Table of Content

25 August 2015, Volume 35 Issue 4

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A Preliminary Study on Phenotype of 5-Lipoxygenase Transgenic Mice

ZHANG Mei-ying, YANG Wei, WU Hong-lian, DONG Wan-wei, ZHOU Sheng-lai,YU Yang, WANG Wei, GUO Xiao-chong, ZHENG Zhi-hong

2015, 35(4):  265-270.  DOI: 10.3969/j.issn.1674-5817.2015.04.001

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Objective To establishment a 5-lipoxygenase (5-LO) transgenic mouse model, which may be used for the research of the pathogenesis of atherosclerosis. Methods RT-PCR and immunohistichemistry was used to detect 5-LO transgenic mice and control mice. The cholesterol and blood pressure was also detected. Result Expression of 5-LO and 5-lipoxygenase activating protein (LFAP) were higher in the kidney and lung of the 5-LO transgenic mice than the control mice. The results of RT-PCR showed that the expression of leukotriene and its receptor were higher in the bone marrow cells, peritoneal cells, spleen and kidney of the 5-LO transgenic mice than the control mice. Compared the 5-LO transgenic mice with the control mice, there were significant differences in the expression of LTA4, LTB4 and Cys-LTR2 in the bone marrow cells and peritoneal cells (P<0.05), there were significant differences in the expression of LTB4, LTC4 and Cys-LTR2 in the spleen and kidney (P<0.05), there was a significant difference in the expression of Cys-LTR1 in the kidney (P<0.05), there were significant differences in the expression of CRP, IL-1b, PDGF and NF-kB in the kidney (P<0.05). The result of blood pressure determination showed that it was higher in the 5-LO transgenic mice than the control mice (P<0.05), and there was no significance in the cholesterol (P>0.05). Conclusion The 5-LO transgenic mouse model has been established.

Establishment of eNOS Knockout Rat Model and Phenotypic Study

YANG Wei, ZHOU Sheng-lai, DONG Wan-wei, WANG Wei, YU Yang, GUO Xiao-chong, ZHANG E-nuo, WANG Hong-yu, ZHENG Zhi-hong

2015, 35(4):  271-276.  DOI: 10.3969/j.issn.1674-5817.2015.04.002

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Objective To establish an endothelial nitric oxide synthase (eNOS) knockout rat model. Methods The encoding eNOS specific Zinc-finger nucleases (ZFN) mRNA was transcripted in vitro and purified, then injected into zygotes. The injected zygotes were transferred into oviducts of pseudopregnant rats. The genotype of knockout rats were identified by PCR and sequencing. The histopathological changes were observed by HE staining. The eNOS expression were detected by Western blot. The body weight differences were detected at 8-16 weeks ages, and the blood pressures, systokic pressures, diastolic pressures and heart rates were determined at 8 weeks age by using a tail-cuff system. Result Total of 441 zygotes were injected and 365 zygote cells were transplanted into oviducts of 12 recipients. Twenty-three offspring were born, and the results of PCR and sequencing showed that 4 offsprings were eNOS knockout rats. The screening result showed No.8 offspring was homozygous, and it exhibited limb defect, abnormal structures of artery. The results of Western blot showed that eNOS were not expressed in the kidney and heart in eNOS knockout rats. The comparison of body weights showed that the effect of eNOS genotype were significant different. Blood pressures, systokic pressures and diastolic pressures were higher in eNOS knockout rats than the SD rats, and there were significant differents. Heart rates were lower in the females eNOS knockout rats, but in the males eNOS knockout rats there were not significantly different. Conclusion eNOS knockout rats have been established successfully. This rat model will contribute to research of hypertension.

Establishment of apoE Gene Knockout Model on SD Rat Using Talen Method

XU Ying-qi, LI Xiao-jing, YANG Wei, ZHOU Sheng-lai, YU Yang, WANG Wei, ZHANG E-nuo, ZHAO Wen, ZHANG Mei-ying, GUO Da-yong, WANG Hong-yu, ZHENG Zhi-hong

2015, 35(4):  277-282.  DOI: 10.3969/j.issn.1674-5817.2015.04.003

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Objective To establish the apoE gene knockout-rat model using Talen method, and provide a basis for further study on the function of the apoE gene, the pathogenesis and treatment of atherosclerotic disease. Methods The Talen sequences were designed and synthesized for the rat apoE gene, and then the linearized and purified fragments were injected into fertilized eggs of SD rat through microsopic injection. Positive rats were detected by PCR-sequencing and TA clone-sequencing. The homozygous type offspring were obtained by siblings mating, and the genotype was identified. Western blot was used to detect the expression level of ApoE protein in some tissues of apoE^-/- rat. The plasma levels of total cholesterol and triglyceride levels have also been detected. The expression of ATP binding cassette transporter A1 (ABCA1) which was related to the lipid metabolism was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results Sequencing analysis indicated that there had a deletion of 1 bp in the second exon, resulting in open reading frame shift mutation in the rat, and obtained the apoE^-/- rats. Western blot test showed that no expression of ApoE protein in the heart, liver, kidney, brain and ovary of apoE^-/- rats, and the apoE gene was successfully knocked out. The total cholesterol and triglyceride levels of apoE^-/- rats were higher than that of wild type SD rats, and the total cholesterol levels have the significantly different (P<0.05). The expression of ABCA1 in the liver of the apoE^-/- rats was decreased, which may play a role of AS promoting. Conclusions apoE^-/- rats were obtained by Talen method. The apoE gene has been knocked out, and the rats can be considered as the model rats showed hyperlipemia and atherosclerosis.

Construction of Golden Hamster Apoa5 Gene Expression Vector and Its Expression in Different Organs

DONG Wan-wei, ZHANG A-nuo, GUO Xiao-chong, WEI Zheng-li, YANG Wei, ZHAO Wen, SHEN Wei, ZHENG Zhi-hong

2015, 35(4):  283-287.  DOI: 10.3969/j.issn.1674-5817.2015.04.004

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Objective To construct apolipoprotein a5 (Apoa5) expressing plamid and describe the gene expression profile of Apoa5 in golden hamster. Methods Total RNA was extracted from liver tissue of golden hamster, cDNA of Apoa5 was cloned by RT-PCR, and then subcloned into plasmid. The plasmid of pEF6/V5-His-Apoa5 was transfected into 293T cells, the expression was examined by RT-PCR and Western blot. The expression of Apoa5 in 8 organs, including brain, heart, lung, liver, spleen, kidney, testis, and ovary of golden hamster were also detected by real time PCR. Results Apoa5 expression vector was constructed. Apoa5 mRNA and APOA5 protein was successfully detected in 293T cells 48 h after transfection. Sequencing results showed that the cDNA of Apoa5 contained 1 243 bp, it encodes 366 aa. The Apoa5 gene in golden hamster has high homology with human, mouse and rat. The Apoa5 gene in golden hamster shares 75%, 84%, 84% amino acids with human, rat, and mouse. Real time PCR results showed that gene expression of Apoa5 was high in liver, brain, testis, but low in heart, lung, and kidney. Conclusion The Apoa5 expression vector was constructed successfully, and Apoa5 expression profile in golden hamster had been described. It may be as the foundation for establishing Apoa5 transgenic golden hamster and researching the function of Apoa5.

Producing Transgenic Pig by Bacteria Magnetic Particles

CHEN Ke-yan, CHEN Zhen-wen, DONG Wan-wei, ZHENG Zhi-hong

2015, 35(4):  288-293.  DOI: 10.3969/j.issn.1674-5817.2015.04.005

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Objective To establish sperm-mediated transgenic pigs models by intratestis injection with bacteria magnetic particles (BMP) and polyethylenimine (PEI) as a vector. Method The Lp-PLA2- GFP plasmid were mixed with BMP-PEI to BMP-PEI- PLA2-GFP, which were delivered into Bama Mini-pigs by intratestis injection. The sperm were collected in 2 weeks, 4 weeks, 6 weeks to 20 weeks after inoculation, and the GFP gene were detected by RT-PCR and immunofluorescence. The positive sperms will be transferred through artificial insemination to sows to establish transgenic pigs. The piglets were detected by RT-PCR to GFP gene and Real-time PCR to Lp-PLA2 gene. Results The GFP gene was first appearance in semen in 4 weeks after inoculation, and sustained through 16 weeks. The GFP protein mainly expressed in the sperm acrosome, sperm tail and neck area were observed by fluorescence microscopy. Through artificial insemination, 21 piglets were born, 9 of which were found with GFP segments, and the expression of PLA2 was obviously higher than that of other pigs, with a total positive rate of 42.86%. Conclusion The BMP combined with PEI can serve as a good non viral vector carrying exogenous gene, and sperm-mediated method with the vector can achieve the purpose of rapid preparation of transgenic pigs, its cost is low, the method is simple and convenient.

Modeling of B16 Melanoma in Dicer1 Transgenic Mice

LI Zhao-yang, JIANG Chuan, GUO Xiao-chong, YANG Wei, DONG Wan-wei, ZHENG Zhi-hong

2015, 35(4):  294-297.  DOI: 10.3969/j.issn.1674-5817.2015.04.006

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Objective To establish a model of B16 melanoma model in Dicer1 transgenic mice. Methods B16 melanoma cell were subcutaneously implanted into Dicer1 transgenic and C57BL/6J mice, the morphology and growth velocity of the tumor were observed. Result Compared with the contrast group, the tumor formation time in Dicer1 transgenic mice is shorter, the tumor growth rate is faster, the tumor volume is bigger (P<0.05). Conclusion The model of B16 melanoma model was successfully established in transgenic mice and the Dicer1 had significant effects on B16 melanoma bearing of mice.

Application of In Vitro Fertilization in Rat Embryos Cryopreservation

ZHOU Sheng-lai, WANG Wei, YU Yang, YANG Wei, ZHANG Mei-ying, ZHENG Zhi-hong

2015, 35(4):  298-301.  DOI: 10.3969/j.issn.1674-5817.2015.04.007

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Objective To investigate the feasibility of in vitro fertilization (IVF) technique in rat embryos cryopreservation. Methods IVF-20 was used as rat sperm capacitation and fertilization medium, mR1ECM as a culture solution for IVF fertilized egg of SD rats; 2-cell embryo were obtained by flushing as a control, and well-developed 2- embryos were cryopreserved, after a week the thawed embryos were transplanted to recipients of same strain rats. Results A total of 463 and 588 embryos were separately collected from IVF group and control group, the development rate of normal 2-cell embryos was 65.01% (301/463) and 63.94% (376/588) respectively, which showed no significant difference in the fertilization rate (P>0.05). In the 150 embryos in each group, morphologically normal embryos were 132 (88.00%) and 130 (86.66%) after the recovery, there was no significant difference (P>0.05); Surviving embryos were transplanted into five recipients of same strain rat, and newborn were 45 and 22. The litter rate (2.14% vs16.92%) showeda significant difference between the two groups (P<0.05). Conclusion IVF technology of rat is feasible in the embryo cryopreservation and breed preservation. It provides technical supports for cryopreservation of genetic manipulated rats in future.

Protective Effect of Melatonin on Pneumonary Edema Induced by Adrenaline in Rabbits

WU Xiao, YANG Xiao-long, WANG Dan

2015, 35(4):  302-304.  DOI: 10.3969/j.issn.1674-5817.2015.04.008

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Objective To investigate the effect of melatonin on adrenaline induced experimental pneumonary edema in rabbits. Methods Eighteen rabbits were randomly divided into normal group, model group, melatonin group. In the melatonin group, rabbits were received an injection of 10 mg/kg melatonin, followed by injection of 1 mg adrenaline (AD) after 30 min. In the model group, after a corresponding bolus of normal saline, AD was administrated. Normal group were injected with a corresponding bolus of normal saline. The effect of melatonin was evaluated by light microscopy, the indications of the animals as breathing and lung index. Results In the model group, respiratory frequency and lung index were significantly higher compared with the normal group. Histopathological alterations presented hemorrhage, edema thickened alveolar septum in alveolar spaces. Melatonin decreased the respiratory frequency remarkably and lung index. Light microscopic analysis disclosed that lung were less deteriorated in the melatonin group than in the model group. Conclusion Melatonin has aprotective effect on adrenaline induced pulmonary edema.

A Method for Establishing Colorectal Orthotopic Transplantable Cancer in Mice

YANG Xi-hua, LI Yao-ping, REN Lian-sheng, ZHAO Li-li, YANG Yong-ming, YAN Lei, BAI Xi-hua

2015, 35(4):  305-307.  DOI: 10.3969/j.issn.1674-5817.2015.04.009

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Objective To develop a self-made inoculator for establishing orthotopic transplantable colorectal cancer in mice. Methods An inoculator for establishing orthotopic transplantable colorectal cancer in mice was designed and made. The inoculator was used for inoculating 1×10⁶ H22 cells into colorectal site of each KM mice, and the tumor formation and metastasis was observed. Result Orthotopic transplanted H22 cells to colorectal site of Kunming mice, the tumor formation rate was 100%, the tumor metastasis rate was 33.3%, and the average lifetime of the mice was 16.5±4.5 d. Conclusion The self-made inoculator could be successfully used for establishing orthotopic transplantable colorectal cancer in mice. The operation was convenient, and the tumor formation rate was high.

Application of Grinding Stone in Mice and Rats Breeding

LANG Xue-nan, CHEN Xue-ting, GUO Xiao-chong, LI Zhao-yang, ZHOU Sheng-lai, WANG Wei, Zhang E-nuo, ZHAO Wen, ZHANG Mei-ying

2015, 35(4):  308-310.  DOI: 10.3969/j.issn.1674-5817.2015.04.010

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Objective To explore the effect of tooth grinding stone in mice and rats breeding. Methods Thirty C57BL/6 mice and 30 SD rats were randomly divided into 2 groups respectively at the age of 3 weeks. Group 1 was as control, and group 2 was given tooth grinding stone in cages, with 3 animals in one cage. All animals had free access to water and everyday weighed feed, the feed consumption were monitored. All animals were tested by open field test 3 months later. Results The feed consumption of group 1 was higher than group 2. Animals in group 2 like playing with the tooth grinding stone. Open field test show that there was no difference between group 1 and group 2 in the activity factor and emotionality factor. Conclusion Tooth grinding stone could save feed and make the life of animals colorful.

Research Progress on Autophagy with Parkinson Disease and Related Models

LI Yuan, ZHANG Mei-ying

2015, 35(4):  335-340.  DOI: 10.3969/j.issn.1674-5817.2015.04.020

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Parkinson disease (PD) is a common neurodegeneration disease, it has seriously affect on quality of life in elder. At present the pathogenesis of PD is unclear, environmental and genetic factors are closely related with it. An increasing number of evidences suggest that mitochondrial dysfunction and autophagy damage may be one of the key molecular mechanisms leading to the onset of PD, thus it would be significant to study the role of autophagy in the pathogenesis of PD by using cellular models and PD animal models. The paper briefly review the research progress of mitochondrial autophagy correlation with Parkinson's disease.

Effects of Nitric Oxide on Embryonic Implantation in Animals

WEI Dong, SUN Quan-wen

2015, 35(4):  341-344.  DOI: 10.3969/j.issn.1674-5817.2015.04.021

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Nitric oxide (NO) is a special biological signal molecule, it is being paid attention to the life sciences in various fields. The NO of the organism is synthesized by catalysis of nitric oxide synthase(NOS). NO is a free radicals in body playing a variety of biological effects, and an important biological messenger producing certain effects in almost all systems of mammals. The recent researches show that NO plays an important role on animal embryo implantation.