Table of Content

28 August 2018, Volume 35 Issue 04

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Written on the 30th Anniversary of the Promulgation and Implementation of the Regulations for the Administration of Laboratory Animals

2018, 35(04):  1. 

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The regulation for the administration of laboratory animals ( herein after referred to as the regulation) is the first administrative regulation for the administration of laboratory animals in China. Over the past 30 years, the regulation has played an important role in standardizing and legalizing the management of laboratory animals, guaranteeing the quality of laboratory animals and animal experiments, promoting the development of science and technology in China and guaranteeing the livelihood of the people. However, with the deepening of scientific and technological progress, social and economic development and legislative work, many new situations and problems have emerged in the scientific research and management of laboratory animals. The role of regulation in the management of laboratory animals cannot fully meet the needs of scientific and technological innovation, economic and social development. Therefore, it is of great significance to standardize the management of laboratory animals, guarantee the quality and also welfare of laboratory animals, maintain public health and safety, support scientific and technological innovation, and promote international scientific and technological cooperation and exchange.

Optimization of Genetic Testing Technique for MHC Haploid Duck

2018, 35(04):  14. 

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Objective Optimization of genetic testing technique for MHC haploid duck. Method Based on the specific pathogen free (SPF) ducks nurtured and preserved in the National Poultry Laboratory Animal Seed Center and polymorphism of duck double-copy gene (Tap1 and Tap2) of the transporter associated with antigen processing (TAP) located in the core region of major histocompatibility complex (MHC). MHC haplotype SPF ducks were selected by using Tap1 genome short tandem repeat (STR), Tap2 peptide binding region sequence and Tap2 genomic sequence genotyping, respectively. Result The STR result of F0 generation and F1 generation ducks were completely consistent. The sequences of Tap1 and Tap2 of F2 and F3 generation duck were completely homozygous. Six consecutive generation Tap2 genome sequence analysis shows that B3 group of ducks have two mutations, B1, B2 and B4 group of ducks have genetic stability. Conclusion Four MHC haploid SPF ducks B1, B2, B3 and B4 were successfully established, which provided laboratory animal support conditions for further research on immune genetics of ducks.

Generation of Human hRas Geneconditional Knockin Mouse Using The Cre-LoxP Strategy

2018, 35(04):  21. 

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Objective Generation of conditional human Rasgene (c-Ha-ras) knock in mouse model by Cre-LoxP strategy for preclinical drug carcinogenicity safety evaluationand related mechanism studies．Method Targeting vector of hRas conditional knock in was constructed and then transfected into embryonic stem (ES) cells by electroporation. ES cells were screened by positive and negative selection, and targeted cells were confirmed by PCR and Southern blot. The targeted ES cells were microinjected into the blastula of C57BL/6 J mice. Chimerical mice were generated by transplanting the blastula into the host mice.hRasfl/+ heterozygous mice were obtained by crossing chimerical founder mice with C57BL/6 J mice. hRasfl/flhomozygote mice were obtained by crossing between hRasfl/+ heterozygous mice. Conventional expression of hRas was induced by crossing between EIIa-cretransgenic mice and hRasfl/fl mice. Quantitative polymerase chain reaction (QPCR) was performed to detect hRas gene expression level in mice embryo at variety of developmental stages. Result Targeting vector based on Cre-LoxP system was constructedand was confirmedby PCR and restriction enzyme digestion.A total of 12 positive ES clones were confirmed by Southern blot by both of 5′-end probeflank and 3′-end flank probe. Clone A11 was chosen and subjected to microscopic blastocyst injection. Forty eight embryos were transplanted into host mice and 9 offspring were obtained, among which six pups were chimeric. Four hRasfl / + heterozygous mice were generated by crossing chimerical mice (chimeric rate>50%) with C57BL/6 J mice.hRasfl / + were intercrossed and 29 offspring were obtained. Fourteen out of 29 offspring were homozygous. In order to obtain hRas- EIIa- cre mice, hRasfl/fl were crossed with EIIa-cre mice for 6 rounds of mating. However, no alive pups were obtained.Further analysis showed that hRas gene expressed during E10.5—15.5 day in the embryo by quantitative polymerase chain reaction (QPCR). Conclusion hRasfl/flhomozygote mice was obtained successfully though we failed to obtain the hRas- EIIa- cre mice due to embryonic lethal. However, establishment of hRasfl/fl mice model provided foundation for generating conditional knockout mice model

Establishment and Application of the Real-time PCR for Detecting Minute Virus of Mouse (MVM) and Mouse Parvovirus (MPV) Simultaneously in Laboratory Mice

2018, 35(04):  29. 

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Objective To establish and apply the real-time PCR for detecting minute virus of mouse (MVM) and mouse parvovirus (MPV) simultaneously in laboratory mice. Method According to the genome sequences of MVM and MPV from NCBI, primers and probes were designed. The linearity, specificity, and sensitivity of the method have been investigated. 178 mouse faeces DNA were detected by the real-time PCR. Result The real-time PCR showed a linear range of 1×109-1×104copies/μL, high specificity, which showed no cross-reaction with other parvovirus. The sensitivity of real-time PCR was 101copies/μL. The result of 178 clean grade mouse faeces by PCR screening were that two out of them were MPV positive. After the whole genome sequencing and blasting with NCBI (NC_001630.1) sequence, the agreement rate was 96%. Conclusion The real-time PCR method detecting MVM and MPV simultaneously were successfully established, which could be applied to routine testing.

Effects of BuShen HuoXue Particle on Sex Hormone Levels, Blood Sugar and Insulin of PCOS Rats

2018, 35(04):  33. 

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Abstract: Objective To observe the effects of BuShen HuoXue Particle on sex hormone levels , blood sugar and insulin of PCOS rats. Method We established the model of PCOS rats using subcutaneous injection of DHEA mode method .Seventy-two Rats were randomly divided into 5 groups: normal control group , model control group , Metformin group, bushen HuoXue of high dose group , middle dose group and 10W dose group of herbs , 12 rats each group. Rats in all groups were treated with corresponding drugs by gastrogavage for 20 days. Determination of 17 - hydroxy serum progesterone ( 17 - OHP) , detected luteinizing hormone ( LH ) , testosterone, follicle stimulating hormone ( FSH ) , fasting plasma glucose( FPG ) and insulin ( INS ) were detected by adopting ELISA method . Result Model group serum 17 -OHP , FSH , LH , T , FPG , INS levels are significantly higher than the normal control group ( P<0. 01 , P<0. 05 ) , Traditional Chinese medicine ( TCM ) in high dose group of lower rat serum LH , T, FSH and INS level is most obvious ( P<0. 01 ) , Metformin group and the Chinese medicine dose group significantly lower FPG ( P t0.01 ) , Chinese medicine in each treatment group can reduce17 -OHP ( P<0.05 ) . Conclusion BuShen HuoXue Particle could effectively improve endocrine levels of PCOS rats, improve the abnormal level, blood glucose and insulin.

The Vascular Tone in Establishing Rat Model of Embolic Middle Cerebral Artery Occlusion

2018, 35(04):  37. 

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Objective To improve the method of establishing embolic middle cerebral occlusion ( eMCAO ) rat model. Method Twenty Sprague-Dawley rats were used to establish the rat eMCAO model , the modified PE tube was fixed and inserted by using the vascular tone. Result The modified PE tube was all inserted, the average time of making the eMCAO model was 30 min , 90 % of the rats was successfully made of eMCAO. Conclusion The vascular tone can be used in the model making of rat eMCAO with reducing the time and labor cost.

The Efficacy of Pseudomonas Aeruginosa-mannose Sensitive Hemagglutinin on H22 Mice Transplanted Tumor

2018, 35(04):  40. 

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Objective The efficacy of pseudomonas aeruginosa-mannose sensitive hemagglutinin on H22 mice transplanted tumor．Method H22 cells were implanted into the subcutaneously of 53 BALB/c mice. Forty eight mice were randomly divided into 6 groups. And set up Control group, Pseudomonas aeruginosa group, Combined cisplatin group, Combined adriamycin group, cisplatin group, adriamycin group, 8 mice/group. Continuous administration for 14 days. Result The tumor inhibition rate of combined cisplatin group and cisplatin group were 49.2% and 46.6%, and the tumor inhibition rate of adriamycin group and adriamycin group was about 32.1% and 19.8%．Conclusion Pseudomonas Aeruginosa Injection can enhance the effect of adriamycin chemotherapy on H22 mice．

Effect of Anshen Umbilical Paste on Autonomic Activity and Serum Levels of GABA and NE in Experimental Insomnia Rats

2018, 35(04):  45. 

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Objective To explore the effect of Anshen umbilical paste on autonomic activity and serum levels of gamma aminobutyric acid (GABA) and noradrenaline (NE) in experimental insomnia rats, in order to reveal its mechanism. Method The model of insomnia rats were established by 4-Chloro-DL-phenylalanine (PCPA) injected intraperitoneally. Forty rats were randomly divided into blank control group, model control group, Anshen umbilicus plaster (2.70 g/kg) group and diazepam (0.90×10-3g/kg) group. The general condition and autonomic activity of the rats were observed, and the contents of serum GABA and NE were detected by enzyme linked immunosorbent assay (ELISA). Result Compared with the model control group, the body weight of the rats in Anshen umbilicus plaster group and diazepam group were significantly increased (P<0.05), and the distance and speed of the movement were significantly decreased (P<0.05 or P<0.01), and the frequency and residence time in the central area also declined. After the treatment with Anshen umbilical paste and diazepam, the content of GABA in serum of rats were increased significantly (P<0.01), and the content of NE were decreased significantly (P<0.01). Conclusion Anshen umbilical paste could improve the general condition of insomnia rats and reduce the autonomic activity. The mechanism of promoting sleep may be related to the increase of GABA secretion and the decrease of NE release.

Construction and Application of Multiple PCR Detection for Four Pathogenic Bacteria in SPF Mice

2018, 35(04):  49. 

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Objective To establish a protocol for simultaneous detection of four common pathogens in SPF environment. Method A multiplex PCR assay was established with primers designed according to invA gene of Salmonella typhimurium, khe gene of Klebsiella pneumoniae, 16S rRNA gene of pasteurella pneumotropica and ecfX gene of Pseudomonas aeruginosa. The specificity and sensitivity of the new multiplex PCR system were also evaluated after optimization. Result The multiplex PCR method could simultaneously amplify the four bacteria in the same sample without cross reaction, and the minimum detection concentration was 10-3ng/μL. The multiplex PCR method could also specifically detect the target pathogens from co-infection disease material. Conclusion The multiplex PCR method , which was rapid, convenient and sensitivity, could provide effective technical for the rapid diagnosis and monitoring of these four pathogens.

Establishment and Application of Real time PCR Method for Bartonella Detection in Experimental Cat

2018, 35(04):  56. 

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Objective To establish an effective and specific real-time PCR assay for Bartonella detection, and use the assay in experimental cat. Method According Bartonella RIBC gene fragments, a pair of specific primers and TaqMan probe were designed and synthesized. Bartonella DNA standards were prepared using some molecular biological method . Then the linearity, sensitivity, specificity and stability of the method were measured. At last, the method was used for testing blood samples from 142 cats. Result We established successfully real-time PCR method for Bartonella detection. The linear range was 1.0×101～1.0×109 copies/μL, the lowest sensitivity was 10 copies/μL, the coefficient of variation (CV) was 0.998. There was no false positive detection from other bacterial strains. 6 positive reactions were detected in the blood samples of 142 cats. Conclusion The method is able to be used to detect and quantify the Bartonella in experimental cats.

Cognitive Function of the ADAM10 Gene Knockout in the Brain of Adult Mice

2018, 35(04):  61. 

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Objective To investigate the effect of deletion of specific ADAM10 gene in brain cells of adult mice on behavior in mice. Method Y maze, elevated plus maze and open field test were used to understand the effect of ADAM10 gene knockout on locomotor activity and inquiry learning ability in mice. Result Behavioral experiments showed that ADAM10 gene knockout mice had decreased autonomic activity compared with the control group, the frequency, time and distance were significantly lower than the control group in the new environment. It indicated that the deletion of ADAM10 gene had certain influence on the autonomic activity and inquiry learning ability of adult mice.

Establishment of a Carbon Dioxide Euthanasia Method Based on Mice Individually Ventilated Cage

2018, 35(04):  67. 

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Objective To establish a carbon dioxide euthanasia method based on mice individually ventilated cage. Method Four to five weeks old BALB/c mice, C57BL/6 mice and 2 weeks old C57BL / 6 mice were respectively killed in their individually ventilated cages with carbon dioxide (the fill rate was 28% chamber volume per minute). Result Four-five weeks old BALB/c mice and C57BL/6 mice lost consciousness whithin 2-3 min and died whithin 5-6 min, which meets the requirement of NIH guidelines. Two weeks old C57BL/6 mice took 6-8 minutes before death, which has significant difference comparing with 4-5 weeks old C57BL/6 mice. Conclusion successfully established a carbon dioxide euthanasia method based on mice individually ventilated cage. This method effectively reduces the stress reaction in mice, and contributes to physical and mental health of operators.

The Comparison of the Methods of Oral Trachea Intubation under Direct Vision after Induction with IsofluraneInhalation in Rats

2018, 35(04):  71. 

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Objective To evaluate the effective method of oral trachea intubation with the isoflurane induction in rats. Method Forty rats were randomly divided into the improved group and the conventional group, a comparison of the success rate of first intubation, intubation frequency, intubation times. Result The time and frequency of intubation were significantly less in the improved group than those in the conventional group. The success rate of the first intubation in the improved group was obviously higher than that of the conventional group (95% vs 50%，P<0. 05).Conclusion The improved method of Oral Trachea Intubation under direct vision is superior to the conventional method , This technology is worthy to be widely used.

The Investigation and Analysis of the Cognition about Laboratory Animal Welfare for Medical Workers

2018, 35(04):  74. 

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The Investigation and Analysis of the Cognition about Laboratory Animal Welfare for Medical Workers

Application of DNA Barcoding in Farm Animals

2018, 35(04):  79. 

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In recent years,DNA barcoding is developing very rapidly as a molecular marker,but its application in farm animals are still few reports.This paper reviews the prospects and limitations of DNA barcoding in farm animals,through the development,principle and technical process of DNA barcoding and its application in maet.It provides a new thought and methed for the identification of the farm animals.

esearch Progress on the Establishment of Animal Model of Asthma

2018, 35(04):  83. 

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By reviewing and sorting out the experimental research on animal models of bronchial asthma in the past ten years, the latest progress in the establishment and evaluation of animal models of asthma is summarized. It is found that the allergic asthma model is still the most classic and widely replicated model type, and the neutrophils based on the phenotype and heterogeneity of asthma. The replication of cellular asthma and refractory asthma has also made some progress.