Abstract:Objective To establish an animal model in which both early-phase asthmatic response (EAR) and late-phase asthmatic response (LAR) can be observed after sensitization and subsequent inhalation challenge with ovalbumin (OVA). Animals were conscious with no narcotic used, unrestricted, and fed ad libitum. Methods Sixty-six SPF 6-8-week old male Brown Norway rats were divided into eleven equal groups. All groups of rats except normal control group were injected subcutaneously with 0.4 mL (sc in back, 2 sites, 0.2 mL/site) OVA or OVA + Al(OH)₃ solution on day 0 and day 5. Four groups were given OVA only at the dose of 0.01, 0.1, 1.0 or 10.0 mg/rat, five groups were given OVA + Al(OH)₃ powder at the dose of 0.1+100, 1.0+100, 10.0+100, 1.0+52 and 1.0+4 mg/rat, one group was given OVA+Al(OH)₃ gel at the dose of 10.0+4 mg/rat. Normal control group was injected subcutaneously with the same volume of saline. All the groups were challenged for 10 minutes with 5 mL 5%OVA aerosol on day 37. Enhanced pause (Penh) was recorded for 16 hours in a whole-body plethysmography system after challenge. Specific IgE of the serum samples on day 0, 7, 14, 21, 28, 35, 38 were measured by ELISA. Pulmonary pathological changes were observed using HE staining. Results Compared with the normal control group, immunized rats except the group given 0.01 mg OVA produced specific IgE (P<0.05), and the content of IgE grew sharply after 7 days, and always kept growing until 5 weeks. The whole course of asthma exacerbation was recorded successfully. The rats developed EAR and/or LAR within 16 hours following OVA challenge. Especially the groups injected with 10 mg OVA and 100 mg Al(OH)₃ or 4 mg Al(OH)₃ gel showed steady pattern of biphasic airway responses and their EAR or LAR peak, and the area under the curve were increased significantly compared with those of the normal control group (P<0.05). Inflammation characterized by eosinophil infiltration was observed in the rat lung of model group (OVA 10.0 & Al(OH)₃ 100 group as a representative case). Conclusions In this work we successfully developed a new model using conscious rats, and the whole time course of asthma exacerbation in this model can be observed after OVA challenge. This model may become a useful tool for further asthma research.

Abstract:Objective To knockout the murine Txnip gene using microinjection of transcription activator-like effector nuclease (TALEN) mRNAs. Methods TALEN knockout site recognizing Txnip was designed by tools on line, then constructed the vectors and assayed its cleavage activity at cellular level. TALEN mRNA was transcribed in vitro and microinjected into C57BL/6J mouse zygotes. F0 mice were verified at DNA level with BamHI and TXNIP-knockout mice were obtained. Results We designed and constructed TALENs which recognized and cut the first exon of Txnip, and got four TXNIP knockout mice, among which two were frameshift mutation, demonstrating that the TXNIP-knockout mice were generated by TALEN technique. Conclusions Microinjection of in vitro transcribed TALEN mRNAs into murine zygotes is a highly effective and convenient way to develop TXNIP-knockout mouse model.

Abstract:Objective To screen strains of minipigs sensitive to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) for evaluation of HP-PRRS live vaccine. Methods Lantang pigs, Juema, Bama and Wuzhishan (white) minipigs were inoculated with virulent strain NVDC-JXA1 of PRRSV, and local binary hybrid pigs were used as control. The animals were continuously observed for 5 weeks on mental status, appetite, survival, etc. after inoculation of virus. The dead pigs were autopsied and the lung tissue samples were collected for detecting virus by RT-PCR. By the end of the experiment, serum of survival animals were collected for detecting PRRSV antibody by ELISA assay. Result The animals showed depression, anorexia, and other clinical signs and death in each group after inoculation. Meanwhile, the testing results were all positive in the RT-PCR and ELISA detection. Bama and Wuzhishan (white) minipigs were the most sensitive to virulent strain NVDC-JXA1 of PRRSV regarding mortality rate. Conclusions Bama and Wuzhishan (white) minipigs are sensitive to HP-PRRSV, and can be used for the inspection of HP-PRRS live vaccine.

Abstract:Objective To investigate the effect of chronic restraint stress on the comobidity behavior of anxiety and depression disorders in mice. Method C57BL/6J mice were randomly divided into 3 groups (n=10 per group): control (normal saline), chronic restraint stress (normal saline), and positive control (citalopram, 10 mg/kg). Citalopram and normal saline were administered by intraperitoneal injection. Chronic restraint stress and individual housing was applied to establish the stress model. The mice were individually housed and restrained for 4 h per day in a 50-mL polypropylene conical tube with ventilation holes. This daily restraint was repeated for 35 consecutive days. Sucrose preference test and forced swim test were applied to evaluate the depressive behavior of the mice. Open field test and elevated plus maze test were used to assess the anxiety effect of chronic restraint stress in the mice. Results The sucrose intake was significantly reduced in the chronic restraint stress models compared with the control mice (P<0.01). The immobility time was increased in the forced swim test (P<0.01). The cumulative duration and distance moved in the center were decreased in the open field test(P<0.01, P<0.05). Chronic treatment with citalopram reversed the above mentioned behavior change. The open arm entry and open arm time were decreased in the elevated plus maze test (P<0.05, P<0.05). Citalopram did not reverse this behavior change. Conclusions Mouse models created by chronic restraint and individual housing stress display both anxiety and depressive behavior making them a potent animal model in the treatment of comorbidity of anxiety and depression disorders.

Abstract:Objective To study the protective effect of vitamin C on UV-induced photoaged skin structure in rats and provide a basis for clinical medicine and health care. Methods Photoaging skin models were set up by chronic ultraviolet radiation in 24 SPF female Sprague-Dawley rats, irradiated twice weekly for 4 weeks. The rat photoaged skin was induced by exposure to a total dose of 123 J/cm² UVA and 5 J/cm² UVB for 4 weeks. Vitamin C was administered by gastric gavage in a dose of 50 mg/kg once daily for 30 days during the model development. We compared the pathological changes in the irradiated skin using HE, Masson's trichrome and Victoria blue B staining, and compared the ultrastructural changes by electron microscopy. Results Rat models with photoaged skin were developed successfully. The vitamin C group showed significant reduction of pathological severity including erythema, ulcers, epidermal cell proliferation, epidermal thickness, dermis vasodilation, inflammatory cell infiltration, fibroblast proliferation, endoplasmic reticulum dilatation, mitochondrial swelling and vacuolization, elastic fibers degeneration and focal accumulation, collagen fibers swelling with uneven thickness,compared with the rats of model group at the irradiation site. Conclusions Vitamin C is effective in reducing the structural damage of UV-induced photoaged skin in rats.

Abstract:Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells. Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis. The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells. The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells. Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface. It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.

Abstract:Objective To construct lentiviral vectors containing peptide P1-GFP fusion genes. Umbilical cord derived mesenchymal stem cells were infected with lentivirus carrying peptide P1 and GFP fusion genes. To inject the targeted umbilical cord derived mesenchymal stem cells into mice and to detect GFP expression in the spleen. Methods Umbilical cord derived mesenchymal stem cells were cultured with adhered tissues of umbilical cord smaller than 1 mm³. Lentiviral vector containing P1-GFP fusion genes with engineering technology was constructed and infected the umbilical cord derive mesenchymal stem cells. Targeted umbilical cord derived mesenchymal stem cells were intravenously injected in the mouse tail vein and after 18 hours GFP expression was detected with immunohistochamical staining of the spleen tissues. Results Harvested umbilical cord derived mesenchymal stem cells grew well in culture medium. Green fluorescence on umbilical cord derived mesenchymal stem cells were observed under fluorescence microscope at 18 hours after infected with lentivirus. Green fluorescence intensity of umbilical cord derived mesenchymal stem cells was increasing over time and reached a peak at 72 hours. Umbilical cord derived mesenchymal stem cells highly expressed CD105 (90.0%)/CD44 (98%) and CD73 (85.0%)/CD90 (98.5%) molecules. GFP expression was detected in the spleen after intravenous injection of targeted umbilical cord derived mesenchymal stem cells in the mice 18 hours later. GFP expressing cells intimately contacted with lymphocytes. Conclusions Targeted umbilical cord derived mesenchymal stem cells contain P1-GFP fusion genes are constructed. Targeted umbilical cord derived mesenchymal stem cells can be targeted to mouse spleen and intimately contact with lymphocytes after intravenous injection. Our results lay the groundwork for further studies.

Abstract:Objective To screen out specific microsatellite markers for use in Tupaia belangeri chinensis genetic testing. Methods Firstly to screen about 700 microsatellite loci from whole genome. Secondly to choose about 100 better loci without defect factors. Lastly 46 primers were designed by 33 tree shrew's microsatellite loci obtained from whole genome and other references. Agarose gel electrophoresis and polyacrylamide gel electrophoresis were used for PCR products, and better loci based on electrophoresis results were chosen. Then STR scan was used to select the microsatellite loci combination for genetic testing.Results Twenty-two microsatellite loci were selected with a significant Stutter peak on STR scanning. Comparing the alternative loci and ultimately selected loci, there were two loci available in the five alternative loci of T. glis. The coincidence rate between T. glis and T.b. chinensis was 40%. There were two loci available in the five alternative loci of T. minor, and the coincidence rate between T. minor and T. b. chinensis was 40%. There were two loci available in the three alternative loci of T. belangeri, and the coincidence rate between T. belangeri and T.b. chinensis was about 70%. Conclusions The 22 microsatellite loci screened in this study are well applied for genetic testing of Tupaia belangeri chinensis, therefore, provide a scientific basis for the genetic quality monitoring of tree shrews.

Abstract:Objective PSGL-1 is specifically expressed in leucocytes. The aim of this study was to explore the changes of myeloid-derived suppressor cells (MDSCs) in the spleen and bone marrow in PSGL-1-deficient mice. Methods PSGL-1^-/- mice were used in the experiment. After identification of the offsprings, flow cytometry was used to test the expression of CD11b and Gr-1 in C57 and PSGL-1^-/- mice. Results Compared with the C57 mice, the expression of MDSCs was up-regulated in the PSGL-1-deficient mice (P<0.001). Conclusion The expression of MDSCs is upregulated in PSGl-1-deficient mice.

Abstract:Objective To develop a duplex PCR assay for detection of rat parvovirus H-1 and KRV and its application. Methods To design specific primers on the basis of H-1(NC_001358) and KRV(U790330) genome sequences published in NCBI and establish a duplex PCR assay using H-1 and KRV DNA as templates. To verify the sensitivity and specificity of the method after optimizing PCR. The rats were infected by oral inoculation. The rats were divided into three groups: H-1 infection, KRV infection and mixed infection groups. To collect feces at the 4th, 6th, 8th and 10th days postinfection. Rats were euthanized on the 10th day and samples from heart, liver, spleen, lung, kidney and cecal contents were collected from each rat, then all the samples were screened with the duplex PCR. Results The 183 bp and 302 bp bands were amplified using H-1 and KRV as templates. The sensitivity test showed that the PCR method can detect as low as 3.8 pg/mL H-1 and 0.73 pg/mL KRV. There were no bands amplified when mouse minus virus, canine parvovirus and feline parvovirus were used as templates, showing that the specificity of the duplex PCR assay is very good. The nucleic acids of H-1 or KRV were detected in all rat feces on the 2th day postinfection and there was no obvious clinical symptoms in all the infected rats. The positive rates of H-1 were as follows: 50% (4/8) heart tissues, 50% (4/8) liver tissues, 62.5% (5/8) spleen tissues, 50% (4/8) lung tissues, 37.5% (3/8) kidney tissues and 62.5% (5/8) cecum contents, and the positive rate of single infection group was higher than that of mixed infection group. The positive rates of KRV were as follows: 0 (0/8) heart tissues, 25% (2/8) liver tissues, 87.5% (7/8) spleen tissues, 12.5% (1/8) lung tissues, 25% (2/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of mixed infection group was higher than that of single infection group. Conclusions The duplex PCR assay for H-1 and KRV established in this study can effectively detect H-1 or KRV infection in rat feces and other tissues, and can be used as an effective supplement to the national standard of lab animals.

Abstract:Objective To establish a rapid, specific and sensitive TaqMan real-time fluorescence quantitative PCR assay for detection of murine polyomavirus (MPyV). Methods The specific primers and TaqMan probe were designed based on genome sequence of MPyV. The primers amplified a 69 bp fragment. After optimizing the reaction system and reaction condition, the standard curve was plotted by detecting recombinant plasmid standards. The specificity, sensitivity and reproducibility of this method were evaluated. In addition, samples of lungs, spleens and feces obtained from experimentally infected mice and 86 clinical samples were used to validate the efficacy of this real-time PCR assay. Results The specificity assay showed that this assay could specifically detect MPyV and the sensitivity for MPyV was about 100 copies/well. The coefficients of variation (CV) of both intra-assay and inter-assay were less than 1.13%. All of the samples from experimentally infected mice were positive for MPyV and 3 out of 86 clinical samples were positive by this TaqMan-PCR detection with a positive rate of 3.5%. Conclusions The real-time fluorescence quantitative TaqMan-PCR assay established in this study has high specificity, sensitivity and stability. It can be used for clinical diagnosis, routine detection and epidemiological investigation of murine polyomavirus infections.

Abstract:Objective To establish a fluorescence quantitative Taqman-PCR method for rapid and accurate detection of mouse poxvirus. Methods After sequence alignment and comparison, ERPV_027 gene was selected as the primer and probe design gene. Furthermore, the specificity, sensitivity, stability and reproducibility of these primers and probes were detected. Results The detection limitation of this method was 68 copies/μL. Data showed that this method has high specificity, which specifically amplifies mouse poxvirus, with no amplification signal of mouse hepatitis virus, Sendai virus, Salmonella and some other viruses and bacteria. This method also showed good stability and reproducibility. Conclusions This study has successfully established a fluorescence quantitative Taqman-PCR method for detection of mouse poxvirus, with high specificity, sensitivity, good stability and reproducibility, and a broad application potential.

Abstract:Due to practical and ethical concerns associated with human experiments, animal models have been essential in cancer research. Vast resources are expended during the development of new cancer therapeutics, and selection of optimal in vivo models should improve this process. Genetically engineered mouse models (GEMM) of cancer have progressively improved in technical sophistication and, accurately recapitulating the human cognate condition, have provided opportunities to accelerate the development of cancer drugs. In this article we consider the different types of animal models used for predicting the results of clinical trials of cancer drugs, and discuss the strengths and weaknesses of each in this regard. In addition, the methods of predicting in vivo models and clinical translation are discussed.

Abstract:Research on laboratory animals is an important issue in biomedicine. Children are a special drug-using population. The selection of suitable experimental animals is a key issue to ensure the scientific quality of research for pediatric drugs. Based on the review of a large number of literature, the authors summarized the application of suckling mice in the pharmacological research and toxicological evaluation of pediatric drugs for the treatment of common diseases in children. We also summarized the existing problems in pediatric toxicology and proposed solutions for providing a reference of test animal application in pediatric drug research.

Abstract:Animal experiments in pathogen studies such as avian influenza viruses are increasing in recent years. Different Animal Biosafety Level (ABSL) facilities have been established completely according to the national standards for biosafety. However, issues of personal protection, experiment management, risk recognition and control, animal care with infectious pathogens, etc. should be strengthened. In this review, suggestions and comments are proposed, and hopefully, are useful for related guideline compilations.