Establishment of an induced pluripotent cell line from Taiwan black silkie chick embryonic fibroblasts for replication-incompetent virus production.

The objective of this study was to establish a versatile cell line for replication-incompetent virus production and inactivation with formaldehyde to generate a model of cell-based vaccine manufacturing process. To achieve this goal, we took advantage of the easily accessed chick embryonic fibroblasts. Nine-day old chick embryonic fibroblasts were obtained and subjected to be transduced with a set of lentivirus to develop a chick induced pluripotent stem (ciPS) cell line. Morphological features, positive periodic acid-Schiff staining as well as strong immunocytofluorescence of alkaline phosphatase, intestinal (ALPI) and POU class 5 homeobox 1 (POU5F1) proteins suggested that these chick embryonic fibroblasts have been transformed into ciPS cells. Further differentiation and immunocytofluorescence assays confirmed that this ciPS cell line possesses capacities and potentials to form embryoid bodies, differentiate into all three embryonic layers: ectoderm, mesoderm and endoderm with evidence of strongly positive and specific molecular markers. Immunoblot analysis next demonstrated that through recombinant DNA technology and the 2nd generation lentiviral transfer system, the goose hemagglutinin gene (H5) gene was packaged into the replication-incompetent virus and highly expressed in a bladder cancer-derived cell line, T24, after transduction. The titer of ciPS-generated replication-incompetent virus is comparable to that from the Phoenix-AMPHO cell line, which is a commercial and high productive retrovirus producer. Our study successfully established a ciPS cell line which is able to produce replication-incompetent virus, providing a new strategy for cell-based vaccine production after virus inactivation.

Effects of Deinococcus spp. supplement on egg quality traits in laying hens.

To counter the ill effects of synthetic dyes, bacterial pigment production as an alternative is now one of the promising and emerging fields of research. This study was conducted to evaluate the applicability of Deinococcus genus on the egg quality traits in laying hens. In study I, 24 single comb White Leghorn layers were fed with various 1 wt % Deinococcus bacterial strains for 10 d. In study II, 84 brown Hendrix layers were fed with one of 4 diets containing 0, 0.2, 1, or 5 wt % Deinococcus sp. GKB-Aid 1995 powder for 12 wk. In study III, 60 White Leghorn laying hens were fed either with or without 1 wt % Deinococcus sp. GKB-Aid 1995 powder, 1 wt % Deinococcus sp. GKB-Aid 1995 granules, or 1 wt % Deinococcus sp. GKB-Aid 1995 oily granules for 10 successive d. In all of the experiments, feeding Deinococcus powder did not affect egg quality traits except for the yolk color. In particular, supplementation with all Deinococcus powder treatments changed the yolk color (P < 0.05) in study I, with the best pigmentation score obtained by D. grandis and Deinococcus sp. GKB-Aid 1995. Moreover, longer supplementation of Deinococcus sp. GKB-Aid 1995 in study II had a significant effect on feed conversion ratio. With these findings under consideration, the present study suggests that the Deinococcus species, especially Deinococcus sp. GKB-Aid 1995, can be an excellent candidate for improving egg yolk color in laying hens.

Passive protection effect of chicken egg yolk immunoglobulins on enterovirus 71 infected mice.

The objective of this study is to evaluate the passive protective efficiency of immunoglobulin in yolk (IgY) specific against human enterovirus type 71 (EV71). The antibody was raised by intramuscular immunization to 10 White Leghorn hens, with inactivated human EV71 serving as the antigen. The titer and specificity of the antibody were analyzed from purified IgY in the egg yolks of immunized hens. Results indicate that the titer of IgY specific against EV71 increased from the third week after the first immunization. The content of total IgY was 190 ± 26 mg/yolk, with an average concentration of specific IgY of 6.34 ± 3.38 mg/yolk in the eggs from 3 to 18 wk after immunization. The results of the neutralization effect of specific IgY in EV71-challenged mice demonstrate that the EV71-specific IgY, either by intraperitoneal injection or oral administration, was able to significantly reduce the morbidity and mortality in EV71 infected mice pups.

Identification of early transcripts related to male development in chicken embryos.

Early transcripts related to male development in chicken embryos and their expression profiles were examined. A total of 89 and 127 candidate male development transcripts that represented 83 known and 119 unknown non-redundant sequences, respectively, were characterized in an embryonic day 3 (E3; Hamburger and Hamilton Stage 20: HH20) male-subtract-female complementary DNA library. Of 35 selected transcripts, quantitative reverse transcription-polymerase chain reaction validated that the expression levels of 25 transcripts were higher in male E3 whole embryos than in females (P < 0.05). Twelve of these transcripts mapped to the Z chromosome. At 72 wk of age, 20 and 4 transcripts were expressed at higher levels in the testes and brains of male than in the ovaries and brains of female chickens (P < 0.05), respectively. Whole mount and frozen cross-section in situ hybridization, as well as Western blotting analysis further corroborated that riboflavin kinase (RFK), WD repeat domain 36 (WDR36), and EY505808 transcripts; RFK and WDR36 protein products were predominantly expressed in E7 male gonads. Treatment with an aromatase inhibitor formestane at E4 affected the expression levels at E7 of the coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316 and EY505812 transcripts across both sexes (P < 0.05), similar to what was observed for the doublesex and mab-3 related transcription factor 1 gene. The interaction effects of sex by formestane treatment were observed in 15 candidate male development transcripts (P < 0.05). Taken together, we identified a panel of potentially candidate male development transcripts during early chicken embryogenesis; some might be regulated by sex hormones.

In vitro culture period but not the passage number influences the capacity of chimera production of inner cell mass and its deriving cells from porcine embryos.

Mammalian embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) of the blastocyst. These cells are able to proliferate continuously without differentiation in vitro under suitable conditions. Their capacity of pluripotency in differentiation will be resumed when they are reintroduced into host embryos, when they will contribute to the embryonic development to form chimeric individuals. Manipulation of ES cells has been mainly established from studies in the mouse, and is powerful in the production of transgenic animals. Porcine ICM-derived cell lines possess the same cellular morphology and in vitro behavior as those of murine ES cells, but have lower efficiency in chimera formation when reintroduced into host embryos. This study was to determine the influences of passage number and the duration of in vitro culture on the capacity of porcine ICM-derived cells in the generation of chimeric embryos. The results showed that when passage number of porcine ICM-derived cells was less than 15, there were no detrimental effects on its integration ability. Extending the culture time up to 6 days in each passage of porcine ICM-derived cells impaired its integration capacity into the host blastocyst. Porcine ICM-derived cells cultured for more than 4 days in each passage should not be used for blastocyst injection if high efficiency of chimera production is to be achieved.