30 June 2008, Volume 28 Issue 6

    

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  Construction of Mouse Testis Tissue Specific Gene RNAi Vector and Study of Its Expression

  DONG Wan-wei, LI Zhao-yang, WANG Wei, YANG Wei, ZHENG Zhi-hong

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 350-355.

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  Objective Construct and screening RNAi vector of mouse testis tissuespecific gene Fankl, to lay a foundation of further study cn gene function of Fankl and gene therapy of male infertility related to Fankl. Methods Designing 2 interference sequence using http://www.dharmacon.com/ online soft according to Fankl cDNA sequence，and clone them to the plasmid vector pSuper-neo-GFP with HI promoter, amplifying Fankl complete sequence from adult mouse testis, and then construct red fluorescent protein fusion vector incorporating Fankl, pdsRED-Fankl. Cotransfecting pdsRED-Fankl with two interference vectors and control to 293T cells respectively using liposome method, to observe the inhibitory effect of siRNA to Fankl and the expression of red fluorescent protein, and to detect the expression of FANK1 mRNA and protein by RT-PCR and Western blot. Results PCR analysis confirmed that recombinant plasmids pSuper-shFankl 631#, pSuper-shFankl 247# and pdsRED-Fankl have been successfully constructed. Sequencing analysis were completely correct. Under fluorescent microscope, shFankl 631、247 significantly inhibit red fluorescent protein expression 36h after tansfection 293Tcell. The inhibitory rates were 95% and 90% respectively. RT-PCR and Western blot showed that Fankl gene was inhibited both on mRNA and protein level (P＜0.05). Conclusions The RNAi vector of mouse testis tissuespecific gene Fankl were constructed successfully, lay a foundation for establishing Fankl knock-down mouse and studying the role of Fankl in spermatogenesis.
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  Study on Gene Mutation and Muscles Subtypes’Expression in DMD Model Mice

  XIAO Nan1，ZHAO Wei2, BA Cai-feng2, SU Rong-jian1, SU Yu-hong1，3

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 356-360.

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  Objective To study muscular characteristics between model mice C57BL/10ScSn-Dmd^mdx/J and background mice C57BL/6J. Methods The Diaphragmatic muscle, latissimus dorsi and gastrocnemius muscles of the CSTBL/lOScSn-Dmd^mdx/J and C57BL/6J mice were collected at the age of 2,3，and 4 weeks，respectively. Each group selected 3 mice for experiments. Dmd gene were amplified and analyzed by RT-PCR. b-dystroglycan expression was detected by Western blot. Results A mutation C — T in 3202th of Dmd gene in model mice was found, and expression of b-dystroglycan was up- regulated in C57BL/10ScSJ. Conclusion During muscle fiber development, breakage and deletion were appeared in C57BL/1 OScSn-Dmd^mdx/J and b-dystroglycan expression was up-regulated.
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  Study on Expression of tau in DJ-1 and DJ-1L166P Transfected NIH3T3 Cells

  ZHANG Mei-ymg，LIN Mei-na, YANG Wei, DONG Wan-wei,LI Zhao-yang, WANG Wei，ZHENG Zhi-hong，WANG Lu-zeng

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 361-366.

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  Objective To explore the relationship between the expression of tau and DJ-i gene in transfected NIH3T3 cell by pEGFP-DJ-1 and pEGFP-DJ-iL166Pplasmid, and it would provide experimental basis to establish DJ-1 and DJ-iL166P transgenic mice and to understand the function of DJ-1 and the pathogenesis of Parkinson’s disease. Methods DJ-1 cDNA sequence were obtained from total RNAof SH-SY5Y cells by RT-PCR. Then one clone of the cDNA was treated with a mutation kit to get a mutation at the site which encodes the 166th amino acids. Recombinant pEGFP-ZV-i and pEGFP-DJ-i^L166P plasmids were constructed in a routine way. NIH3T3 cells were transfected into pEGFP-ZV-i，pEGFP-DJ-I^L166P, or pEGFP-C3 plasmids, and then the cells were selected with G418. The successfully transfected cells were analyzed by RT-PCR and Western blot to detect the expression of tau gene. Results We gained 6，2, and 9 clones of pEGFP-DJ-i, pEGFP-DJ-I^L166P, and pEGFP-C3 transfected cells. The results of RT-PCR and Western blot show that: the expression of tau in pEGFP-DJ-i transfected cells is lower than in pEGFP-C3 transfected cells, and the expression of tau in pEGFP-DJ-I^L166P, transfected cells is higher than in pEGFP-C3 transfected cells. The experimental result analyzed by statistics method is significant (P<0.05). Conclusion DJ-1 gene down-regulates the expression of tau on both transcription and translation levels, and DJ-I^L166P, up-regulats the expression of tau. The expression of tau is associated with DJ-i gene in NIH3T3 cell.
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  Cloning of Murine IFN-Y and its Effect as a Molecular Adjuvant

  LIU Yong，WANG Hai-yan，ZHANG Jia，WANG Yu-zhou, ZHANG Bmg

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 367-371.

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  Objective Study the molecular adjuvant effect of MuIFN-T by co-expressing MuIFN-T and EMCV structural protein VP1. Method Mature peptide gene of MuIFN-T was cloned by using RT-PCR. VP1 of pig EMCV was subcloned from pGEX-6p-l/vpl, and constructed into the express plasmid pET32-a /VPl/Rosetta(DE3) and co-expression plasmid pET32-a/VPl/MuIFN-T, respectively. BALB/c mice were inoculated with the VP1 and VIuIFN-Y/VPl proteins in 'E.coli, respectively, for three times at two week intervals, serum IgG were detected by ELISA every two weeks post-inoculation. Result In Ecoli Rosetta(DE3), expression and co-expression were succeeded IPTG (Isopropyl β-D-l-Thiogalactopyranoside) induction, with 60 MW of VP1 protein and 73 MW of co-expressed protein. The serum IgG of mice post-inoculated by VP1 and MuIFN-T/VP 1 are 1 : 32000 and 1 : 128000. Conclusions VP1 had good antigenicity which could stimulated humoral immune response in inoculated mice, the detected serum IgG of MuIFN-γ/VP 1 inoculation is higher than VP1. Moreover, immune enhancing effects was found in mice inoculated with co-expressed MuIFN-T/VPl .LMuIFN-T was a satisfactory immune adjuvant.
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  Experimental Study on Mouse Model Infected with Mycoplasma suis

  ZHOU Xiao-mm, BA Cai-feng, FENG Hui-quan

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 372-376.

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  Objective To establish a My coplasm suis Kunming mouse model with blood from the My coplasm suis infected pigs to explore the laws，the occurrence and development and outcome, of this disease in rodents to reveal the pathogenic mechanism of Mycoplasma suis further. Methods The Kunming mouses were inoculated with blood from the Mycoplasm suis infected pigs, the rate of infection and histopathological changes were determined in different stages after challenge. Results The Kunming mouse model was established successfully and the tissues and organs of different infection stages were detected. It was demonstrated that different pathological changes were in different infection stages. Conclusion Investigating the rate of infection and histopathological changes of the Kunming mouse model is beneficial to reveal Mycoplasma suis pathogenic mechanism.
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  Cloning of TNNT2，a Key Gene during Heart Function and Its Expression in Rat

  LI Zhao-yang, YANG Wei, DONG Wan-wei, ZHOU Sheng-lai,LV Xiang-chuan，YU Yang，WANG Wei, ZHENG Zhi-hong

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 377-382.

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  Objective To Clone the gene of TNNT2 and analyze its expression. Methods Total RNA was extracted from the heart of adult SD rat and first strand cDNA was obtained by reverse transcription. Primers was designed according to the offered sequence in Genebank. TNNT2 cDNA was cloned by PCR and confirmed by sequencing，RT-PCR was applied to examine the expression pattern of TNNT2 in rat tissues. Results The TNNT2 cDNA sequence was cloned and submitted it to Genebank (Genebank acc.No.EU295527), the TNNT2 was expressed in many tissues and had the highest expression level in heart. Conclusions Rat TNNT2 cDNA sequence was first cloned(Genebank Acc.No.EU295527), TNNT2 was expressed in many tissue and had the highest expression level in heart.
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  The Effect on Tension of Denervated Skeletal by Transplantation of Skeletal Muscle Satellite Cell in Rats

  ZHAO Jian-wen1，YIN Huan1，SU Qiu-xiang2

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 383-386.

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  Objective To explore if the muscle satellite cells in rats can delay the skeleton muscular dystrophy. Method Sixteen male Wistar rats randomly assigned into the experimental and the control group.The tibial nerve of the right leg of each rat was cut with lcm lesion, and the model of denervated skeletal muscle was made. Equal amount of muscle satellite cells suspension or physiological saline was injected into the denervated gastrocnemius of the experimental group and the control group respectively. Four weeks after transplantation,both groups were stimulated with different strength of electric impulses to measure the lension of denervated skeletal muscle. Result The differences of tension of skeletal muscle between two groups was statistically significant. Conclusion The transplantation of rat muscle satellite cells into denervated muscle can maintain the muscular tension, and thus alleviate the rate of atrophy of skeleton muscle, and help the recovery of the muscle’s function. This may provide a clinical effective way to prevent and treat denervated skeleton muscular dystrophy.
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  Study on Relationship Between Synaptophysin in Adrenal Glands and Postmortem Interval in Rats

  GU〇 Xiao-chong1-3，HE guan-ymg2，WU Xu1，ZHANG Wei-dong2，LI Ru-bo1，WANG Chang-liang1，YU Tian-shui1，ZHANG Guo-hua1

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 387-389.

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  Objectives To explore the changing of synaptophysin after death, and describe the relationship between the changing of synaptophysin and postmortem interval (PMI). Methods Rats were killed by cervical dislocation. Adrenal glands were taken out at different time after death. The synaptophysin in adrenal glands was detected by immunohistochemistry，and the average gray value of synaptophysin was measured by image analyzing system . The average gray values of different group were analyzed by SPSS. Results Synaptophysin in adrenal glands was stable during the first 12h after death, and then it decreased rapidly from 16h to 24 h after death. Conclusion Degradation of synaptophysin in adrenal glands varies with PMI, synaptophysin might be a new indicator for estimating PMI.
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  Changes in Baroreflex Sensitivity in Middle Pregnant SD Rats

  SHEN Jie1，FENG You-ji1, SHEN Lm-lm2, QIAO Wei-wei3，LI Xiao-tian1

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 390-393.

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  Objective To determine the changes of cardiovascular autonomic control in unrestrained conscious pregnant SD rats. Method Blood pressure, heart rate and their variabilities were measured in conscious, unstrained rats and middle pregnant rats. BRS of heart rate control(BRSHR) was measured by using a bolus intravenous injection of phenylephrine(5 jug/kg). Results HF of HRV as well as spontaneous BRS and BRShr attenuated (P<0.05) in pregnant SD rats compared with control group. Conclusion BRS reduced and regulating function of vagus decreased in middle pregnant SD rats. Measurement of HRV, BPV and BRS by spectral analysis is credible.
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  Comparative Study on Superovulation and Embryo Freezing in SD Rats

  ZHOU Sheng-lai，YU Yang，WANG Wei, WANG Lu-zeng, ZHANG Zhi-hong

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 397-400.

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  Objective To improve superovulation and establish a method of embryo freezing for rats. Methods Different doses of PMSG-HCG (15 IU，20 IU，25 IU/rat) were injected (i.p.) into three groups(Group A，B，C) of 5 weeks SD rats, and each treated female was coupled with fertile male, the effect of each dose was compared to determine the best dose for superovulation. Two-cell was collected by clysising fimbriae tubae. The embryos were divided into 2 groups. Embryos in group 1 were frozen for 1 minute, and embryos in group 2 for 5 minutes. All the embryos of rats were frozen according to the standard method for mouse embryos frozen. The embryos were resuscitated, and the rate of resuscitation were compared to assess the two method. Results The average number of zygotes obtained from each rat in group A，B，C was 13 土 12.7，31 土 14.2，and 41 土 25.4. There were significant differences among the groups (P＜0.05). The resuscitation rate of embryos in group 1 is 88.3%, and 84.9% in group 2. Of 152 rat embryos were frozen for 1 minute, 139 embryos were successfully resuscitated, and cultured for 20 minutes.The normal cultured 105 embryos were transplanted into pseudopregnant rats, and 39 offspring were obtained. Conclusion The method for rat superovulation has been successfully improved, and a method of embryo freezing for rats has been established.
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  Comparative Study on Different Embryo Collecting Method for Rat Microinjection

  YU Yang，ZHOU Sheng-lai, WANG Wei, LU Xiang-chuan,ZHANG Mei-ymg，WANG Lu-zeng, ZHENG Zhi-hong

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 401-403.

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  Objective To improve embryo collecting method for rat microinjection. Methods PMSG-HCG(ip) were injected into 5 weeks SD rats. Embryos were collected by tearing intumescences of oviduct or clysising fimbriae tubae. The amount of embryos, rates of fertilization, and survival rate of embryos after injection were analyzed to compare the two methods. Results The amount of embryos, rates of fertilization, and survival rates of embryos after injection of the embryos collected by clysising fimbriae tubae were higher than those collected by tearing intumescence of oviduct. There are significant differences between the two methods. Conclusion Clysising fimbriae tubae is a better embryo collecting method for rat microinjection.
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  The Construction, and Application of Animal Models for Spinal Cord Injury

  ZHOU Tmg-tmg，ZHENG Zhi-hong

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 411-415.

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  For the reasons that the genesis and development of human being’s diseases are complicated, and many medical researches are not permitted to be applied on human bodies, the kinds of animal models, which are replicable for human diseases, are playing a more and more indispensable role in medical research. The mechanism of spinal cord injury and regeneration is a difficult problem in current neuroscience research. However, the construction and development of the animal model for spinal cord injury provides a powerful support for breakthrough. This review aims at introduce how to choose animals, the means of constructing each kind of spinal cord injury model, the characteristic and the application of each kind of spinal cord injury model.
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  Progress in Methods for Conditional Control of Protein Function in Mouse

  WANG Jm-xia, ZHENG Zhi-hong

  Laboratory Animal and Comparative Medicine. 2008, 28(6): 416-420.

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  The spatiotemporal and controlled gene expression has always been a problem that people hope to solve. In the mouse, this has been accomplished by using Cre/LoxP, GAL4/UAS, conditional RNA interference and other systems at the DNA, transcriptional, posttranscriptional and posttranslational level. This paper reviews several methods and the latest progress of conditional control of gene expression in the mouse.