus strains isolated in Japan

The E2 regions of 177 bovine viral diarrhea virus (BVDV) strains isolated in Japan between 1957 and 2006 were analyzed for genotyping. The strains were classified into 8 genotypes (1a, 1b, 1c, 1d, 1e, 1f, So and 2a) based on the phylogenetic analysis. The restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products using 6 selected enzymes (Apo I, Mly I, BstAP I, Pvu II, Ear I, EcoR V) disclosed the cutting patterns classified into 11 groups (I-XI), each of that consisted of strains belonging to a single genotype. Namely, groups-I and -II were composed by genotype-1a strains, groups-III and -IV by 1b strains, and groups-V and -VI by 1c strains. Other groups-VII, -VIII, -IX, -X and -XI comprised genotypes-1d, -1e, -1f, -So and -2a strains, respectively. The results suggest that the RFLP analysis can simply and rapidly differentiate the 8 genotypes of BVDV strains.     

Susceptibility to N-methyl-N-nitrosourea-induced retinal degeneration in different rat strains

To evaluate the potential role of genetic background in the susceptibility to retinal degeneration induced by N-methyl-N-nitrosourea (MNU), female rats of the Sprague-Dawley (SD), Long-Evans (LE) and Copenhagen (CH) strains were administered 50 mg/kg MNU or saline at 7 weeks of age. Retina morphology and morphometric analysis of all rats was performed 7 days after MNU administration. Atrophy of both the peripheral and central outer retina occurred in all rat strains exposed to MNU. Decreased photoreceptor cell ratio and increased retinal damage ratio were observed. The severities of the retinal atrophy were similar among all three rat strains. In conclusion, MNU-induced photoreceptor degeneration developed consistently in all three strains regardless of the absence (SD rats) or presence (LE and CH rats) of melanin in the retina, suggesting that genetic and melanin factors did not affect photoreceptor cell death after MNU.     

Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse

In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses only one odorant receptor (OR) gene in a monoallelic and mutually exclusive manner. Such expression forms the genetic basis for OR-instructed axonal projection of OSNs to the olfactory bulb of the brain during development. Here, we identify an upstream cis-acting DNA region that activates the OR gene cluster in mouse and allows the expression of only one OR gene within the cluster. Deletion of the coding region of the expressed OR gene or a naturally occurring frame-shift mutation allows a second OR gene to be expressed. We propose that stochastic activation of only one OR gene within the cluster and negative feedback regulation by that OR gene product are necessary to ensure the one receptor-one neuron rule.     

Maintenance of mouse trophoblast stem cells in KSR-based medium allows conventional 3D culture

Mouse trophoblast stem cells (TSCs) can differentiate into trophoblast cells, which constitute the placenta. Under conventional culture conditions, in a medium supplemented with 20% fetal bovine serum (FBS), fibroblast growth factor 4 (FGF4), and heparin and in the presence of mouse embryonic fibroblast cells (MEFs) as feeder cells, TSCs maintain their undifferentiated, proliferative status. MEFs can be replaced by a 70% MEF-conditioned medium (MEF-CM) or by TGF-ß/activin A. To find out if KnockOut^TM Serum Replacement (KSR) can replace FBS for TSC maintenance, we cultured mouse TSCs in KSR-based, FBS-free medium and investigated their proliferation capacity, stemness, and differentiation potential. The results indicated that fibronectin, vitronectin, or laminin coating was necessary for adhesion of TSCs under KSR-based conditions but not for their survival or proliferation. While the presence of FGF4, heparin, and activin A was not sufficient to support the proliferation of TSCs, the addition of a pan-retinoic acid receptor inverse agonist and a ROCK-inhibitor yielded a proliferation rate comparable to that obtained under the conventional FBS-based conditions. TSCs cultured under the KSR-based conditions had a gene expression and DNA methylation profile characteristic of TSCs and exhibited a differentiation potential. Moreover, under KSR-based conditions, we could obtain a suspension culture of TSCs using extracellular matrix (ECM) coating-free dishes. Thus, we have established here, KSR-based culture conditions for the maintenance of TSCs, which should be useful for future studies.     

根区温度对水培生菜生长和矿质元素含量的影响

在水培条件下,研究了5个根区温度对生菜(Lactuca sativa L.)生长和矿质元素含量的影响。结果表明:随着根区温度的升高,生菜单株产量呈先增加后减少的趋势,在25℃时达到最大值,且干物质含量最高;根区温度对生菜叶片净光合速率和矿质元素含量的影响与对产量的影响相似,即在25℃时达到最大值。35℃根区温度处理下,根叶生长和地上部矿质元素的积累受到严重阻碍,生菜叶片硝酸盐含量显著下降。在5个根区温度处理中,25℃对水培生菜的生长及矿质元素的吸收最为适宜。     

Pigmentation of cultured red sea bream, Chrysophrys major, using astaxanthin from Antarctic krill, Euphausia superba, and a mysid, Neomysis sp.

Pigmentation enhancement in cultured red sea bream, Chrysophrys major, was investigated using Antarctic krill, Euphausia superba, and a mysid, Neomysis sp., as a source of astaxanthin. Diets fortified with processed Antarctic krill (krill meal) and its acetone extract, containing 0.82–4.92 mg carotenoids/100 g dry weight, and raw krill and raw mysid supplemented diets, containing about 2.00 mg carotenoids/100 g wet weight, were formulated and tested for carotenoid deposition. The rate of carotenoid deposition in fish fed with raw krill and raw mysid was significantly higher and resulted in distinct pigmentation. The groups fed with the krill meal and acetone extract diets showed varied concentrations of skin carotenoids and resulted in faint pigmentation. Pigmented fish then fed on a carotenoid-free diet for the same length of time showed no apparent differences in the skin pigmentation although the detectable amounts of carotenoids varied. The bream converted some of the dietary astaxanthin to skin tunaxanthin.     

Improvement of porcine oocytes with low developmental ability after fusion of cytoplasmic fragments prepared by serial centrifugation

We have shown in pigs that oocytes denuded of cumulus cells at 24 h of in vitro maturation culture and subsequently matured for a total of 46 h (DO24 oocytes) have lower cytoplasmic maturity than those matured with cumulus cells for 46 h and then denuded (DO46 oocytes). In the present study, DO24 zona-free oocytes were fused with one (1C) or two (2C) cytoplasmic fragments produced by serial centrifugation ("centri-fusion") of DO46 oocytes (DO24+1C and DO24+2C oocytes, respectively). Groups of (1) DO46 (a control), (2) DO24, (3) DO24+1C and (4) DO24+2C oocytes were partheno-activated by an electrical pulse or fertilized in vitro and subsequently cultured for 6 days. In the fused groups, female pronucleus (FPN) formation rates were higher than that in the DO24 group after parthenogenetic activation (PA); however, the blastocyst rates were intermediate between those of the control and DO24 groups. After in vitro fertilization, the male pronucleus (MPN) formation rates in the fused groups were similar to that in the control group and higher than that in the DO24 group; the normal fertilization rate in the DO24+2C group was higher than that in the DO24 group and similar to that in the control group, resulting in significantly higher blastocyst rates in the DO24+2C and control groups than that in the DO24 group. These results suggest that centri-fusion using ooplasm from fully matured DO46 oocytes can offer a potentially novel approach for restoration of cytoplasmic maturity to oocytes with low developmental ability and subsequent improvement of fertilization and developmental competence.     

Signature-tagged mutagenesis of Vibrio vulnificus

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.     

Pluripotent cell derivation from male germline cells by suppression of Dmrt1 and Trp53

Diploid germ cells are thought to have pluripotency potential. We recently described a method to derive pluripotent stem cells (PSCs) from cultured spermatogonial stem cells (SSCs) by depleting Trp53 and Dmrt1, both of which are known suppressors of teratomas. In this study, we used this technique to analyze the effect of this protocol in deriving PSCs from the male germline at different developmental stages. We collected primordial germ cells (PGCs), gonocytes and spermatogonia, and the cells were transduced with lentiviruses expressing short hairpin RNA against Dmrt1 and/or Trp53. We found that PGCs are highly susceptible to reprogramming induction and that only Trp53 depletion was sufficient to induce pluripotency. In contrast, gonocytes and spermatogonia were resistant to reprogramming by double knockdown of Dmrt1 and Trp53. PSCs derived from PGCs contributed to chimeras produced by blastocyst injection, but some of the embryos showed placenta-only phenotypes suggestive of epigenetic abnormalities of PGC-derived PSCs. These results show that PGCs and gonocytes/spermatogonia have distinct reprogramming potential and also suggest that fresh and cultured SSCs do not necessarily have the same properties.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.