Laboratory Animal Science

Establishment of SyncytinA Conditional Knockout Mouse by CRISPR-Cas9

2015, 32(05): 1-6.

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Abstract: Objective To establish syncytinA conditional knock out mice to investigate the function of syncytinA in the placental development and preeclampsia pathology． Method After successful incorporation of loxp synA exon by homologous recombination in ES cells，we used CRISPR-Cas9 technology to construct the syncytinA-loxp mice．We construct syncytinA-loxp transgenic vector and sgRNA，they were injected with Cas9 into fertilized eggs of C57 by prokaryotic microinjection methods，and these eggs were transplanted into pseudopregnant mice． The genotype of transgenic mice was identified by PCR． Then loxp positive mice were mated with WT ( wild type) to get syncytinAloxp mice． In order to detect whether syncytinA knockout． SyncytinA－ / － was obtained by mating with prime1cre and zp3cre． SyncytinA － / － was identified by PCR and q-PCR． Conclusion SyncytinA conditions knockout mouse was successfully constructed in present study．

Development of Splints Closed Fracture Model in Rats Tibia

2015, 32(05): 7-10.

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Abstract: Objective By establishing a standard rat model of closed tibial fracture，to explore the physiological fracture healing mechanisms to provide experimental basis． Method 14 male 8 weeks SD rats received midshaft tibial closed fracture induced by self-designed fracture model stent，and self-made splint for restoration and fixation，the fracture types and healing were observed by X-ray at 0，7， 14， 21， 28 days after surgery． Result X-ray shows13 for transverse or oblique fracture，1 for comminuted fracture，and is typical of the secondary healing process．Conclusion The closed tibial fracture model which induced by a self-designed fracture modeling stent and splints for external fixation can be obtained easily，avoiding or reducing the effects of hard or soft tissues damage on fracture artificially，according with the requirement of standard closed fracture model．

Establishment and Application of a PCR Method for Detection of Murine Cytomegalovirus

2015, 32(05): 11-14.

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Abstract: To establish a PCR assay for detection of murine cytomegalovirus ( MCMV) ． Provide help for the purification of MCMV． Sequence of MCMV was obtained from NCBI Genbank． One pairs of primers were designed based on this sequence． The primers was determined through amplifying the major strains in China． Eighty-seven mice serum were tested with this PCR assay． The positive amplified fragments were sequenced to verify the reliability of this method． A PCR method for detection of MCMV is successfully established，with a high specificity and sensitivity． Among the tested 87 blood samples， 13 positive cases were detected． Sequencing of the samples are consistent with the amplification results，and verified the applicability of this detection method． The establishment of this method provides the basis for detection of MCMV in mice，and application of this assay in mouse．

Measurement of Some Immunological Parameters in GFP Transgenic Nude Mice

2015, 32(05): 15-17.

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Abstract: Objective To measurement some immunological parameters of transgenic nude mice with Green Fluorescent Protein． Analyze the advantages and disadvantages of GFP transgenic nude mice and other mice． To provide basis for future research． Method ①6 ～ 8 weeks mice in each，were used in this experiment． Live weigh，anatomy and take the spleen weighing． The proliferation ability of spleen lymphocyte was determined． ②Blood samples were measured using an automatic analyzer and ELISA． Result ①The body weight ，spleen index，T and B lymphocyte proliferative response of GFP transgenic nude mice were similar to BALB /c nude mice． But there was a significant difference between the two and BALB /c mice． ②The levels of leukocytesl ymphocytes in the peripheral blood of two nude mice，and the proportion of IgG and IgM were much lower than that of BALB /c mice． Conclusion The immune system of GFP transgenic nude mice and BALB /c nude mice were weaker than that of BALB /c mice，and the level of immune system in GFP transgenic mice was the lowest．

Establishment and Preliminary Application of the Real-time PCR for Detecting Minute Virus of Mice ( MVM)

2015, 32(05): 18-21.

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Abstract: Objective To establish and preliminarily apply the real-time PCR for detecting minute virus of mice ( MVM) ． Method According to the genome sequences of MVM( NC_001510) from NCBI，one pairs of primers and probes were designed． The linearity，specificity，and sensitivity of the method were investigated． 178 mouse faeces DNA were detected by the real-time PCR． Result The real-time PCR for detecting MVM showed a linear range of 1x109 ～ 1x104copies /μL，high specificity，which showed no cross-reaction with rat parvovirus H1，KRV and porcine parvovirus． The sensitivity of the real-time PCR was 101copies /μL． The real-time PCR detecting results of 178 clean grade mouse faeces were that all of them were MVM negative． Conclusion The real-time PCR method is good in specificity，sensitivity，which can make a powerful technical support for MVM epidemiological investigation and detection．

Preliminary Discussion on H-1 Parvovirus Infection Mechanism in Rat Glioma Cells C6

2015, 32(05): 22-25.

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Abstract: Objective To investigate the changes of cytokine levels after the infection of rat glioma cells C6 by H-1 parvovirus． Method C6 cells were taken after infection H-1 parvovirus at different time points，while the normal C6 cells were taken in these time points，and the ELISA． Methods were used to detect the expression levels of TNFα，IL-6，TGF-β1 and IL-17A． Result The concentrations of TNF-α，TGF-β1 and IL-17A in C6 cells were increased during culture，and which of IL-6 was no significant change; The concentrations of TNF-α，TGF-β1 and IL-17A in H-1 parvovirus infected C6 cells were no significant change，while IL-6 concentration was significantly increased over time． Conclusion H-1 parvovirus infection leads to changes in C6 cytokine expression levels．

Designation and Activity Verification of High Efficiency sgRNAs for Hipp11 Locus

2015, 32(05): 26-30.

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Abstract: Objective Designed and verified sgRNAs for Hipp11 locus，providing working foundation for gene knock-in in Hipp11 locus． Method Predicted sgRNAs for Hipp11 locus by software． Two sgRNAs with lowest offtarget effect were selected for vector construction． CRISPR/Cas9 activity kit was used to test relative activity of sgRNAs in vitro． sgRNA with relative activity of 51. 7% was subjected to in vitro transcription and was microinjected with Cas9 mRNA into zygotes． The cleavage activity of new born mice were tested to obtain sgRNA activity in vivo． Result The activity of two sgRNAs was 19. 2% and 51. 7% respectively in vitro． 2# sgRNA with higher in vitro activity was used from microinjection． Eight mice were born，among which 62. 5% ( 5 /8) of the cleavage sites had base pairs insertion /deletion． Conclusion A sgRNA with high activity targeted Hipp11 locus was obtained，providing working foundation for gene knock-in by CRISPR/Cas9 technique in this locus． The activity testing in vitro can efficiently predict the result in vivo． So verification activity of sgRNA in vitro was an efficient method for sgRNA screening．

Forest Encephalitis Inactivated Vaccine Rabbits and Guinea Pigs systemic Allergic Muscle Stimulation Test

2015, 32(05): 31-34.

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Abstract: Objective The aim of the study is to compare the muscle stimulationand systemic allergic reactions of encephalitis inactivated vaccine before and after changing production sites in rabbits and guinea pigs and evaluate the safety of production． Method The experiments used 3 batches encephalitis inactivated vaccine produced in both production sites． Their muscle stimulation was tested in 12 rabbits which were randomly divided into two groups with equal number of male and female． Using adjuvant as control group and the Mori brain vaccine as experimental group，in the same body adjuvant and Mori brainvaccine were injected into left and right quadriceps intramuscularly． Three days after the animals were killed and necropsied， gross and routine pathological examinations were performed． Systemic allergy test was performed in six guinea pigs ( 3 animals in each group) ， and each was given 3 times intraperitoneal injection of 0. 5 mL sample，every the other day，and the behavior and characteristics of each guinea pig were recorded． At the 14th and 21st days，1 mL sample was injected intravenously． Result The injections of Mori brain vaccine and adjuvant hadthe similar muscle irritation and production sites change did not significantly alter the pathological appearance in local muscle tissue． Conclusion Mori brain vaccine after changing production site is safe in the animal experiments．

Chracteristics of Mineral and Bone Disorder in 5 /6 Nephrectomy Mouse Model

2015, 32(05): 35-39.

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Abstract: Objective We produced the 5 /6 nephrectomy ( 5 /6 Nx) mice model of chronic kidney disease ( CKD) in order to observe FGF23，calcium phosphorus metabolism，and contribute to study the pathogenesis of CKDMBD．Method 28 C57 male mice were adapted for 1 week． Then the model group underwent unilateral renal ablation by 5 /6 Nx． Upper and low poles of the left kidney were cut． The right kidney was removed 1 week later．All the mice were executed at the 16th week． Blood，urine and kidney samples were collected． Then we detected FGF23，PTH，1，25( OH) 2D3，calcium，phosphorus and observed the renal histopathology． Result Comparedwith the sham group and the control group，the model group lost weight，had higher serum FGF23，PTH， phosphorus，urine output，urine protein and less 1， 25( OH) 2D3． Histopathology results found the kidney after 5 /6 Nx had proliferative mesangial matrix，increased collagen deposition，fibrosis and compensatory hypertrophy．Conclusion 5 /6 Nx mice had obvious characteristics of CKD-MBD，and is a good CKD-MBD model．

Ten-Years Review: Development of Laboratory Animal Science Discipline in Capital Medical University

2015, 32(05): 40-42.

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Abstract: Mainly based on the constructing progress of laboratory animal science discipline in Capital Medical University，we analyzed the necessity and superiority of establishing a laboratory animal science department in a medical university or school． We also displayed the contents and achievements of laboratory animal science department of Capital Medical University． These experiences would be valuable for development of laboratory animal science discipline in medical university．

The Effects of Three Common Anaethetic Drugs on Mongolian gerbils

2015, 32(05): 43-46.

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Abstract: Objective To observe and analyze the effects of three kinds of common anaethetic drugs． Method The adult Mongolian gerbils were divided into three groups and injected with the physiological saline solution of chloral hydrate，urethane，ornembutal by intraperitoneal injection，respectively． Then induction time，duration and recovery time of anesthesia，and heart rates and respiratory changes after anesthesia were measured． The same experiments were also carried out in rats and mice． Result The duration of chloral hydrate is the longest，and that of urethane is the shortest． Chloral hydrate inhibited respiratory and heart rates less than urethane and nembutal．Relatively to urethane，the anaethetic duration of the other two drugs in gerbils were longer than that in rats and mice． Conclusion The anaethetic effects of three kinds of drugs are obviously different in gerbils． The influence in heart rates and respiratory changes is also different． We should choose these drugs according to the requirement of experiments． Compared to rats and mice，Mongolian gerbils are more sensitive to these drugs．

Emergency Drill of Significant Laboratory Animal Epidemic Disease

2015, 32(05): 47-50.

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Abstract: The biological safety of laboratory animals is not only the major premise of the development of experimental animals，but also the bottom line for the management of laboratory animal science． After decades of development，the laboratory animal industry in China has made great progress，however，the bio-safety disposal system of experimental animals is not perfected especially in the face of emergency major laboratory animal disease， and the long-term mechanism of emergency management is in absence． In order to strengthen our country＇s sudden major laboratory animal epidemic emergency disposal system，Beijing administration office of laboratory animal organized an emergency drill of significant laboratory animal epidemic disease on November 6，2015． This drill proved that the composed draft of emergency drill of significant laboratory animal epidemic disease worked effectively，showing that the draft is rigorous and scientific． On the other hand，the drill reflects that the laboratory animal epidemic emergency disposal system in China is still to be proved．

Tests for the Hemolysis Test of Medical Infusion Set Injector and Halogenated Butyl Rubber Stopper

2015, 32(05): 51-54.

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Abstract: Objective To test the hemolysis of the halogenated butyl rubber stopper for injection，medical injectors and medical infusion sets producing，operating or using in Hebei，in order to evaluate their biological safety．Method According to the national standard，using static in vitro direct contact hemolysis，by the experimental animal rabbit heart blood，the hemolysis ratios of infusion sets for single use，sterile injector for single use and halogenated butyl rubber stopper with injection of 10 batches of sample were tested respectively．，and analyzed and compared in vitro hemolysis degree of the three categories of products． Result The hemolysis rates of the tested samples ＜ 5%． Conclusion Hemolysis of the tested samples is all in line with the provisions of relevant product standards，show that the three kinds of products have definite biological safety in hemolysis．

Research Progress and Thoughts of the Suture-Occluded Method for Cerebral Ischemia-Reperfusion in Rats

2015, 32(05): 55-58.

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Abstract: The application of the suture-occluded method for cerebral ischemia-reperfusion in rats is widely used， but there are many factors that affect the success rate． The cerebral ischemia animal models are very important in the course of the study of ischemic cerebrovascular disease，and at present the method has been extensively used for studying ischemia-reperfusion injury． The choice of rats，the use of narcotic drugs，the use of suture and operation method is the key factors in the process of model making． In this paper，the method and characteristics of the model of local cerebral ischemia in rats by suture-occluded method are summarized and introduced．

The Advantages and Disadvantages of Human PDX Transplantation Model

2015, 32(05): 59-62.

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Abstract: The basic features and application in biomedical research of PDX models ( patient-derived tumor xenograft ) are introduced． We compare the advantages of this model applied to cancer research and the existing problems． The purpose is to use PDTX models to carry out the research work and analyse experimental results correctly，which can speed up the process of cancer drug development．

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