Table of Content

28 June 2022, Volume 39 Issue 3

Previous Issue   

Breeding, Reproducing and Genotype Identification for GalR2 Gene Knockout Mice

MA Yanhua, HE Ni, PEI Shaofei, LV Dian, LI Wenjun, FAN Baomin, ZENG Guangzhi, LIN Chengyuan

2022, 39(3):  1-5.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 001

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Objective To breed and culture galanin receptor 2 knockout mice, and mouse genotypes were analyzed and identified, which was also verified for the applicability of the detection method. Method F1 generation were obtained via GalR2 knockout heterozygotes male mice and wild-type female mice bred at 1 ∶ 2 ratio, or heterozygous female mice and wild-type male mice bred at 1 ∶ 1 ratio. The obtained F1 generation heterozygous mice were bred for sibling mating to obtain F2 generation homozygous, heterozygous and wild-type mice. The tails (2-5 mm) of 2- week-old mice were cut to obtain genomic DNA, PCR was utilized to amplify the target gene fragments and genotype result were determined by agarose gel electrophoresis. Result The breeding and reproducing were successful with a number of knockout mice were stably bred. Three genotype offspring of F1 generation including homozygous ( GalR2- / - ) , heterozygous ( GalR2+ / - ) and wild-type ( GalR2+ / + ) were identified using the PCR method. Conclusion GalR2 knockout mice can be bred and cultured in a scientific way and genotypes can be identified with PCR method.


Establishment of Beagle Dog Infection Model with RH Strain of Toxoplasma gondii

WEN Wanxin, CHEN Hongyan, HAN Lingxia

2022, 39(3):  6-10.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 002

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Object To establish a infection dog model with RH strain of Toxoplasma gondii. Method Three specific pathogen-free 76 day-old Beagles were intraperitoneal injected with 1. 5 × 107 tachyzoites of Toxoplasma gondii. The dogs had been growing in positive pressure isolators under barrier environment and fed with artificial milk. The experiment was carried out under negative pressure barrier environment. After inoculation, anal temperatures, oral and anal swabs were collected twice daily to detect B1 gene of T. gondii by PCR. At 8 days post inoculation ( dpi ) all animals were euthanized, and nucleotide copies of Rep529 nt at various tissues were quantified by real-time PCR method. Histopathology and immunohistochemical stains changes were observed. Result The result showed that the three dogs had fever and diarrhea at 4 dpi-5 dpi; much ascites was appeared when the abdomens were opened; B1 gene of T. gondii was detected in oral or anal swabs at 2 dpi - 6 dpi by a routine PCR method, and the PCR result positive rate of the anal swabs was higher than that of the oral swabs totally, there was obvious inflammatory reaction in the liver tissue in histopathological observation with HE stain. Among all the detected tissues sampled on autopsy using the Taqman real-time PCR, the copy number of the Rep529 nt was high in cecum and liver. Conclusion The infectious experiment shows that there merely causes a mild infection model on SPF Beagles when inoculated with RH strain of T. gondii, with transient increase of body temperature and diarrhea, liver degeneration and necrosis.

Evaluation on the Detection Capacity of Esterase 1 and Tansferrin in the Proficiency Testing of Laboratory Animals Quality Control

WANG Hong, WEI Jie , GUANG Jiaona, ZHOU Jiaqi, YUE Bingfei

2022, 39(3):  11-15.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 003

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Objective To help the laboratories of quality control for laboratory animals find problems and improve the level of quality control of laboratories through the proficiency testing. Method To carry out the proficiency testing according to the document CNAS-CL03, and samples passed the homogeneity test and stability test. The participating laboratories should submit the testing result and original record. To compare the laboratory result and the standard result. Result A total of 9 laboratories participated in the program. 7 laboratories presented satisfactory experiment result. The satisfaction rate of the program is 77. 78%. Conclusion The overall testing level of esterase 1 and transferrin in the laboratories of quality control was relatively high. There is room for improvement in a few laboratories.


Construction of Human CSF-1R Gene Knock- in Mouse Model

LIU Susu, WUYong, GU Wenda, CAO Yuan, ZHAI Shijie, ZHAO Haoyang, FAN Changfa

2022, 39(3):  16-21.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 004

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Objective The CSF-1R knock in mouse model was established by CRISPER / Cas9 technology. Method The partial sequences of exons 3 to 11 cDNA of humanized CSF-1R gene, together with TGA codon and WPREPolyA original, were inserted into downstream of the 20th alanine of exon 3 on mouse CSF-1R gene. The gene expression characteristics were further analyzed by RT-qPCR and sperm cryopreservation was carried out for transgene positive mice. Result The result showed that the trans gene expressed in heart, liver, spleen, lung, kidney and brain of CSF-1R gene knock-in mice. The average in vitro fertilization rate of frozen sperm was 67. 74%, and the average birth rate was 16. 06%. Conclusion A humanized CSF-1R gene knock-in mice model was established successfully,which provides a novel tool for evaluating the in vivo efficacy of antibodies and drugs of this target.


Effect of Emodin on Nonalcoholic Fatty Liver Disease and the Expression of 11β-hydroxysteroid Dehydrogenase Type 1 in Liver

SUN Hongshuang, LI Penglin, LIU Yongshuang, NIE Chuncheng, CHONG Baogui

2022, 39(3):  22-26.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 005

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Objective To observe the effect of emodin on nonalcoholic fatty liver disease ( NAFLD ) and the expression of 11β-HSD1 in liver. To investigate the expression changes of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in the liver of rats with nonalcoholic fatty liver ( NAFLD) and the intervention effect of emodin. Method 40 male Wistar rats were randomly divided into four groups: control, model, low-dosage emodin treatment ( E1) and high-dosage emodin treatment ( E2) groups. The control group was feed with normal diet, the model and treatment group were feed with high-fat diet. The treatment group was given emodin from the 9th week, and the experiment was completed at the end of the 14th week. Result Compared with the control group, rats in the model group had significant NAFLD, and the expression of 11β-HSD1 in liver was increased. Compared with the model group, emodin treatment could reduce liver index, blood lipid and liver lipid deposition, improve liver function and pathological changes, and significantly inhibit the expression of 11β-HSD1 gene and protein in liver tissue. All emodin treatment effects were dose-dependent. Conclusion Emodin has a significant protective effect on NAFLD, which maybe related to the inhibition of the 11β-HSD1 activity.


Immunomodulatory Effects of Bifidobacterium lactis V9 in Mice and its Mechanism

MA Liyan, SUN Wei

2022, 39(3):  27-32.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 006

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Objective Probiotics play an important role in the immune regulation of the body, and different strains often have different regulatory functions on immune cells. The present study aimed to evaluate the immunomodulatory effects of Bifidobacterium lactis V9 in mice and its mechanism. Method Normal C57BL / 6J mice were randomly divided into 4 groups ( n = 10) , and intragastrically administrated distilled deionized water, 0. 5 mg / kg, 1. 0 mg / kg, and 3. 0 mg / kg of Bifidobacterium lactis V9 for 30 days. After that, the mice were killed and the organ / body weight ratio of mice was measured. The cellular immunity was evaluated by splenic lymphocyte transformation assay and delayed hypersensitivity. The function of monocyte macrophage was evaluated by carbon clearance test and mouse peritoneal macrophage phagocytosis of chicken red blood cells. The humoral immunity of mice was evaluated by antibody-producing cell assay and serum hemolysin assay. NK cell activity was evaluated by NK cell activity assay. Result Bifidobacterium lactis V9 showed no significant effects on organ ratios of the mice compared with the negative control(P> 0. 05). Bifidobacterium lactis V9 significantly increased the transformation ability of mice splenic lymphocytes, murine macrophage carbon clearance function, mouse peritoneal macrophage phagocytosis of chicken red blood cells, hemolytic activity and NK cell activity ( P < 0. 05 ) . Conclusion Bifidobacterium lactis V9 exhibited potent to enhance the immunomodulatory effects, and provide a new idea for its further development and utilization.

Proficiency Testing of Laboratory Virus Detection Capacity and Detection of Murine Norovirus in Feces Samples#br#

FU Rui, WANG Shasha, WANG Ji, LI Xiaobo, WANG Shujing, YUE Bingfei

2022, 39(3):  33-39.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 007

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Objective Implemented virus detection Proficiency Testing plan organized by ICLAS PEP, which contained four blind samples, and further improve laboratory testing quality. Method Analyzed and extracted nucleic acid of the samples, selected appropriate primers targeted on the promising virus to proceed polymerase chain reaction. Based on the result of polymerase chain reaction, obtain the sequencing analysis of positive result and further verify the virus in blind samples. Mixed the different dilutions of cecum contents exclude murine norovirus infection and murine norovirus culture with different concentration in the same volume, subjected to RNA extraction applied by two disparate kits and quantitative polymerase chain reaction analysis. Compared the RNA copy number derived from each experimental group with the control group. Result The four blind samples were identified as Theiler ’ s mouse encephalomyelitis virus, murine norovirus, mouse rotavirus and mouse lactate dehydrogenase hypertrophic virus separately which were agreed with the ICLAS standard result. Comparing the result of fluorescent quantitative PCR detection of norovirus in cecal contents with different dilutions, it was found that when nucleic acid was extracted with viral DNA / RNA extraction kit, the detection result showed different degrees of deviation compared with the virus control when the dilution was not higher than 1 ∶ 16, and the detection result were similar to the virus control when the dilution was 1 ∶ 32. When nucleic acid was extracted with the viral RNA extraction kit and the dilution was not less than 1 ∶ 8, the detection result was similar to the viral control. Conclusion The international comparison shows that our laboratory is reliable in virus detection, after pollution experiment research, stool samples 1 ∶ 8 diluted use viral RNA extraction kit nucleic acid for faeces, such as the best virus detection method to determine the cecum contents in samples.


Study on Different Influence Factors in Malic Enzyme-1 and Isocitrate Dehydorgenase-1 Standard Samples#br#

WEI Jie, WANG Hong, GUANG Jiaona, ZHOU Jiaqi, FU Rui, YUE Bingfei

2022, 39(3):  40-44.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 008

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Objective To analyze the influencing factors of temperature-time, concentration, freeze thawing, transportation conditions and different strain sources in malic enzyme-1 and isocitrate dehydorgenase-1 standard samples. Method C57BL / 6, BALB / c and CBA inbred mice were used to prepare standard samples. After homogeneity test, random samples were collected and tested according to GB / T 14927. 1—2008. We used 4 ℃ , room temperature ( RT) and 37 ℃ as horizontal ordinate, and day 1, day 2, day 3, day 7, day 14 and day 21 as ordinate to analyze temperature-time factors. At each coordinate point, two tubes are randomly selected from each strain. The concentration factors were measured by the gradient of original, 1 / 2, 1 / 4 and 1 / 8 concentration samples in 1 tube per strain. In the factors of freeze thawing analysis, we tested samples (3 tubes per strain) for 3 times under the conditions of 2 hour storage at - 20 ℃ and thawing at room temperature. The transportation condition were 3 round-trip test from Beijing to Shenyang, Xi’ an and Chengdu using dry ice in cold chain. Result There were no significant differences in temperature-time, concentration, freeze-thaw and transportation conditions among the standard samples from different strains. Each standard sample could exist stably in condition of 4 ℃ , RT for 21 days, and only 2 days at 37 ℃ . After dilution to 1 / 8 concentration, 3 times freeze-thaw and dry ice transportation, the samples can still be stable. Conclusion The malic enzyme-1 and isocitrate dehydorgenase-1 standard samples are homogeneous, stable and comparable, whIch can meet the requirements of the proficiency testing program.


Anergy Saving Effect of Ethylene Glycol Heat Recovery Device in Laboratory Animal Facility Operation#br#

YAN Chen , SHANG Xiaosong , SUN Bo , LIU Xin , LI Xueyong

2022, 39(3):  45-50.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 009

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Objective To statistically analyze the energy-saving effect of ethylene glycol heat recovery device in the annual operation of laboratory animal facilities. Method Using the automatic control software Kingview provided by the facility, the original data such as the temperature of the fresh air, the relative humidity of the fresh air, the air temperature after heat exchange, and the relative humidity of the air after heat exchange were recorded. And through the analysis of the heat exchange data, the enthalpy value H1 ( kJ / kg) of the fresh air, the enthalpy value H2 ( kJ / kg) of the air after heat exchange, the heat recovery value at each time point and the heat recovery value at each time point are calculated. The power consumption value corresponding to the value. Result When the indoor dry bulb temperature was kept at (24. 0±2. 0) ℃ and the indoor relative humidity was kept at 40% -70%, when the outdoor daily average temperature was higher than 26. 15℃ or lower than 17. 30℃ , the energy saved by the device was higher than its own energy consumption; when the outdoor daily average temperature is 17. 30 ℃ to 26. 15 ℃ , the energy saved by the device is lower than its own energy consumption. Conclusion The application of ethylene glycol heat recovery device in the ventilation and air conditioning system of experimental animal facilities can indeed achieve energy-saving operation, but when the energy saved is lower than its own energy consumption, the operation of the device should be stopped, so as to effectively reduce the operation cost.


Studies on Oral Toxicity of Mixed Lactobacter Freeze-dried Powder in Rats

FANG Xiao, DUAN Rongshuai, ZHANG Xiaoyuan, ZHANG Yanyan, SUN Xiaokang, CHEN Lei

2022, 39(3):  51-56.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 010

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Objective The 28 days oral toxicity of mixed Lactobacter freeze-dried powder ( zhang +R9639+P9+V9+P-8+XLTG11) in rats was studied to provide safety evidence for its application. Method The mixed freeze-dried powder were given to male and female rats intragastrically for 28 days according to the method of GB 15193. 22— 2014. The general status, body weights, organ indexes, food consumption, hematology, blood biochemistry, urinalysis, and histopathological examination of the rats were recorded and evaluated. Result Compared with the control group, no significant differences were found in general status, organ indexes, food consumption, hematology, blood biochemistry, urinalysis, and histopathological examination in rats administrated with different doses of mixed Lactobacter powder. Conclusion Theses result showed that the mixed Lactobacter freeze-dried powder had no significant toxicity in the 28 days oral toxicity assay.


Key Points of Ovariectomy and Post-operative Nursing in Osteoporosis Animal Model

MA Yanfang , ZHANG Xin, LI Hui , CHENG Dong , SUN Changhua

2022, 39(3):  57-62.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 011

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Objective To establish and discuss the key points of ovariectomy and postoperative care in osteoporosis rats, and to provide reference for the successful establishment of animal models. Method Animal models were constructed by surgically applying ovariectomy to rats. The design of animal models should conform to the principles of similarity, reliability, repeatability, applicability, controllability, feasibility and economy. Result and Conclusion The establishment of animal models is extremely important in the study of osteoporosis, and the rational selection of animal models plays a key role in the result of experiments. Rats are the most commonly used animal for osteoporosis animal models.


Statistical Analysis of Rabbit Euthanasia Methods Reported in Animal Experiments#br#

LI Shuolei, LIU Jichao, ZHANG Menglei, NIU Yidong

2022, 39(3):  63-66.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 012

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Objective To assess the euthanasia method used for rabbits in domestic animal experiments, and to provide references and basis administrative authorities of laboratory animal management to understand the implementation of euthanasia method in China, to formulate corresponding policies and implement management. Meanwhile, to strengthen the review of relevant academic journals in euthanasia method for effective implementation of animal welfare in China. Method the CNKI database was retrieved from 2017 to 2021 for literatures containing the searching terms “ rabbit” “ rabbit” and “ execution” “ euthanasia” , in the text, and the excel program was used to perform statistical analysis. Result Among the 367 literatures investigated, 315 literatures ( 85. 83%) did not clearly describe the euthanasia method in rabbits. Only 52 literatures ( 14. 17%) clearly proposed the euthanasia method, and air embolization was reported in 32 literatures ( 61. 54%) ; excessive anesthesia was reported in 12 literatures ( 23. 08%) ; cervical dislocation was reported in 6 articles ( 11. 54%) ; carbon dioxide asphyxiation method was used in 1 article (1. 92%) . Among the 52 articles, only 11 articles (21. 15%) described the method of execution corresponding with the method of euthanasia. Conclusion At present, the implementation of "
euthanasia" in rabbits is not optimistic, and the rabbit execution method have been seriously neglected in the article writing, and description of the euthanasia method is not standardized. The animal welfare awareness of Chinese researchers needs to be improved, and the laws and regulations related to " euthanasia " need to be strictly implemented.


Methods and Evaluations of Making Germ-free Rats and Mice Model

LI Jianxiang, WANG Junjun , HUANG Tianma, ZHANG Zijian , GUO Weifeng

2022, 39(3):  67-69.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 013

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The successful breeding technology of germ-free animals, especially germ-free rats and mice, has become the key technology to study the relationship between microorganisms and human life phenomena. Strict germ-free animal means that no living body can be detected in any part of the host and living environment. In a broad sense, germ-free animals also include animals that clear most of the intestinal flora, also known as pseudo germ-free animals. At present, the method of making germ-free rat or mice mainly include the method of caesarean section and aseptic feeding, and the method of antibiotic cocktail. In this paper, the method of making germ-free rats and
mice model were summarized and evaluated.


Research Progress on Inhibition of Heart Transplantation Rejection in Mice

GUO Lei , TANG Yixin, WEI Ran , XIN Benkai, ZHU Cuilin , ZHANG Jizhou

2022, 39(3):  70-73.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 014

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Heart transplantation is the only way to treat end-stage heart failure, which requires long-term immunosuppressive therapy, but cannot effectively prevent the occurrence of rejection after transplantation. Heart transplantation model in mice is an important method to study the immunology of transplantation. Immunosuppressive therapy after heart transplantation in mice can prolong cardiac allograft survival, but at the same time, it is found that the use of immunosuppressive therapy has serious side effects. In this article, we will review the different immunosuppressive treatments after heart transplantation in mice.


Research Progress on the Effect of Sodium Butyrate on Apoptosis and Laboratory Animals

WANG Haoran, LIAO Yi, YAO Jiao, ZHAO Deming, ZHOU Xiangmei

2022, 39(3):  74-78.  DOI: 10. 3969 / j. issn. 1006-6179. 2022. 03. 015

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Sodium butyrate is processed into sodium salt form of butyric acid. Sodium butyrate has the effects of anti-inflammation, improving immunity, stabilizing gut microbiota, etc. In this paper, in combination with the research progress at home and abroad in recent years, it focuses on the induction of body cells induced by sodium butyrate. The specific mechanism of apoptosis includes sodium butyrate activating death receptor pathway, mitochondrial pathway and endoplasmic reticulum stress pathway. The reasons why mechanism of sodium butyrate in improving animal model of disease was discussed. Hope to provide new ideas for sodium butyrate in disease prevention and treatment.