Influence of Microgravity on the Concentration of Circulating Primordial Germ Cells in Silky Chicken Offspring

This study was performed to examine the implications of microgravity on circulating primordial germ cells (PGCs) of the offspring of silky chickens at stages 13 to 17. China's ShenZhou-3 unmanned spaceship was launched on March 25, 2002, at 22: 00 Beijing time (14: 00GMT). The spaceship carried nine fertilized silky chicken eggs (F0) to test the reliability of the life-support system in the space environment. One female and two male chickens were born from these eggs. The three chickens mated naturally and F1 fertilized eggs were collected. Blood was collected from the dorsal aorta or the marginal vein of the embryos at stages 13 to 17, and the number of circulating PGCs of F1 offspring was counted. A similar experimental protocol was performed for the control group (C1 and C2 group). No differences were observed except at stage 15, when the F1 offspring of the flight group (F0 group) showed higher PGC concentrations than the other treatment groups. These results indicated that microgravity may have little effect on the migration and concentration of PGCs in F1 offspring, perhaps because the flight chickens were raised to maturity on Earth under a gravity of 1×g and had sufficient time to recover. Thus, microgravity appeared to have little effect on the PGC concentrations of F1 offspring of silky chickens during circulating stages 13 to 17

Expression of Green Fluorescent Protein Gene in Somatic Chimeric Chickens Produced by Transplantation of Transfected Chicken Embryonic Fibroblasts

To investigate whether the genetically modified somatic cells can survive and express foreign gene for a long time after being transplanted into avian embryo, chicken embryonic fibroblasts (CEFs) as a cell vehicle for delivering foreign protein were transfected with the green fluorescent protein (GFP) gene, and were introduced into early chicken embryos via blood vessel microinjection at 65-70h of incubation at 38.5°C. The manipulated eggs were continued to incubate at same condition. The chimerisms of the transplanted CEFs were preliminarily observed under fluorescence microscopy at the different stages in the embryos and the hatchlings. Meanwhile, the chimeric positions of the donor CEFs and the expression of GFP were further examined by polymerase chain reaction (PCR) and immunohistochemistry. The results showed that fluorescent-labeled CEFs embed in the different organs of the recipients including brain, heart, liver, intestine, muscle, etc. and can survive in post-hatch chickens for at least 134 days and the GFP gene can be expressed normally. Long-term survival of the donor CEFs in recipient and normal expression of the GFP gene imply that this approach can be explored for continuous production of target protein in the host chicken

Specific Inhibitory Effect of κ-Carrageenan Polysaccharide on Swine Pandemic 2009 H1N1 Influenza Virus

<div><p>The 2009 influenza A H1N1 pandemic placed unprecedented demands on antiviral drug resources and the vaccine industry. Carrageenan, an extractive of red algae, has been proven to inhibit infection and multiplication of various enveloped viruses. The aim of this study was to examine the ability of κ-carrageenan to inhibit swine pandemic 2009 H1N1 influenza virus to gain an understanding of antiviral ability of κ-carrageenan. It was here demonstrated that κ-carrageenan had no cytotoxicity at concentrations below 1000 μg/ml. Hemagglutination, 50% tissue culture infectious dose (TCID₅₀) and cytopathic effect (CPE) inhibition assays showed that κ-carrageenan inhibited A/Swine/Shandong/731/2009 H1N1 (SW731) and A/California/04/2009 H1N1 (CA04) replication in a dose-dependent fashion. Mechanism studies show that the inhibition of SW731 multiplication and mRNA expression was maximized when κ-carrageenan was added before or during adsorption. The result of Hemagglutination inhibition assay indicate that κ-carrageenan specifically targeted HA of SW731 and CA04, both of which are pandemic H1N/2009 viruses, without effect on A/Pureto Rico/8/34 H1N1 (PR8), A/WSN/1933 H1N1 (WSN), A/Swine/Beijing/26/2008 H1N1 (SW26), A/Chicken/Shandong/LY/2008 H9N2 (LY08), and A/Chicken/Shandong/ZB/2007 H9N2 (ZB07) viruses. Immunofluorescence assay and Western blot showed that κ-carrageenan also inhibited SW731 protein expression after its internalization into cells. These results suggest that κ-carrageenan can significantly inhibit SW731 replication by interfering with a few replication steps in the SW731 life cycles, including adsorption, transcription, and viral protein expression, especially interactions between HA and cells. In this way, κ-carrageenan might be a suitable alternative approach to therapy meant to address anti-IAV, which contains an HA homologous to that of SW731.</p></div

Induction of Immunological Tolerance in Chicken (Gallus domesticus) to DT40 Cells Stably Transfected with GFP

Genetically modified cells expressing xenogeneic proteins may trigger strong immunological responses when introduced into animals; however, these responses may be prevented by inducing immunological tolerance. In this study, we have introduced allogeneic cells into chicken embryos via embryonic blood vessel microinjection and induced immunological tolerance to them. Chicken DT40 cells stably transfected with the GFP gene (DT40-GFP cells) were produced and used as antigens to induce immunological tolerance by this method at 65-70h of incubation for preimmunization. Enzyme-linked immunosorbent assays and lymphocyte proliferation assays were performed to determine the serum antibody and lymphocyte responses in hatched chickens after reinjection of the cells at 6 weeks post hatching. Our findings indicated that humoral and cellular immunological responses to allogeneic DT40 cells and DT40-GFP cells expressing xenogeneic proteins can be suppressed by preimmunization during embryogenesis; moreover, an effect on lymphocyte proliferation was observed

Time of addition of κ-carrageenan on SW731 replication.

<p>MDCK cells were infected with SW731 at an MOI of 3. κ-carrageenan was added to the cells at 0 h (adsorption), 1 h, 2 h, 4 h, 6 h, and 8 h. After 24 h, viral titers were determined using TCID₅₀ assays. Results were analyzed with the independent sample t-test (n = 3). Values are means ± SEM. Significance: *<i>P <</i> 0.05 vs. nondrug treated control group; **<i>P <</i> 0.005 vs. nondrug treated control group. ^<i>$</i><i>P <</i> 0.05 vs. 8 h group. Results are representative of two independent experiments.</p

Inhibition effect of κ-carrageenan on SW731 mRNA and protein expression.

<p>(A) MDCK cells were infected with SW731 (MOI = 3) for 1h, and then treated with 250 μg/ml of κ-carrageenan or PBS after virus internalization. After 6 h, total RNA was extracted and analyzed using qRT-PCR. (B) MDCK cells were infected with SW731 (MOI = 3), and then treated with 250 μg/ml of κ-carrageenan or PBS after virus internalization. After 8 h, Western blot was performed using anti-NP protein antibody and HRP-labeled second antibody. (C) MDCK cells were infected with SW731 (MOI = 3), and then treated with 250 μg/ml of κ-carrageenan or PBS after viral internalization. After 8 h, immunofluorescence staining was performed using anti-NP protein antibody and FITC-labeled second antibody, and NP positive cell percentage was measured. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM. Significance: *<i>P <</i> 0.05 vs. nondrug treated control group; **<i>P <</i> 0.005 vs. nondrug treated control group. Results are representative of two independent experiments.</p

Cytotoxic effect of κ-carrageenan on MDCK cells.

<p>MDCK cells were treated with the indicated concentrations of κ-carrageenan. After 48 h of incubation, metabolic activity was measured via MTT assay. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM (n = 3). Significance: *<i>P <</i> 0.05 vs. nondrug-treated control group; **<i>P <</i> 0.005 vs. nondrug-treated control group. Results are representative of two independent experiments.</p

Specific inhibition effect of κ-carrageenan on SW731 HA.

<p>(A–C) Twenty-five microliters of serially diluted SW731, SW26, CA04, PR8, WSN, LY08, and ZB07 viruses were incubated with the same volume of PBS (Mock) or serially diluted κ-carrageenan, at RT for 20 min. Then each sample was mixed with 50 μl of 1% chicken red blood cells (RBC). After 30 min, the concentrations of κ-carrageenan inhibiting HA activity were measured. (D) Phylogenetic trees of the HA genes were constructed with sequences from GenBank based on the open reading frame sequences. The tree was generated using the neighbor-joining method in MEGA 6.0, with 1000 bootstrap trials performed to assign confidence to the grouping. Results were analyzed with the independent sample t test (n = 3). Values are means ± SEM. Significance: *<i>P <</i> 0.05 vs. nondrug treated control group. **<i>P <</i> 0.005 vs. nondrug treated control group. Results are representative of two independent experiments.</p