Table of Content

25 December 2013, Volume 33 Issue 6

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Generation of Trp53 Gene Knockout Mouse and Its Application in Carcinogenicity Assessment of Pharmaceuticals

LIN Dan, ZHENG Jin-hua, ZHUANG Hua, SHI Ji-jing, LIN Yue-lin, WANG Zhu-gang, KUANG Ying

2013, 33(6):  418-424.  DOI: 10.3969/j.issn.1674-5817.2013.06.002

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Objective To establish Trp53 gene knockout mouse. Methods Using ET cloning, homologous recombination, microinjection technology, the Trp53 gene knockout mouse with the deletion of exons 2 to 11 was established. PCR, RT-PCR and Western blot were used to detect the expression of Trp53 in Trp53^+/+, Trp53^+/- and Trp53^-/- mice. The phenotype of the knockout mice was analyzed by spontaneous tumors observation and histopathological examination. The susceptibility of Trp53 heterozygote (+/-) and wild type (+/+) mice to N-methyl-N-nitrosourea (MNU) was investigated to assess the application of the knockout mouse in carcinogenicity assessment of pharmaceuticals. Results PCR, RT-PCR and Western blot confirmed the deletion of Trp53 gene in the knockout mouse. Trp53^-/- mice appear normal but are highly susceptible to genesis of a variety of tumors at a very early age-13 weeks of age and all have developed tumor and died by 36 weeks of age. These mice developed many deferent types of tumors, but the most frequently observed tumor is malignant lymphoma, occurring in more than 60% of the mice. Mortality from N-methyl-N-nitrosourea (MNU)-induced lymphomas was much greeter in Trp53^+/- group. After a single intraperitoneal injection of MNU at dose of 75mg/kg in twenty male and female Trp53^+/- and Trp53^+/+ mice, eighteen male Trp53^+/-mice or female mice developed tumors, especially the malignant lymphoma of thymus, Trp53^+/- in mice 86%. Twenty Trp53^+/- mice and Trp53^+/+mice per sex receiving a single intraperitoneal injection of citrate buffer appeared normal and no tumor was found. Conclusion The Trp53 gene knockout mice have been established , which can be use in the function study of Trp53 gene and the carcinogenicity assessment of pharmaceuticals.

Generation of Sumf2 Conditional Gene Knockout Mouse Model and Its Preliminarily Phenotypic Analysis

KUANG Fang-zhe, ZHUANG Hua, ZHOU Tao, KUANG Ying, WANG Zhu-gang

2013, 33(6):  425-432.  DOI: 10.3969/j.issn.1674-5817.2013.06.003

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Objective To establish the sulfatase modifying factor 2 (Sumf2) gene conditional knock-out (CKO) mouse model and to analyze the biological functions for further in vivo study. Methods Use the technology of bioinformatics, ET cloning, homologous recombination, and microinjection, etc to establish the CKO mouse model. Mating transgenic (TG) homozygous EIIa-cre males with CKO heterozygous females, Sumf2 gene knock out (KO) mouse model was established. The deletion of Sumf2 gene in the KO mouse was confirmed by PCR, RT-PCR and western-blot analysis. Results There are about 20% - 19 ES cells positive clones, 2 chimeras males and about 57% of 28 Aguoti mice-16 heterozygous obtained. Based on this result, mating TG homozygous EIIa-cre males and CKO heterozygous females, Sumf2 gene KO mouse model has been established. Analyzed by PCR, RT-PCR and Western Blot, it is verified for the deletion of Sumf2 gene at different levels. Preliminarily phenotypic observations show that the growth and development of Sumf2 KO mice were generally normal, and abnormalities were not found from the data of routine blood tests and some of serum biochemical tests in comparison with those of wild type mice. Conclusions The establishment of Sumf2 CKO mouse model and Sumf2 KO mouse model has paved the way for further study of the functions of this gene.

Development of Liquid Suspension Chip of Rabbit Immune Globulins and Flexible Multi-analyte Profiling

CAI Yue-qin, XU Jian-qin, XU Xiao-ping, WANG De-jun, HUANG Yu, WU Wei, CHEN Min-li

2013, 33(6):  433-438.  DOI: 10.3969/j.issn.1674-5817.2013.06.004

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Objective To establish flexible multi-analyte profiling (xMAP) in rabbit immune globulins using the Luminex liquid suspension array system. Methods The rabbit IgG, IgM and IgA affinity capture antibody was conjugated to different fluorescent encoded microspheres, respectively. The conjuctional capacity of rabbit IgG, IgM and IgA coupling antibody was optimized, and the liquid suspension chip of rabbit IgG, IgM and IgA was developed. The simultaneous measurement of multiple immune globulins in WHBE and JW rabbit was semi-quantitative detected by xMAP in liquid suspension array system. And verify the rabbit immune globulins results by ELISA analysis. Results (1) The optimal conjuctional capacity of rabbit IgG, IgM and IgA coupling antibody was 10 μg, 20 μg and 15 μg, respectively. (2) IgG, IgM and IgA in WHBE rabbit and JW rabbit were simultaneously detected by xMAP technology. IgG and IgM in WHBE rabbit were significantly lower than JW rabbit (P<0.05), and IgA in WHBE rabbit was significantly higher than JW rabbit (P<0.01). Meanwhile, IgG, IgM and IgA in WHBE rabbit and JW rabbit was respectively analyzed by ELISA method. IgG and IgM in WHBE rabbit were significantly lower than JW rabbit (P<0.05, P<0.01), and IgA in WHBE rabbit was significantly higher than JW rabbit (P<0.01). Conclusion The xMAP for semi-quantitative detecting rabbit immune globulins has been successfully established. And all the results were consistent with the ELISA results.

Expression of Inflammatory Cytokines in the Brain, Blood Serum and Periodontal Tissue in Alzheimer's Disease Rat Model Induced by a Combined Treatment Procedure

YIN Xiu-hua, JIANG Zhen, LIU Tian-yun, PANG Ping, QIN Ting, XIA Chun-lin, SUN Mao-min

2013, 33(6):  439-443.  DOI: 10.3969/j.issn.1674-5817.2013.06.005

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Objective To establish rat Alzheimer's Disease by a combined treatment with Amyloid protein1-42 (Aβ1-42) and D-galactose (D-gal) andinvestigate the levels of CRP,IL-1,IL-6,TNF-α in the brain, blood serum and periodontal tissue. Methods Twenty-four two-month old SD male rats were randomly divided into two groups: peritoneal injection with D-gal combined with lateral ventricle injection with Aβ1-42 to establish AD animal model, and injection of physiological saline served as the control group. The learning and memory function of the rats were evaluated by Morris water-maze test, the number and morphology of neuron in hippocampus and cortex were observed, and the expressions of CRP,IL-1,IL-6,TNF-〈 in the brain (hippocampus and cortex), serum and periodontal tissue were measured by ELISA. Results Comparing with the control group, the number of neurons stained in AD animal model reduced significantly (P<0.05), but no morphological change was observed. The expression of CRP, IL-1, IL-6, TNF-〈 in the brain (hippocampus and cortex), serum and periodontal tissue were increased in model group. Conclusion This model can better imitate inflammation in central nerve system; periodontal inflammation may involve in the progress of AD.



Cleansing of Gerbils Through Cesarotomy and Foster Nursing with Rat and Mice

WEI Yong-fang, LIANG Chen-jie, LIU Han -ying, FENG Yuan-yu

2013, 33(6):  444-447.  DOI: 10.3969/j.issn.1674-5817.2013.06.006

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Objective To Explore methods of foster nursing in the cleansing of gerbils through cesarotomy. Methods Pregnant gerbils for cesarotomy in super-clean bench and the newborn litters were fostered by mother NIH mice and SD rat which have 1-2 litters, skillful suck in barrier system. The litters interval ,the survival rate and the weaning rate of litters which reared by foster nursing were detected, the growth curve of litters for 1-4 weeks were drawn. The baby gerbils were detected at 60-day-old following the state standard for clean mouse. Results The litter interval of gerbils were 30.14±4.40 (22~38) d, the survival rate and weaning rate of litter gerbils by foster nursing were over 50%. There were no significant difference in the growth curve between normal suckled gerbils and gerbils by foster nursing for 1~4 weeks. These gerbils were in accordance with the state standard for clean mouse. Conclusion The cleansing of gerbils can be obtained through cesarotomy and foster nursing by using mother ICR mouse and SD rat.



Monitoring on Some Indicators of Micro-environment Inside the IVC Cages

TANG Jia-ming, WU Wen-bin, SUN Xiao-qin, ZHANG Chao-chao, MI Jin-xia

2013, 33(6):  448-451.  DOI: 10.3969/j.issn.1674-5817.2013.06.007

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Objective To study the micro-environment indicators inside the IVC raising with mice or rats, providing the basic data for enact national IVC standard. Methods The temperature, relative humidity, and ammonia concentration were detected inside the cages raising with mice or rats, and the relations between the ventilation rates and temperature, relative humidity, and ammonia concentration inside the cages were analyzed. Results For the mouse cages, the temperatures were 1.5~2.2℃ higher and the relative humidity was 10%~15% higher inside the cages than those of outside the cages after changing bedding ; while the ammonia concentrations raised gradually the day after day and higher than national standard on the third day after changing bedding. For the rat cages, at rate of 20 times per hour ventilation, the temperature and relative humidity inside the cages raised gradually the day after day, 3℃ and 20% higher than those outside the cages respectively at the third day after changing bedding. The ammonia concentration inside the rat cage was significantly higher than the national standard at the third day after changing bedding. Increasing the ventilation rate can slightly lower the temperature and relative humidity and significantly decrease ammonia concentration inside the cages. Conclusion The temperature, relative humidity, and ammonia concentration inside the IVC cages are apt to be influenced by the animal numbers, animal body sizes, and bedding-change interval, thus affecting the results of animal experiments. In order to decrease the ammonia concentration inside the cages, the ventilation rate of rat IVC cage should be more than 60 per hour. In order to ensure the health and safety of animals raising inside the IVC cages, it is necessary to monitor the ammonia concentration inside the cage periodically.

The Breeding, Husbandry, Technique and Welfare of Zebrafish

CHANG Jia, PAN Xue-ying, LU Liang, CHANG Yan, MA Jing

2013, 33(6):  452-457.  DOI: 10.3969/j.issn.1674-5817.2013.06.008

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Zebrafish as an ideal animal model was widely used in scientific experiments. In recent years, with the increase in its research and application, the number of zebrafish used is increasing. But the zebrafish‘s breeding management, operation and the welfare has less been reported, and also there is no SOP. To solve this problem, this article reviews the relevant laws and regulations, guidelines and literature, zebrafish‘s breeding management(Transportation, environmental conditions, equipment maintenance), operation (Breeding, identification mark, anesthesia and euthanasia) and the welfare has been discussed respectively. This paper aims to provide a relevant reference for other researchers, to protect and improve the welfare of zebrafish.

Measurement of Haematology, Serum Biochemistry and Routine Urinalysis of Naked Mole Rat

ZHAO Shan-min, SUN Wei, TANG Qiu, YU Chen-Lin, XU Chen, LIN Li-fang, YUAN Zi-yan, ZHANG Lu, CAI Li-ping, XIAO Bang, SUN Zhi-yuan, CUI Shu-fang

2013, 33(6):  469-472.  DOI: 10.3969/j.issn.1674-5817.2013.06.012

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Objective To determine the blood physiology, serum biochemistry and routine urinalysis of the naked mole rat. Methods The blood, serum and urine samples from naked mole rats were analyzed by using automated hematology analyzer, biochemistry analyzer and urine analyzer, respectively. Results Haemal physiological parameters, biochemical values and routine urinalysis were determined in naked mole rats of different ages (3 months, 6 months and 9 weeks of age) and gender. Some haemal physiological parameters were influenced by age and gender, showing significant differences. Conclusion Haemal physiological, biochemical values and urine values of naked mole were determined, laying a foundation for future research of the naked mole rat.

Measurement on Growth and Development Indicators and Organ Coefficient in Naked Mole Rats

ZHANG Lu, YUAN Zi-yan, ZHAO Shan-min, LIN Li-fang, TANG Qiu, SUN Wei, YU Chen-ling, CAI Li-ping, XU Chen, SUN Zhi-yuan, CUI Shu-fang

2013, 33(6):  473-476.  DOI: 10.3969/j.issn.1674-5817.2013.06.013

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Objective To measure the growth and development indicators and organ coefficient of naked mole rats in different growth stages. Methods Measure the growth and development indexes and viscera coefficient of the naked mole rats, which were bred by the laboratory animal center in the past three years. Results The difference of indicators in growth is not obvious between male and female naked mole rats. Conclution Measurement of the growth and development indicators and organ coefficient of naked mole rats puts an effort to the standardization of the naked mole rats and provides important reference to the scientific research.



Preliminary Studies on Biochemical Loci of Naked Mole Rat

LIN Li-fang, WANG Hong, ZHAO Shan-min, XIAO Bang, SUN Zhi-yuan, ZHANG Lu, YUAN Zi-yan, YU Chen-lin, CAI Li-ping, XU Chen, TANG Qiu, SUN Wei, CUI Shu-fang

2013, 33(6):  477-480.  DOI: 10.3969/j.issn.1674-5817.2013.06.014

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Objective A elementary detection method on biochemical marker loci was established to study the characteristics of naked mole rat biochemical loci. Methods With inbred mouse as a reference, 11 isozymes of naked mole rat were detected according to the methods for biochemical markers of mice. Results The esterase-1, transferrin of naked mole rat showed slower bands; glucose phosphate isomerase-1, malic enzyme-1, isocitrate dehydrogenase-1 showed intermediate bands; hemoglobin chain, peptidase-3, alkaline phosphatase-1, phosphate glucose mutase-1, esterase-3, hydrogen peroxide enzyme-2 showed faster bands. Conclusion Biochemical locis of 7 naked mole rat performed consistent bands, showed homozygous, no polymorphism.



Establishment of Primary Culture Method of Fibroblast Derived from Naked Mole Rats

LIN Li-fang, ZHAO Shan-min, XIAO Bang, TANG Qiu, ZHANG Lu, YUAN Zi-yan, SUN Zhi-yuan, YU Chen-lin, CAI Li-ping, XU Chen, SUN Wei, CUI Shu-fang

2013, 33(6):  481-486.  DOI: 10.3969/j.issn.1674-5817.2013.06.015

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Objective To established the primary culture method of fibroblasts derived from naked mole rat, and research the growth characteristics of the cells. Methods The fibroblasts derived from naked mole rat and mouse were cultured by method which combined traditional enzymes digestion with explant culture.A comparative study between fibroblasts derived from two kinds of species on growth rate and cell cycle was conducted. Fibroblasts derived from liver of naked mole rats were inoculated with density gradients, then counted the cells after 24 h, 72 h, 96 h, 144 h. Results Fibroblasts derived from naked mole rat and mouse had a good growth behavior with the primary culture method combining enzymes digestion and tissue block culture. Compared to mouse, fibroblasts derived from naked mole rats had higher proportion in the logarithmic growth phase and proliferation rate significantly. Fibroblasts derived from naked mole rat can reach logarithmic phase within two days, seeded with the concentration of 1.5×10⁴/ml. Conclusion The method which combined enzymes digestion with tissue block culture improved efficiency of cell culture. Moreover, the proliferation ability of fibroblasts derived from naked mole rat were significantly higher than mice.



Comparasion on Structures and Aerobic Power of Skeletal Muscle between Naked Mole Rat and C57BL/6J Mice

YUAN Zi-yan, ZHANG Lu, ZHAO Shan-min, LIN Li-fang, TANG Qiu, SUN Wei, YU Chen-ling, CAI Li-ping, XU Chen, SUN Zhi-yuan, CUI Shu-fang

2013, 33(6):  487-492.  DOI: 10.3969/j.issn.1674-5817.2013.06.016

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Objective To compare the the structure and aerobic power of skeletal muscle between the naked mole rat and C57BL/6J mice. Method The musculus gastrocnemius were isolated from anaesthetic animals and made to paraffin sections and frozen section. The microstructure and ultrastructure of skeletal muscles were observed under optical microscope and transmission electron microscope. Meanwhile, the skeletal muscles were immuohistochemical stainined with CD34 antibody, which revealed the microvessel density in skeletal muscles. At last, myoglobin in skeletal muscles were measured by spectrophotometry. Result Comparasion with C57BL/6J mice ,the skeletal muscle fibrils of NMR are larger, and connective tissuse are more abundant; The number of mitochondrial in NMR is larger, but the volume of mitochondrial is smaller; The microvessel density of NMR skeletal muscle both is much higher; and the myoglobin is 1.5 times higher. Conclusion NMR is more muscular then C57BL/6J mice, and rich in connection tissuse ,which lead skeletal muscle to be more softer and more flexible then C57BL/6J mice. The most importantly, NMR has much more amount mitochondrial, microvessel and myoglobin,which revealed that the aerobic power of NMR skeletal muscle is stronger then C57BL/6J mice.



Making of Skeleton Specimens and Morphological Observation in Naked Mole Rat

ZHAO Shan-min, XIAO Bang, YU Chen-Lin, LIN Li-fang, CAI Li-ping, ZHANG Lu, YUAN Zi-yan, SUN Zhi-yuan, SUN Wei, TANG Qiu, CUI Shu-fang

2013, 33(6):  493-498.  DOI: 10.3969/j.issn.1674-5817.2013.06.017

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Objective The traditional technique for making skeleton specimen was applied to study the morphology of the skeleton of the naked mole rat. Methods Skeleton specimens of the naked mole rat were made and compared with inbred mice strains C57 BL/6J. Then the three-dimensional structure of the femur was observed in the naked mole rat. Results Skeletons of naked mole rats were made without damaged. The traits of skull, spine, ribs, sternum and limbs of the naked mole rat, which are adapt to their fossorial habits, are discovered. Conclusion Skeletons of the naked mole rat have typical characteristics of adaptation to their fossorial habits.