Laboratory Animal Science

Establishing Technology of in-situ PCR Genotype Identification in Single-celled Levela

ZHU Xuan, SHAO Zhu-Xiong, ZHU Bao-Chang

2012, 29(01): 1-5.

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Abstract: Purpose Spermatogenesis is a fine control process with mitosis and meiosis， which is from spermatogonial stem cells began in male seminiferous tubule． By the phenomenon proportion of made to female in mammals，which is 1 ∶ 1，we can infer that the fertility rate of sperm with X gene and Y gene should be 1 ∶ 1． ·4· 第1 期朱璇等: 在单细胞水平建立原位PCR 基因鉴别技术平台———以精子基因型鉴别为例 However，after differentiation，X and Y sperm for 1 ∶ 1 ratio is not known through spermatogenesis． Not all spermatocytes can develop into sperm during spermatogenesis eventually． However，to study the male and female birth ratio need to first study that if the spermatogenesis is strictly in accordance with 1∶ 1． Therefore，the present study，using a rapid and reliable platform of In-situ PCR， can further support the studies on regulation in spermatogenesis． Step Using HSL gene exploration steps，we can obtain an optimal fluorescence in-situ PCR experiment scheme，including proteinase K effect time and concentration，sperm depolymerization method and degree，etc，then applies to SRY gene． Result By Nikon fluorescence microscopy，we can clearly see the sperm head with fluorescent signals． Sperm head with green fluorescent signals target to SRY gene is the Y chromosome sperm ( Y sperm) ． Our lab tested 493 Kunming mice sperm altogether and there were total 273 signals． X sperm ∶ Y sperm = 55. 37% ． The experimental results approached to draw after the chi-square test． Conclusion We can use in-situ PCR technology to quickly appraisal the sperm“gender”in single-celled level，which is also suitable to detect any kind of gene in whatever the cell type is．

Senescence Accelerated Mouse SAM-P /8 Appears Syndrome Similar to Parkinsons Disease

LIU Yun-He, MIAO Zhen-Hua, LI Zhen, CHENG Hai-Yan, QU You, LIU A-Qing, XU Wen-Bin, WEN Ming-Fei, SHANG Lei, CHEN Lu, HAN Jing-Xian, YU Jian-Chun

2012, 29(01): 6-8.

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Abstract: Objective To explore the relativity between the rotational behavior of Parkinsons disease mice model and the spontaneous rotational behavior to the same direction in senescence accelerated mouse SAM-P /8． MethodsThe mice were divided into 2 groups : 1． Rotation group includes 6 Male SAM-P /8 at 7-month old that appear rotational behaviors2． Normal control group includes 6 Male SAM-R /1 at 7-month ． First applies “the scores of aging”to study the aging symptoms; then evaluate their characteristics of behavior of rotation to the same direction by PD mice model standard． Results Compared with SAM-R /1 control group，SAM-P /8 group appeared obvious aging sign and symptoms at 7 months old; SAM-P /8 appear steady rotational behavior to the same direction，the frequency is 60 rotations per minute． This kind of rotation behavior coincides with PD mice model standard． Conclusions Senescence accelerated mouse SAM-P /8 appears syndrome similar to PD． This discovery will make sense to the research of PD．

Study on Primer Selection and Tm Optimization for MicrosatelliteDNA Polymorphism of Inbred Mousea

LIU Xian-Ju, WANG Yan-Rong, GAO Zi-Qi, DAI Xiao-Wei, TONG Wei, ZHANG Li-Fang, LIU Yun-Bo

2012, 29(01): 9-13.

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Abstract: Objective To find the method of genetic monitoring on mouse microsatellite DNA polymorphisms by select the microsatellite DNA polymorphism primer and optimize the Tm for the inbred mouse strains． Methods Analysis of microsatellite DNA polymorphisms and genetic monitoring of seven inbred mouse strains including C57BL /6、C3H、BALB / c、DBA /2、129、FVB and SCID were investigated by polymerism chain reaction( PCR) with 34 pairs of microsatellite primers loci on different chromosomes of mouse． Results 32 pairs of microsatellite primers could be amplified stably，of which 9pairs of primers display monomorphic among the various inbred strain， 25 pairs of primers display polymorphisms among the different strains，8 pairs of primers had more significant polymorphism． Conclusion Microsatellite DNA displays significant polymorphism，which could be used for various inbred strains identification and genetic monitoring．

Comparison of Donor-recipient in BALB / c and C57BL /6 Mice on Acute Cardiac Allograft Rejection Model

FAN Ping, ZHUO Fei-Ya-·Ke-Yi, MU Li-Tao, HOU Yue-Mei

2012, 29(01): 14-17.

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Abstract: Objective To establish and compare of donor-recipient in BALB / c and C57BL /6 mice on acute cardiac allograft rejection model． Methods Making BALB / c and C57BL /6 mice with donor-recipient on cardiac transplantation model and assessment of the immune rejection situation and complications，the survival time was observed and cardiac allograft was harvested for histopathological evaluation． Result Success rate was 94. 7% ． A group ( BALB / c → C57BL / 6) and B group ( C57BL /6 → BALB / c) have no significant difference in survival time． Two groups have typical pathological changes of acute rejection，and have no significant statistical difference． Two groups of heart function after heart transplantation also have no significant difference． Conclusion Inbred BALB / c and C57BL /6 mice have genetically stable characteristic，different donor -recipient combinations between BALB / c and C57BL /6 inbred mice are suitable for acute rejection model． C57BL / 6 as recipient are rational choice because the transplantation time was significantly shorter and the higher the success rate in model．

Construct Eukaryotic Expression Vector and Study the Function of BMP7 Gene

CHENG Jin-Jin, REN Ping-Ping, GUO Chun-Lei

2012, 29(01): 18-21.

Asbtract ( 1947 )

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Abstract: Objective To clone BMP7 gene and to construct eukaryotic expression vector． And observe the changes of cell proliferation of the H22 transfected of BMP7 gene． Methods Use RT-PCR technique successfully amplified BMP7 gene 1293bp cDNA sequence coding region from 293T cells total RNA and in the upstream and downstream of BMP7 gene adding BamHI and Hind Ⅲ restriction sites，then connected with pEGFP-C1 expression plasmid which contains BamH I and Hind Ⅲ restriction enzyme digestion sites． The transfection of BMP7 gene into HL-7702 cells was done by liposome-mediated method． RT-PCR was used to detect the expression of mRNA，and western-blot was used to detect protein expression levels of BMP7． We injected H22 which has transfected of BMP7 in mice subcutaneous． Results Sequencing analysis revealed that the orientation of the ligations and the reading frame were correct． Digested by BamH I and Hind III，two fragments of 4. 7kb and 1. 3 kb respectively were formed in eukaryotic expressing vector． Electrophoretic results were completely coincident with theoretical calculation． After transfected，the tumor volume increased significantly． Conclusion BMP7 were successfully cloned and eukaryotic expressing vector were successfully constructed． Over expression of BMP7 gene maybe is one of the reasons of carcinogenesis

Breeding Reproducing and Identifying for p53 Gene Knockout Mice

QIAO Lu-Xin, XU Meng, CHAI Meng-Yin, QIAO Xin, CHEN De-Xi

2012, 29(01): 22-24.

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Abstract: Objective To breed and identify p53 gene knockout mice，Heterozygote mice were bred and reproduced． Methods Genome DNA extracted from the mice’s tails were subjected to PCR test for genotype identification． Results Heterozygous were used to acquire baby mice for Protection species． Conclusion The breeding and reproducing were successful and Homozygous genotype mice were acquired． Appropriate methods of feeding，breeding and identifying are important for obtaining gene knockout mice and protecting species．

A Model of Controllable Small Intestinal Obstruction Using New Zealand Rabbit

TIAN Qing-He, YUAN Guo-Hong, WANG Zhong-Kai, BAI Yang, FU Wei

2012, 29(01): 25-29.

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Abstract: Objective To establish a stable rabbit model of controllable small bowel obstruction，the experiment can present good time-dependent relation and unification between the intestinal incarceration time and the degree of intestinal necrosis and recovery． Methods 52 adult healthy male New Zealand rabbits weighing 2. 5 ～ 3. 0 kg were divided into control group ( 4 rabbits ) and model group ( 48 rabbits ) randomly． The model group imitated controllable small bowel obstruction by making incarcerated abdominal wall hernia，which was that a midline fascial defect was made，the length of the defect was set by the 100 percent of the diameter of incarceration 10 cm- length bowel． By different intestinal incarceration and recovery time，the model group was divided into 8 groups randomly， including incarceration 2 h、4 h、6 h、8 h groups and releasing obstruction from the time point of irreversible intestinaldamage 0 h、24 h、48 h、72 h groups． Using the sham operation group as the control group，the grade of intestinal injury among the groups was compared by necrosis degrees，and the statistic method was Ridit analysis． Results Comparing with the control group，t he different degrees of intestinal necrosis were found in model group． The mean Ridit value of control group was 0. 071; the mean Ridit value of each strangulated obstruction group from 2 h to 8 h was 0. 327、0. 545、0. 735、0. 890 respectively． Using LSD test of variance analysis，microscopic intestinal injury in each obstruction group was significantly different from the control group ( P ＜ 0. 05) ; the significantly difference of intestinal injury was found between the obstruction groups ( P ＜ 0. 05 ) ; Besides，the mean Ridit value of each releasing obstruction from the time point of irreversible intestinal damage( 6h after obstruction) group from 0 h to 72 h was 0. 774、0. 595、0. 443、0. 222 respectively． Using LSD test of variance analysis，the significantly difference of intestinal injury was found between the releasing obstruction groups ( P ＜ 0. 05 ) ． Conclusion This model of rabbit could present the process of controllable intestinal obstruction typically and show a time-dependent recovery degree when the block was released on time．

Sterility Testing of Animal Drinking Water for Beijing Experimental Animal Housing and Identification of Pseudomonas aeruginosa Strains in Water

XING Jin, FENG Yu-Fang, GAO Zheng-Qin, YUE Bing-Fei, LI Gen-Ping, HE Zheng-Ming

2012, 29(01): 30-33.

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Abstract: Objective To grasp the microbial control of animal drinking water in laboratory animal facilities of Beijing． Methods According to Method for Examination of Environment and Faeces of GF animal，the drinking water samples of the animal housing were isolated and cultured． 16SrRNA of Pseudomonas aeruginosa ( PA) isolates that were amplified，and sequenced，then draw phylogenetic tree． Results In the 27 participates of this testing， sterile rate of the pre-treated water is 41% ，and the treated water is only 29% in which PA positive is 30% ，mainly RHH13 and ZAQ22 two strains． Conclusion The detection of experimental animal’s drinking water treatment effects is in general less than ideal． PA contamination will directly influence on the quality of laboratory animals．

The Effects of Bedding Materials on Body Weight，Blood Biochemical Parameters， Organ Weights and Coefficients of Mice and Rats

WANG Yan-Rong, SUN Shu-Hua, YANG Xu, MENG Jin-Ping, LIU Yun-Bo

2012, 29(01): 34-39.

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Abstract: Objective To investigate the effects of bedding materials from poplar，corn and pine on the body weight，the blood biochemical parameters，the organ weights and coefficients of mice and rats． Methods The 60KM mice were divided into three groups randomly，20 in every group，half male and half female，used the bedding materials from poplar，corn and pine respectively． The 60 Wistar rats were divided into three groups randomly，20 in every group，half male and half female，using bedding materials from poplar，corn and pine respectively． The body weight were Weighed weekly，the blood biochemical parameters，including ALT，TP，ALP，LDH，T-SOD，ADH，were measured，a nd the organ weight were weighed after 4 weeks，then organ coefficients were calculated． Results 1. The body weight: Comparing with animals used poplar bedding，the weight of the male mice used pine bedding is reduced，P ＜ 0. 001． Comparing with animals used corn bedding，the weight of the male rats used pine bedding is reduced from third week，P ＜ 0. 05． 2. The blood biochemical parameters: mice: Comparing with the mice used poplar bedding，ALT of male mice used corn bedding is raised very significantly，P ＜ 0. 001; LDH of female mice used pine bedding is reduced significantly，P ＜ 0. 01，ADH of male mice used corn and pine bedding is reduced significantly，P ＜ 0. 001，P ＜ 0. 05． rats: Comparing with the rats used poplar bedding，ADH of male rats used pine bedding is raised significantly，P ＜ 0. 01。3. Organ weights: Mice: comparing with the mice used poplar bedding，the liver and brain weight of male mice used pine bedding is reduced significantly，P ＜ 0. 01，P ＜ 0. 05; the kidney weight of male mice used pine bedding is raised，P ＜ 0. 05，and the adrenal gland weight is reduced，P ＜ 0. 05，and the testiculus weight is raised，P ＜ 0. 05． Rats: comparing with the rats used poplar and pine beddings，the liver weights of rats used corn beddings are raised significantly respectively; P ＜ 0. 001，P ＜ 0. 05; comparing with the rats used poplar beddings，t he kidney and brain weights of rats used pine beddings are raised significantly respectively， P ＜ 0. 01，P ＜ 0. 01; comparing with the rats used corn and poplar beddings，the prostate weights of rats used pine beddings are raised significantly，P ＜ 0. 01． 4. Organ coefficients: Mice: Comparing with the mice used poplar bedding，the liver and brain coefficient of male mice used pine bedding is reduced significantly，P ＜ 0. 01，P ＜ 0. 05，and the kidney and testiculus coefficient of male mice used corn and pine bedding is raised significantly，P ＜ 0. 01． Rats: Comparing with the rats used poplar bedding，t he liver coefficient of male rats used pine bedding are raised significantly，P ＜ 0. 001． Conclusion The poplar material is the ideal beddings for mice and rats，and the corn maybe a backup bedding material，but the pine bedding is prudential usage as mice and rat bedding．

Reform on Veterinary Clinical and Diagnostic Science of Laboratory Animal Science

WU Yan-Hua, RUAN Wen-Ke

2012, 29(01): 42-44.

Asbtract ( 2030 )

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Abstract: Veterinary clinical and diagnostic science of laboratory animal science is a subject about diagnostic methods and theory of laboratory animal science disease． It is the basis and premise of the quality of experimental animals and disease diagnosis． In this article，we introduce the reform of teaching content，teaching methods and examination forms which make students master the basic theory of knowledge and apply the learned knowledge in clinical practice，thus fundamentally improve the teaching effectiveness of veterinary clinical and diagnostic science

Analysis of Current Situation on License of Staff Engaged in Laboratory Animal

HU Juan-Feng, ZENG Lin, ZHAN Da-Wei

2012, 29(01): 45-47.

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Abstract: Laboratory animal sciences in our country has entered into a fast developing period，which depended upon staff professional engaged in laboratory animal． Administration of licensee of staff engaged in Laboratory animal is an important part of work in normalization，and is also an important means for improving competence of staff on laboratory animal，speeding up process of normalization． The aim of this work is to review and analysis of current situation on License of staff engaged in Laboratory Animal and to provide reference to laboratory animal administration．

Application of Fluorescent Microsphere Technology on Laboratory Animal Quality Control

ZHAO Hua-Yang, CHEN Li-Chao, LI Wei, BAI Yu, YIN Liang-Hong, LU Sheng-Ming, WANG Xiao-Ke

2012, 29(01): 57-60.

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Abstract: For the past several decades，ELISA and IFA have been used as the primary assays for viral or bacterial antibody detection． However，these traditional methods are either time-consuming or labor-intensive． Microspherebased suspension array technologies， such as the Luminex xMAP  system， offer a new platform for highthroughput biomolecule detection which has been used in a variety of research fields． Using Luminex  Corporation’s xMAP  technology， the Charles River Laboratories has developed Multiplexed Fluorometric ImmunoAssayTM ( MFIATM ) profiles for sero-surveillance of various laboratory animal species，which has promoted the level of laboratory animal quality control significantly．

Effects of Storage Time on Blood Routine Test of Venous Blood Sample in SD Rats

WANG Xue-Wen, GAO Jun-Feng, HU Jian-Guo, LI Yu-Wei, KANG Ai-Jun, TIAN Feng

2012, 29(01): 63-66.

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Abstract: Objective To investigate the effect of storage time on blood routine test of venous blood sample in SD rats at room temperature． Methods Sixteen SD rats( 200 ～ 220) g were selected ，which female and male was half and half，which were adopted Venous blood ． Venous blood samples were detected at 0 min，30 min，1 h，2 h，4 h， 8 h，24 h and 48 h respectively at room temperature( 18 ～ 25℃) by using MEK-6318K automatic blood cell analyzer．We compared the effect of different time on blood cells and blood platelet in the same venous blood sample． Results The HCT and LYM% at 48 hours were significantly higher than those at 0 min ( P ＜ 0. 05) ． The MCV at 24 hours and 48 hours were very significantly higher than those at 0 minute ( P ＜ 0. 01 ) ． Comparing the RDW and GRN% at 0 minute，the difference was significant ( P ＜ 0. 05) at 24 hours and very significant ( P ＜ 0. 01) at 48 hours． In contrast with 0 minute，the PLT at different time was significantly higher than the one at 0 minute ( P ＜ 0. 01) ． Comparing the PCT at 0 minute，the difference was significant ( P ＜ 0. 05) at 30 min、1 h、2 h and 4 h， very significant ( P ＜ 0. 01) at other times． The difference of MID was significant ( P ＜ 0. 05) at 24 hours and 48 hours compared to the one at 0 minute． Other hematological parameters in different periods showed no significant differences ( P ＞ 0. 05 ) ． Conclusion At room temperature，the blood routine test of venous blood in SD rats should be detected within 30 minutes to 24 hours．

Effect of Kuanmaikang Collocystis on the Animal Model of Pneumatorexis and Myocardial Ischemia

2012, 29(01): 66-68.

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Abstract: Objective To copy the animal model of myocardial anoxia and myocardial ischemia，observe the protect effect of Guanmaikang collocystis on animal model of myocardial anoxia and myocardial ischemia． Methods The rats ranine vein were injected through hypophysin to induce the model of acute ischemic myocardium，and the change of ECG T-wave of all the groups injected Pituitrin ( Pit) were observed at different intervals． Closed container induced oxygen deficient circumstances in normal atmosphere，the ability of Guanmaikang collocystis on mouse to endure pneumatorexis was observed． Results Guanmaikang collocystis can antagonize the change of Twave by Pit，a nd lengthened the survival time of mouse in Pituitrin． Conclusion Guanmaikang collocystis have the effect of anti-myocardial ischemia and the ability of enduring oxygen deficient circumstances in normal atmosphere． kuanmaikang collocystis; myocardial ischemia;

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