CpG methylation modulates tissue-specific expression of a transgene in chickens

The use of genetically modified germ cells is an ideal system to induce transgenesis in birds; the primordial germ cell (PGC) is the most promising candidate for this system. In the present study, we confirmed the practical application of this system using lentivirus-transduced chicken gonadal PGCs (gPGCs). Embryonic gonads were collected from 5.5-d old Korean Oge chickens (black feathers). The gPGC population was enriched (magnetic-activated cell sorting technique) and then they were transduced with a lentiviral vector expressing enhanced green fluorescent protein (eGFP), under the control of the Rous sarcoma virus (RSV) promoter. Subsequently, the eGFP-transduced PGCs were transplanted into blood vessels of 2.5-d-old embryonic White Leghorn (white feathers). Among 21 germline chimeric chickens, one male produced transgenic offspring (G₁ generation), as demonstrated by testcross and genetic analysis. A homozygous line was produced and maintained through the G₃ generation. Based on serum biochemistry, there were no significant physiological differences between G₃ homozygotes and non-transgenic chickens. However, since eGFP transgene expression in G₃ chickens varied among tissues, it was further characterized by Western blotting and ELISA. Furthermore, there were indications that DNA methylation may have affected tissue-specific expression of transgenes in chickens. In conclusion, the PGC-mediated approach used may be an efficient tool for avian transgenesis, and transgenic chickens could provide a useful model for investigating regulation of gene expression.     

Seasonal prevalence of mosquitoes collected from light traps in the Republic of Korea, 2007

Adult mosquito surveillance was conducted using New Jersey light traps from 1 May to 15 October during 2007 at 14 US Army and three Air Force installations and two US Army training sites located in six provinces in the Republic of Korea to determine threshold levels to initiate pesticide applications based on trap indices. A total of 24 013 adult mosquitoes (19 763 [82.3%] females and 4250 [17.1%] males) comprising 16 species (including five members of the Anopheles Hyrcanus Group) and belonging to seven genera were collected. Females of the most commonly collected species were members of the Anopheles Hyrcanus Group (52.1%), followed by, Culex tritaeniorhynchus Giles (20.6%), Culex pipiens Coquillett (13.8%), and Aedes vexans nipponii (Theobald) (11.6%). Trap indices varied widely for species over their range, due in part, to geographical distribution and degree of association with urban or rural communities.     

Zebrafish as a new model for phenotype‐based screening of melanogenic regulatory compounds

Although many hypo‐pigmenting agents are currently available, the demand for novel whitening agents is increasing, in part due to the weak effectiveness and unwanted side effects of currently available compounds. To screen for novel hypo‐pigmenting agents, many methodologies such as cell culture and enzymatic assays are routinely used. However, these models have disadvantages in terms of physiological and economic relevance. In this study, we validated zebrafish as a whole‐animal model for phenotype‐based screening of melanogenic inhibitors or stimulators. We used both the well‐known melanogenic inhibitors (1‐phenyl‐2‐thiourea, arbutin, kojic acid, 2‐mercaptobenzothiazole) and newly developed small molecule compounds (haginin, YT16i). All the tested compounds produced inhibitory effects on the pigmentation of zebrafish, most likely due to their inhibitory potential on tyrosinase activity. In simultaneous in vivo toxicity tests, a newly developed melanogenic inhibitor YT16i showed massive abnormalities in terms of deformed morphologies and cardiac function. Together, these results provide a rationale in screening and evaluating the putative melanogenic regulatory compounds. We suggest that the zebrafish system is a novel alternative to mammalian models, with several advantages including the rapidity, cost‐effectiveness, and physiological relevance.     

Deletion of quinolinate phosphoribosyltransferase gene accelerates frailty phenotypes and neuromuscular decline with aging in a sex-specific pattern

Decline in neuromuscular function with aging is known to be a major determinant of disability and all-cause mortality in late life. Despite the importance of the problem, the neurobiology of age-associated muscle weakness is poorly understood. In a previous report, we performed untargeted metabolomics on frail older adults and discovered prominent alteration in the kynurenine pathway, the major route of dietary tryptophan degradation that produces neurotoxic intermediate metabolites. We also showed that neurotoxic kynurenine pathway metabolites are correlated with increased frailty score. For the present study, we sought to further examine the neurobiology of these neurotoxic intermediates by utilizing a mouse model that has a deletion of the quinolinate phosphoribosyltransferase (QPRT) gene, a rate-limiting step of the kynurenine pathway. QPRT^−/− mice have elevated neurotoxic quinolinic acid level in the nervous system throughout their lifespan. We found that QPRT^−/− mice have accelerated declines in neuromuscular function in an age- and sex-specific manner compared to control strains. In addition, the QPRT^−/− mice show premature signs of frailty and body composition changes that are typical for metabolic syndrome. Our findings suggest that the kynurenine pathway may play an important role in frailty and age-associated muscle weakness.     

Identification of Heat-Stable Enterotoxin-Producing Strains of Yersinia enterocolitica and Vibrio cholerae Non-O1 by a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay

Using a mouse monoclonal antibody (MAb) 2F raised against Vibrio cholerae non-O1 heat-stable enterotoxin (NAG-ST) which also recognizes a shared epitope of Yersinia enterocolitica heat-stable enterotoxin (Y-ST), a competitive enzyme-linked immunosorbent assay (ELISA) was developed for independent detection of NAG-ST and Y-ST. There was good concordance between the Y-ST ELISA and the suckling mouse assay (SMA) for detection of Y-ST from test strains of Y. enterocolitica, and the Y-ST ELISA can effectively replace the SMA for routine detection of Y-ST. On the contrary, evaluation of the NAG-ST ELISA and the SMA using 139 strains of V. cholerae non-O1 showed discordant results and this was attributed to the presence of the suckling mice active factor(s) such as El Tor hemolysin and to the production of low amounts of NAG-ST. Concentration of culture supernatants of V. cholerae non-O1 followed by heating at 100 C was essential to obtain reproducible results by both the NAG-ST ELISA and the SMA. The ELISA developed in this study can be used for the identification of biologically active strains. While recently genetic methods such as polymerase chain reaction became available and were very reliable and simple techniques, the ELISA in this study has an advantage in detecting biologically toxic gene products of the strains. The genetic methods cannot differentiate silent STa genes which we often encounter in the case of Y. enterocolitica.     

Production of monoclonal antibodies against human chorionic gonadotropin by hybridoma cultures in calcium alginate capsules

A new method of making microcapsules with calcium alginate gel was developed and the cultivation of the encapsulated hybridoma cells producing monoclonal antibodies against human chorionic gonadotropin was investigated. A high cell density of 2.0×10⁸ cells/cm³ in the capsules led to a high dilution rate of 0.68 per hour and resulted in the high volumetric monoclonal antibody productivity of 652.8 mg/l/day, which was 20–30 times higher than those of traditional continuous suspension cultures. However, long-term continuous culture was not achieved with this capsule system probably because of the limitation in nutrient supply and the accumulation of waste products. Also the analysis of oxygen transfer in this system showed that oxygen supply was not enough to support such a high cell density.