Laboratory Animal Science

Established Tyr03，Axl Receptor Tyrosine Kinase Conditional Transgenic Mouse Based on Cre／Loxp System

ZHANG Shu-Jing, JIANG Qi-Hui, LU Qing-Jun, WANG Ning-Li, CHEN Zhen-Wen, LU Qing-Xian

2011, 28(02): 1-05.

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Abstract：Objective Use escherichia coli B·galactasc gene LacZ as report gene，established Tyr03，Axl receptor tyrosine kinases transgenic mouse containing loxp sites，mating with different tissue specific Cre mice to obtain space．time expressions of transgenic mice，make a foundation for research Tyr03 and Axl in each organization functions．Methods Constructing Tyr03，Axl receptor tyrosine kinase plasmid containing loxp sites，sequencing correctly，through microscopic injection technology to insert the Geo-STOP—msAxl and Geo—STOP。msTyr03 gene containing CAG promoter upstream into BDFl fertilized eggs，Transplant to ICR receptor mouse，Stay after breeding， using double primers PCR to appraisal Fo and Fn generation mouse genotype，using RT·PCR，X—gal staining to observe LacZ genes’express in mice organization，using WB to detect the protein expression in transgenic mice and wild type．Result Adopt double primer PCR to appraisal new born mice，get modified positive transgenic mouse， use the RT．PCR conformed the exist of LacZ gene；the x—gal staining，observed blue precipitation in the brain， testicular and the thymus of Geo—STOP—msTyr03。observed blue precipitation in brain，heart，ovarian，kidney of Geo· STOP．msAxl，indirectly illustrate the expression part of aim gene；For identification of double primers positive Geo· STOP．msTyr03。Geo—STOP．msAxl transgenic mice and wild type mice，we use the WB to validate the Axl and Tyr03 gene expressing no difference，and explain the exist of STOP sequence，so the Axl and Tyr03’8 expression is restrained．Conclusions Successful got Geo—STOP．msAxl and Geo·STOP—msTyr03 tyrosine receptor transgenic moUSe．

Reproduction and Genotype Identification of Complement 3 Gene Knockout Mice

ZHENG Jing, LIU Yang, HU Song, LI Min-Hui, LI Li-Mei, YANG Tai, YANG Shu-Xia, ZOU Qiang

2011, 28(02): 7-9.

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Objective This study was to explore the method to breed and identify complement 3 gene knockout mice．Methods The heterozygote of complement 3 gene knockout mice(C3+／一)were bred and reproduced． Wild genotype(C3+／4-)，heterozygote genotype(C3+／一)，and homozygote genotype(C3一／一)would appear in offspring．The genotypes were characterized using ELISA and PCR．Result The heterozygote mice bred 1 35 offspring．38 mice are wild type，68 mice are heterozygote，and 29 mice are homozygote．Byx2text，tlle segregate ratio of offspring was fitted to the Mendelian ratio．Conclusion Appropriate methods for breeding，reproducing，and identifying are the effective way for acquiring complement 3 gene knockout mice from heterozygote．

The Studies on Heterozygote of 8 Biochemical Markers of Inbred Mice

WANG Hong, LIU Shuang-Huan, WANG Shu-Jing, YUE Bing-Fei

2011, 28(02): 10-14.

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Objective To explore heterozygote of 8 biochemical markers of inbred mice in order to monitor correctly and efficiently the genetic quality of inbred mice．Methods Filial generation 1 CCBFl were obmined by intercrossing of CBA／Ca and BALB／c；and filial generation 1 B6CBFl were also obtained by intercrossing of CBA／ Ca and C57BIJ6．Then CCBFI and B6CBFl were monitored by biochemical markers．Results Heterozygote of 8 biochemical markers of inbred mice was obtained．Conclusion Alleles of 8 biochemical markers are co-dominant， and their heterozygote maps can be criterion of monitoring the genetic quality of inbred mice．

Effects of Vitamin E on the Transcription of Cytokines Genes of the Spleen in Mice Treated with Endosuifan

JIA Jing, ZHOU Huan-女, ZHOU Xian-Qing, WANG Ying

2011, 28(02): 15-19.

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To investigate the effects of vitamin E on the transcription of spleen IL-2，IL-6 and IFN一-y genes in mice treated with endosulfan．The treatment was assigned 3 groups．and the level of both endosuIfan and vitamin E were 0 mr,／(kg·d)and 0 mg／(kg·d)(control group)，0．8 ms／(kg·d)and 0 mg／(kg·d)(endosulfan-treatment group)，0．8 mg／(kg·d)and 100 mg／(kg·d)(experimental group)，respectively．Spleens were collected to be analyzed the mRNA of IL-2，IL-6．and IFN-．y genes by Real-Time PCR method．The results showed that the mRNA levels of IL-2 and IL-6 gene in the endosulfan-treatment group were significantly higher than that of control group； although IFN-^r mRNA level in endosulfan-treatment group had an increasing tendency compared with control group，there was no significant difference between those two groups．The mRNA levels of IL-2 and IL-6，in the experimental group were significantly lower than that of endosulfan—treatment group，while the mRNA level of IFN-1 gene had a decreasing tendency in experimental group compared with endosulfan-treatment group，but there was no significant difference between those two groups．The results indicated that endosulfan can induce the gene transcriptions of spleen IL-2 and IL-6，but there is no significant effect on the gene transcription of spleen IFN一1 in mice，while vitamin E can alleviate or partly alleviate the endosulfan actions．

Study on in vitro Culture and Differentiation of Mouse Spermatogonia

YANG Dan-Tong, YANG Xiao-Mei, QIU Yi, JIA Yi-Fang

2011, 28(02): 20-22.

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Abstract： Objective To study on the isolation and purification and in vitro culture of mouse spermatogonia． Methods The combined enzymatic digestion was used to separate and purify the germ cells from the testes of neonatal mouse，which were then in vitro cultured in DMEM／F1 2 medium with rFSH and testosterone．Reverse transcription—PCR(RT—PCR)was used to investigate the expression of transition protein 2(Tnp2)，a spermatid specific gene，to identify the culture result．Results The round spermatids were generated after 5-day culture，and Tnp2 expression was detected in the collected cells after 7-day culture．Conclusion The mouse spermatogonia could perform meiosis and differentiation to generate spermatids after in移如rD culture．

Compare the Rat Model of Hyperuricemia Made by Adenine and Oxonic Acid

YANG Gui-Mei, HUANG Sheng-Hua, LI Jiang, ZHAO Jin-Tao, LIAO Yun-Juan, LIAN Xi-Yan

2011, 28(02): 23-26.

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0bjective To discuss how to establish a durative rats model of hyperurieemia through comparing the rat model of hypemricemia made by adenine and oxonie acid．Methods To inject into stomach with adenine or oxonic acid either in high or low dose separately．Administer serum uric acid，urea nitrogen，creatinine，compared with the control group，and make histopathologic observation of kidney．Results Adenine groups have serious kidney damage that is earlier than the increase of serum uric acid．The serum uric acids of oxonic acid group’8 increase clearly and stably and there are not significant renal damage in a long time．Conclusion Adenine is only suitable to make rat model of renal failure．Oxonic acid is more appropriate for the production of rat model of hyperurieemia．

Effects of Endosulfan on the Blood Cell Immune Factors in Mongolian Gerbil

YANG Lei-Lei, ZHANG Ying, ZHOU Xian-Qing, WANG Ying

2011, 28(02): 27-30.

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Objective To investigate the effects of organ chlorine pesticide--endosulfan--on the blood cell immune factors in Mongolian gerbil we designed this experiment．Methods 50 Mongolian gerbils were randomly divided into 5 groups．They were garaged endosulfan at levels of 0，0．4，0．8，1．6，and 6．4 ms／(kg·d)， respectively．twenty one days later，blood was then collected to determine 3 of blood cell immune factors．Results (1)The expression levels of CD59 on erythrocyte membrane and CD35 on B lymphocyte membrane had no differences among the 5 groups(P>0．05)after treatment；(2)The levels of CD58 on erythrocyte membrane gradually decreased with the increase of endosulfan dosage，and the three groups given endosulfan level at 0．8，1．6，6．4mg／(kg·d)were significantly lower than that of the control group(P<0．05)．Conclusion rI'IIe results suggested that endosulfan can inhibit the express of CD58 on erythrocyte，but have no effect on the expression levels of CD59 on erythrocyte membrane and CD35 on B lymphocyte membrane．

Establishment of Genetic Standards of Closed Colony Gerbil

LI Wei, JIANG Qi-Hui, DU Xiao-Yan, SHANG Shi-Chen, CHEN Zhen-Wen

2011, 28(02): 31-36.

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Objective To establish genetic quality standards of closed colony gerbil and its detecting methods． Methods Based on investigating a large amount of documents and data，genetic standards of closed colony gerbil were established according to laboratory animal··Genetic quality control of mammalian laboratory animals GB 14923·· 2001 and theory of population genetics．The method WSS established on microsatellite marker$，which is selected from 536 mouse microsatellite markers chosen from Gene Bank to identify polymorphic dinucleotide repeat loci in the gerbil by cross-ampli-cation，and then by agarose gel electrophoresis assay and STR loci gene scan analysis． Finally，microsatellite loci in closed colony gerbil were selected in the light of allele number and polymorphism，and detecting methods were established．Conclusion To some extent，Genetic standards of closed colony experimental gerbil 0 and its detecting methods have been established．

Foster Nursing by ICR Micc in the Cleaning of Gerbils Through Cesarotomy

QIAO Xin, LI Sheng-Li, JIAO Kun, LU Jing

2011, 28(02): 37-39.

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Objective To optimize the technique of cleaning the gerbils through ces缸otomy．Methods To clean the gerbils through cesarotomy in super·clean stable and the youngest animals were suckled by ICR mice which can skillful suck in barrier system．The rate of livability was dealt and the growth carve was drown．The baby mice were detected at 30一day—old and 60一day-old following the state standard for clean mouse．Results The livability of gerbils by foster nursing was over 50％and these animals were up to the mustard of state standard for clean mouse． There we陀no difference between the growth carve of normal gerbils and gerbils by foster nursing．Conclusion Clean gerbils can be obtained thmughcesamtomyand by foster nursing using ICR mouse．

Development of Rabbit Liver Cirrhosis by Carbon Tetrachloride Subcutaneous Injection

YU Xiao-Hong, JIN Bo, QIU Hui-Bin, FU Shan-Feng, LU Ping

2011, 28(02): 40-43.

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Objective To establish a liver cirrhosis animal model in rabbit through carbon tetrachloride subcutaneous injections．Methods Total 50 male rabbits were divided into 2 groups randomly．The experimental group(n=35)were injected 50％CCl4-olive oil subcutaneously with the dosage 0．23 ml／kg in the gluteal region， twice a week．The same dosage of olive oil was injected in the control group by a same way．The liver function test and the histopathology were investigated at the end of 4，8，1 2weeks．Results The hepatic fibrosis progressed gradually ag continuous injection of the toxin．The ALT，AST，GGT increased obviously．At the end of 12 weeks，the albumin／globulin ratio was reversed．A typical pathology of liver cirrhosis was detected．In the control group the liver funetion and histology were normal．The mortality is 20％at the end of experiment and the achievement ratio of animal model is 83％．Conclusion The rabbit liver cirrhosis model can be established by subcutaneous injection of carbon tetrachloride．This animal model can be used in liver cirrhosis experiment．

Determination of Values of Physiological and Biochemical Parameters in HBK-SPF Ducks

HAN Ling-Xia, LIU Xiao-Lei, CAI Wen-Bo, YU Hai-Bo, LI Shu-Lan, QU Lian-Dong

2011, 28(02): 44-46.

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Objective To determine the values of physiological and biochemical parameters in of HBK-SPF ducks at age of 7 and 56 days old and comparative analysis of the differences between male and female．Methods The values of physiological and biochemical parameters in of 7 and 56··day old HBK··SPF ducks at age of 7 and 56 days were measured and the results were t-tested was made to analyze the results．Results There was a very significant difference between NE，PLT，ALT，ALP，ALB，BUN，Na，K and Mg values in male and female chickens at the age of 7 days days(P<0．01)among NE，PLT，ALT，ALP，ALB，BUN，Na，K and Mg values，and a significant difference of e between MO and P values sexually in male and female chickens at the same age of 7 days(P<0．05)；while at age of 56 days，a very significant difference difference existedbetween at WBC，HCT，HGB，ALT，ALP，LDH， BUN，P and Mg values in male and female chickens at age of 56 days(P<0．01)，a significant difference between at MCV，MCH，PLT，and Cl values in male and female chickens at age of 56 days(P<0．05)． Conclusion The values of the physiological and biochemical parameters were different between male and female HBK—SPF ducks at age of 7 and 56 days．

An Approach to the Methods of Observing Behavioral Indexes of Experimental Minipigs in Basic Research on Traditional Chinese Medicine

ZHANG Yu-Qin, TIAN Yu-Ying, DING Yu-Long, HUANG Tao, JIA Shu-Yong, ZHANG Wei-Bo

2011, 28(02): 47-51.

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Abstract：Objective To probe into methods of observing behavioral indexes of experimental minipigs in the basic research on traditional Chinese medicine(TCM)．Methods The special cages and electronic surveillance system were adopted in this series to observe and record the changes in the behavior of minipigs before and after pathological modeling．Results The study established the methods for observing and recording the changes in the behavior of big animal in the basic research on traditional Chinese medicine，and the special cages and behavioral surveillance system adopted in this series played an important role in completely and accurately collecting and processing data．Conclusion This method has been successfully used in the study on behavioral science in the experimental minipigs with blocked meridian，and could also be used in other basic researches in TCM．More conclusions still remain to be observed and investigated．

Hepatitis C Vaccines in the Future

SHAO Hui-Xun

2011, 28(02): 64.

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Hepatitis C is an important infectious disease threatening human health．Because of the most heterogeneous and high mutational rate of the HCV genome，the virus can easily escape from the immune recognition of the host，thus it is difficult to develop a vaccine，as theory and technique of molecular biology rapidly develop， gene immune technique now is applied into HCV vaccine development．Recombinant plasmid pEF-HCV—C constructed by inserting a highly conserved HCV—core gene fragment with the highly expressed eukaryotic expression vector pEF．By direct intramuscular immunization，DNA-based immunization has successfully induced specific humeral immune response against HCV-core protein．Nucleic acid vaccine have a number of advantages over conventional vaccines including the ability to induce a wider range of immune response types，this vaccine expressing HCV—core is a potential candidate for a prophylactic vaccine for humans．Dendritic cells are capable of eliciting a vigorous antiviral response in native T cells．The administration of DC offers a potential approach to induce high—level immunity against hepatitis C virus．Vaccination with protein-transduced DC may constitute an important antiviral strategy for hepatitis C virus．

Establishment of Model of Rats Infected by Ureaplasma Urealyticum

WANG Zhe, LU Cheng-Rong, LUO Yuan, DUAN Lian-Ning, HU Hai-Xiang

2011, 28(02): 64.

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Abstract：Objective This study observed the effects of Uu on the morphology of the testis of rats infected· Methods Rats were inoculated the Uu(105ccU／mL)by injecting broth into the urinary bladder following laparectomv．The teste$of rats were used to detect Uu．Uu were obtained by PCR．The testes were observed with light micm8cope．Results 8 positive Uu rats were tested out in model group，the tested rate is 66·7％；no one was tested out in normal group，the weight of testis in model group is lower than that in normal group(尸<0·01)；The testi8 seminiferous tubules in normal group were normal，and germ cells’array was regular，and sperms could be ofren跎en in the tubules．The changed seminiferous tubules，and the reduced germ cells in some area could be seen under light microscope in model group．Conclusion It has been shown that Uu interfere with spermatogenesis·Uu might be one of the causes of male infertility·

Histopathology of Spontaneous Lesions in Beagles Used for Toxicity Studies

ZHU Kui-Cheng, LIU Na, WANG Chun-Yao, JIN Shu-Xing

2011, 28(02): 64.

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A histopathological study was carried out on spontaneous lesions in 40 beagle dogs employed in toxicity studies．Lesions detected included acidophilic crystalline intraocular inclusion in the liver and kidney，atrophy and cyst formation in the thymus,siderofibrotic nodules in the spieen，and parafoUicular cell hyperplasia in the thyroid． In addition．fatty and vacu。lar degeneration，and nodular hyperplasia in the adrenal gland also be found

Prevalence of Experimental Beagles with Giardia Infections

BI Tian-Tian, ZHANG Jin, WEN Run-Lai, HAN Jie, FU Qiang, WANG Xiao-Ke, LU Sheng-Ming

2011, 28(02): 64.

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In order to provide high quality of experimental Beagles to institutes of scientific research in both China and foreign countries．the presence of Giardia infections is periodically detected by our monitor lab·This study will carried out a sun，ey for the pfesence of Giardia infections in Beagles using a Synbiotics OVASSAY Plus kit·Nine hundred and ten dogs were examined for Giardia infections in four age groups．The result also shows that：Slightly more female dogs(14．76％)than male(7．86％)dogs were affected；there was no significant difference between tIIe two groups．The overall positive rates of Giardia infections were I I．98％，there is no significant difference between different gender，and X² analysis revealed a significantly higher prevalence in the<2-month。old group than in the other groups．Through periodical detection，Giardia infection situations can acquire，immediately diagnose and control can canrry out．which further ensure the high quality of experimental Beagles that our company provided·

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