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Development and implement of a disaster plan in a laboratory animal care and use program

Jiaqi LU ; Jihong LIU ; Yan ZHOU ; Lijun GE ; Jianfei WANG

Chinese Journal of Comparative Medicine.2014;(4):78-82. doi:10.3969/j.issn.1671.7856.2014.004.017

As a vital component in an overall laboratory animal care and use program , development of a disaster plan plays a critical role for every research institution .Currently, most of domestic institutions would draw up an“emergency operation plan , EOP”, but ignoring a practicable “business continuity plan , BCP” in establishing a disaster plan.In this article, we will discusse about the definition of disaster , how to set up an EOP, and how to establish a thorough BCP , in order to show an integrated and professional disaster plan in laboratory animal care and use program .

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Analysis of the routine blood counts and blood biochemical parameters and hemorheology in the rats with congenital cataract

Xiaoyun TIAN ; Jingwei YOU ; Longxiang XU ; Zhigang ZHAO ; Li CHEN ; Shifeng YUN

Chinese Journal of Comparative Medicine.2014;(3):20-24. doi:10.3969/j.issn.1671.7856.2014.003.005

Objective To measure the range of routine blood counts and blood biochemical parameters ,to analyze hemorheology of the rats with congenital cataract .Methods Blood samples were taken from 90 rats with congenital cataract weight about 185 ~211 g.Routine blood analysis was performed and blood biochemical and hemorheology paramenters were determind using an automatic blood biochemical and hemorheology analyzer .Results There were no significant difference ( P >0.05 ) between the cataract rats and the normal rats in Blood test results; but there were significant difference between the microphthalmos cataract rats and the normal same -sex rats ( P <0.01 or P <0.05 ) . The biochemical results is the cataract rats and the normal rats were different significantly in ALB group ( P <0.01 or P<0.05), and the female microphthalmos cataract rats compared with the control rats had significant difference in Ure (P<0.01) , the female cataract rats ompared with the normal rats were very significant difference in Cr group ( P <0.05 or P <0.01).The erythrocyte counts of the male cataract rats and male microphthalmos cataract rats were significantly lower than that in the female ones, respectively(P <0.05, P <0.01).The platelet counts of the male cataract rats and the male microphthalmos cataract rats were significantly higher than that in the female ones , respectively(P <0.01), and the creatinine of the male cataract rats and the male microphthalmos cataract rats were significantly lower then that in the female ones, respectively(P <0.01).There were no significant difference in every group on hemorheology .Conclusions There were significant differences in some blood indexes between the congenital cataract rats and the normal rats .These data may become useful reference for biomedical researcher in this field .

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Long-time observation of blood glucose and pathological phenotype of leptin knockout obese rats

Li ZHANG ; Feifei GUAN ; Xu ZHANG ; Wei CHEN ; Caixian SUN ; Lianfeng ZHANG

Chinese Journal of Comparative Medicine.2014;(3):45-49. doi:10.3969/j.issn.1671.7856.2014.003.010

Objective To obtain more physiological data of Leptin knockout SD rats available for the user , the long-term observation of fasting blood glucose and pathological phenotypes were performed .Methods The protein expression levels in liver tissues were determined by western blot .Body weight of Leptin knockout rats ( Leptin-/-) and littermate lean rats (Leptin+/+) were weighed up at 1,3,6,8 months of age.Fasting blood glucose of Leptin +/+rats and Leptin-/-rats at 1,3,6,8 months of age were measured using One Touch? brand blood glucose monitoring systems.Pathological changes of pancreas and livers of Leptin -/-rats were observed by the method of HE staining and Immunohistochemistry (IHC).Results Short null Lepin proteins were expressed in liver tissues from Leptin -/-rats. Leptin-/-rats become heavier than Leptin +/+rats since they were one month old .The body weight of Leptin -/-rats at 8 months of age was twice as heavy as Leptin +/+rats, female Leptin-/-rats weighing 884g, and male Leptin-/-rats weighing 1200g.Overt hyperglycemia was observed during the first month after birth .Compared with Leptin+/+female rats,the fasting blood glucose of Leptin -/-female rats was increased by 40%-26%from 1 to 6 months old. After that, blood glucose values decreased and eventually become nearly normal at 8 months of age.Pathological examination indicated that Leptin -/-rats at 8 months of age had a fatty liver , more pancreas islets with lager volume and more beta cells with increased insulin secretion .Conclusion Leptin-/-rat were characterized by obesity , fatty liver, islet cell hyperplasia and early hyperglycemia .

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IL-33 transgenic mice increase the myeloid differentiation in hematopoietic stem cells

Lin BAI ; Guiying SHI ; Shan GAO ; Yajun YANG ; Kun GAO ; Lianfeng ZHANG

Chinese Journal of Comparative Medicine.2014;(3):39-44. doi:10.3969/j.issn.1671.7856.2014.003.009

Objective To study the influence of IL-33 on the Hematopoietic stem cells and progenitor cells . Methods Cells from the peripheral blood , spleen, thymus and bone marrow were stained with indicated antibodies and analyzed by flow cytometry . The LT-HSCs were sorted and culture using in vitro clonogenic assay . Results The percentage of B cells and T cells was decreased and the percentage of M cells was increased in the peripheral blood from IL -33 transgenic mice .Compared with the wildtype mice , the number of HSCs , MPPs and CLP was decreased;meanwhile the number of CMP and GMP was increased in the bone marrow from IL-33 transgenic mice .An in vitro clonogenic assay showed that LT-HSCs increased the ability to self-renew from IL-33 transgenic mice .And the percentage of S-G2-M stage hematopoietic stem cell was increased from IL-33 transgenic mice .Conclusion IL-33 increase the myeloid differentiation in hematopoietic stem cells .

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Generating insulin receptor substrate 1 (Irs1) knockout rat using CRISPR/Cas9

Yuanwu MA ; Jing MA ; Yingdong LU ; Wei CHEN ; Xu ZHANG ; Lei YU ; Lianfeng ZHANG

Chinese Journal of Comparative Medicine.2014;(3):55-60. doi:10.3969/j.issn.1671.7856.2014.003.012

Objective To study the relationship of insulin receptor substrate-1 (Irs1) and metabolic disease, we generated Irs1 gene knockout rat by CRISPR/Cas9 system.Methods Two sgRNA targeting sites were designed for Irs1 targeting.The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro.Cas9 mRNA and sgRNA mixtures were pooled and microinjected into one-cell fertilized eggs of SD rats to generate rats with targeted mutation .Results Five rats with the mutations were detected with the efficiency of 83%.Conclusion The Irs1 gene knockout rats generated in this study can be transmitted by germline .

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Effective of anticancer mouse monoclonal antibody against hMIC-1 targets pancreatic tumor for nude mice in vivo

Zhaoyang LIU ; Xiaobing WANG ; Wei ZHANG

Chinese Journal of Comparative Medicine.2014;(3):14-19. doi:10.3969/j.issn.1671.7856.2014.003.004

Objective To investigate a antitumor effects of mouse original monoclonal antibody against hMIC -1 as intravenous administration with human pancreatic tumor in vivo and providing experimental data .Methods The fourty-eight mice were randomized into eight groups for loaded with two pancreatic tumor cell lines panc -1 or sw1990 respectively , and individual tumor growth was observed , antitumor efficacy was evaluated after using mouse original monoclonal antibody against hMIC-1 by intravenous administration .The pathological change with formalin fixed , paraffin embedded tissues section was viewed .Results There was a significant difference in tumor volume and weigt in intravenous injection of mouse original monoclonal antibody against hMIC-1 on load pancreatic tumor with nude mice group compared with that in the control group after four week treatment , and the mouse original monoclonal antibody against hMIC-1 demonstrated a close association between inhibition of tumor volume growth and dose-effective in the two xenograft models examined .Under examined microscope , the pancreatic tumor tissue was destroyed evidently in mouse original monoclonal antibody against hMIC-1 group.Conclusions The antitumor effect of intravenous injection for mouse original monoclonal antibody against hMIC-1 is better than that of systemic using gemcitabine .

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The study of screening breast tumor suppressor microRNA in mice

Li ZHANG ; Xiaoying LI ; Caixian SUN ; Xu ZHANG ; Wei CHEN ; Lianfeng ZHANG

Chinese Journal of Comparative Medicine.2014;(3):72-77. doi:10.3969/j.issn.1671.7856.2014.003.015

Objective To investigate the functional role of 25 microRNAs in breast cancer ,and to find new tumor suppressor microRNAs that may serve as specific targets of new gene therapies . Methods Twenty-five microRNAs expression vectors were constructed and stably transfected into mouse mammary tumor cells 4To7 by Lipofectamine2000. Cells were selected with G418 and sorted by Flow cytometry.The cells in logarithmic phase were collected and 2 ×105 cells/mouse was inoculated into BALB/c mice via tail vein .Lungs were harvested 14 days after tumor cell inoculation , and the number of metastasis foci was counted .Results Mice inoculated with mir-449a-expressing 4To7 cells via tail vein developed reduced lung metastases compared with mice inoculated with negative control cells .Mice inoculated with mir-1935-expressing 4To7 cells via tail vein developed increased lung metastases compared with mice inoculated with negative control cells .Other twenty-three microRNAs neither promoted nor inhibited lung metastases of breast cancer .Conclusions Two of twenty-five microRNA were identified to be associated with breast cancer metastasis .MiR-449a may play a tumour suppressor role in the regulation of migration and metastasis in breast cancer .miR-1935 transgenic over-expression promoted tumor growth and metastasis .

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Establishment of two cardiac-specific human cardiac troponin C mutation transgenic mice and comparative analysis

Shan GAO ; Wei CHEN ; Ning LIU ; Wenping GE ; Xiang GAO ; Dan LU ; Lianfeng ZHANG ; Wei DONG

Chinese Journal of Comparative Medicine.2014;(3):67-71. doi:10.3969/j.issn.1671.7856.2014.003.014

Objective To established cardiac-specific transgenic mice of the cTnC D145E and cTnCG159D and compare the HCM and the DCM.Methods The cTnCD145E and cTnCG159D were generated by site-directed mutagenesis and the transgenic plasmids were constructed by insertion of the mutant genes under the control of α-MHC, which is a myocardium specific promoter.The transgenic mice were generated by microinjection and were all maintained on a C57BL/6J genetic backgroud .The cardiac structure and function of the transgenic mice were compared and analysized by echocardiographic and pathological observation at different ages .Results The cTnCD145E and cTnCG159D transgenic mice were established and developed to HCM and DCM, respectively, with aging.The left ventricular end-systolic volume (ESV) and left ventricular end-diastolic volume ( EDV) decreased and ejection fraction ( EF) and left ventricular end-systolic posterior wall thickness (ESPWT) increased in the cTnCD145E transgenic mice, while EDV and ESV increased and EF and ESPWT decreased in the cTnCG159D transgenic mice at 12 months of age.Conclusions Cardiac-specific human cTnCD145E transgenic mice showed HCM phenotypes , and cardiac-specific human cTnC G159D transgenic mice showed DCM phenotypes , which can be used as different models for comparative study of the pathogenesis of cardiomyopathy .

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Preconditioning effect of isoflurane on mitochondria free calcium concentrate and mitochondria permeability transition pore after liver ischemia-reperfusion injury in rat brain

Jie SHEN ; Changhui CONG

Chinese Journal of Comparative Medicine.2014;(3):25-29. doi:10.3969/j.issn.1671.7856.2014.003.006

Objective To investigate the protective effect and the mechanism of isoflurane preconditioning on rat brain tissue suffering from rat liver ischemia-reperfusion injury .Methods 75 SD rats were randomly divided into five groups, sham group (S group);ischemia-reperfusion group (I/R group): liver ischemia for 60 min, reperfusion for 120 min;isoflurane preconditioning group ( ISO group ): 60 min before liver I/R, ISO pretreatment for 30 min; CsA +ISO group;CsA ( MPTP specific blocker ) 50 mg/kg intravenous injection , the same as ISO group after 30 min; CsA group:CsA 50 mg/kg intravenous injection .30 min before I/R.The animals were killed 24 h after ischemia and their brain were excised for measurement of mitochondrial permeability transition pore ( MPTP) and calcium content in mitochondria .The measurement of content of S-100βprotein in serum before ischemia and 120min after reperfusion through the application of Elisa kit.Results The mitochondrial Ca2+concentration of I/R group(287.32 ±26.17)was higher than that in S group (198.54 ±21.02) and the ISO group (209.74 ±29.49) ( P <0.05), and Ca2 + concentration of CsA +ISO group (267.31 ±37.52) was higher than the ISO group (P <0.05);there was not statistical significance between I/R and CsA group(288.63 ±23.15)(P <0.05).MPTP were more opened in I/R group(△S:1.73 ±0.24)than that in S group (△S:2.36 ±0.35)and ISO group(△S:2.11 ±0.32)(P <0.05),but MPTP were more opened in CsA +ISO group than that in ISO group (P <0.05).The content of S-100βprotein in serum was significantly higher in I/R group than that in sham and ISO groups (P <0.05),and the content of S-100βprotein in serum was significantly higher in CsA +ISO group than in the ISO groups ( P <0.05 ) .Conclusion The liver ischemia-reperfusion may injury the brain of the rat and isoflurane preconditioning can protect the brain on the rat .The reduce of mitochondria calcium overload and inhibition of MPTP opening are involved in the mechanism .

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Establishment of sperm specific Sleeping Beauty transposase-expressing transgenic mouse

Yingdong LU ; Xu ZHANG ; Jing MA ; Lianfeng ZHANG ; Yuanwu MA

Chinese Journal of Comparative Medicine.2014;(3):34-38. doi:10.3969/j.issn.1671.7856.2014.003.008

Objective To establish the sperm specific Sleeping Beauty ( SB ) transposase-expression transgenic mouse for the study of the genetic modification mediated by transposon system in mouse .Methods Prm1 promoter was cloned from mouse genomic DNA to drive the expression of SB transposase .The transgenic mice were generated by microinjection .The gene type of transgenic line was identified by PCR .The expressing level in testis was determined by western blot and immunohistochemistry (IHC) staining.Results Five lines of transposase transgenic mice were obtained by microinjection and three can be germline .One mouse line with higher expression level of transposase in the testis was obtained.Conclusion One transgenic mouse model with Sleeping Beauty transposase - expression was successfully established .This model will greatly contribute to the research of genetic modification mediated by transposon in mouse.

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