Table of Content

28 February 2009, Volume 29 Issue 2

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Construction and Identification of ssp411 Targeted Recombinant Plasmids by RNA Interference

HUA Min-min1，LI Yu-hua2，LI Run-sheng2，SHI Hui-juan2，OU Ling1

2009, 29(2):  74-80. 

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Objective To identify the role of new sperm-specific gene ssp411 in sperm maturation, fertilization, and embryo development, the expression vectors of ssp411 for RNA interference as siRNA-ssp411s were designed and contructed. Methods The interfering sequences of ssp411 were designed according to the sequence of ssp411 of GenBank.Three pairs of oligonucleotides were synthesized and inserted into plasmid pRNAT-U6.1 to generate siRNA expression vector, and were identified by PCR and sequence analysis. The recombinant plasmid siRNA-ssp411s and ssp411 expression plasmid ssp411-pDsRed was cotransfected into the cultured 293T cell line. The interfering efficiency was detected by real-time PCR. Results Three ssp411 siRNA frames were successfully inserted into the plasmid vector pRNAT-U6.1，and the recombinant plasmids were sent to the sequence analysis. The result of real-time PCR indicates that the siRNA expression vector which was designed by SSP411 protein coding area 3-22 got the best interfering efficiency as 71%. Conclusion The siRNA recombinant can be constructed successfully by RNAi technique for inhibiting ssp411 expression.

Primary Study on Effect of EGF on Cell Cycle and Apoptosis in Epithelia of Dorsal Lingual Mucosa from Mice and Rabbits

ZHANG Jun-feng，ZHAN Zhen

2009, 29(2):  81-85. 

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Objective To investigate the effect of EGF on cell cycle and apoptosis in epithelia of dorsal lingual mucosa from mice and rabbits in vitro. Methods Dispase II and trypsin were combined to digest the lingual dorsum epidermis, and the epithelia were obtained from healthy mice and rabbits. After the lingual dorsal epithelia were incubated in 10% RPMI-1640 withl, 5，10 )ag/L EGF (control group 0 jug/L) by 16 h，32 h, 48 h，flow cytometric analysis was used to analyze the cell cycle distribution and apoptosis. Results Compared with the control group, cell cycle distribution and apoptosis showed distinct after the period of time with EGF. EGF could inhibit the apoptosis of the epithelia from mice at 32 h and 48 h, as well as inhibit the apoptosis of the epithelia from rabbits at 16 h. In addition, the effect of anti-apoptosis showed dose-dependent in the epithelia from mice at 48 h. Conclusion EGF could inhibit the apoptosis of the lingual dorsum epithelial cells from mice and rabbit by altering the cell cycle distribution.

The Rapid Construction of Rabbit HPRT Promoterless Gene-targeting Vector with PCR Product

GUO Yi1，ZHANG Chuan-shan2，GU Rui-huan1，YAO Gang2，CHEN Xue-jin1

2009, 29(2):  86-92. 

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Objective To construct a knockout HPRT targeting vector and a promoterless knockin targeting vector,which can be used for making rabbit HPRT gene knockout models and transgenic rabbit in the future. Methods The rabbit full length HPRT gene BAC clone LBNL1-304M19 is used as the template. A 12.5kb rabbit HPRT gene fragment，which does not include promoter, is cloned into pKS-plasmid to form pKS-rHPRT recombinant plasmid via Gap-Repair by Red recombination system. Then, the PCR was used to obtain the IRES-eGFPCre-Frt/Neo/Frt fragments flanking with 50bp homologous arms for homologous recombination of rabbit HPRT gene on the basis of pKS-rHPRT and pIGCN21 plasmids. A knockout HPRT taigeting vector by replacing the last three exons with a IRES-eGFPCre-Frt/ Neo/Frt cassette and a promoterless knockin targeting vector by inserting a IRES-eGFPCre-Frt/Neo/Frt cassette in the 3’UTR of the HPRT gene were rapidly constructed. Results The identification of the vectors with PCR,enzyme restriction and sequence showed two vectors were constructed successfully. Conclusions We rapidly constructed a knockout HPRT taigeting vector and a promoterless knockin targeting vector, the efficiency of deleting or inserting DNA fragment by homologous recombination technology has also been studied.

Identification and Susceptibility Test of Bacteria Isolated from the Trachea and Ileocecal Junction of Grey Hameters from Xinjiang Area of China

GAO Zheng-qin1，HE Zheng-ming1，ZHANG Qiang2，YUE Bing-fei1，XING Jin1，SHI Ji-chun1，FENG Yue-fang1，WANG Shan-shan1，YE Qiang1

2009, 29(2):  93-99. 

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Objective To investigate the prevalence and the antibiotic resistance of bacteria isolated from the trachea and ileocecal junction of grey hamsters (Cricetulus migratorius) which came from Xinjiang area of China. Methods Twenty two bacterial strains were isolated, which were identified by biochemical assays and were evaluated for resistance to thirty six kinds of antibiotics. Results The identification results showed that the twenty two strains belonged to Escherichia (Escherichia coli), Pseudomonas (Pseudomonas aeruginosa), Staphylococcus (Staphylococcus sciuri), Pasteurella (Pasteurella pneumotropica, Pasteurella aerogenes and Pasteurella multocida) and Bacillus (Bacilluspumilus, Bacillus alvei, Bacillus licheniformis and Bacillus cereus). These bacteria were susceptible to tetracycline and levofloxacin. However, their resistence to ampicillin and penicillin G were found. Conclusion Microbial population carried by grey hamster in China had characters of diversity. Results of this study provided scientifical accordance for the microorganism monitoring of grey hameters in China.

Effects of hVEGF Genetically Engineering Membrane on Expression of Fn and Integrin α5 During Healing Process of Full-thickness Cutaneous Defect of Rats

ZHAO Lei1，WANG Li1，JIANG Xu-cheng1，ZHEN Lin1，ZHANG Fan1，ZHAO Han-fang2，XU Wei-rong2

2009, 29(2):  100-104. 

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Objective To study the effects of hVEGF on the expression of Fn and integrinjA5 during the healing process of rats，full-thickness cutaneous defect which covered by genetically membrane planted by fibroblast integrated with hVEGF recombinant. Methods Wounds with full-thickness cutaneous defect were produced on the dorsum of the SD rats. The rats were randomly divided into four groups. For the rats in the experimented group，the wounds were covered with hVEGF genetically engineering membrane. For the three control groups，the wounds were covered with membrane planted by the fibroblast integrated with blank plasmid，blank membrane and Tegaderm membrane respectively. Specimens were obtained at the 3,7,14 and 29 days after injury, immunohistochemistry was employed to detect the changes in Fn and integrina5 expression, image analysis was used to determine changes of these expression level by detecting the mean optical density value of their expression. Results The expression level of Fn in experimental group was significant higher than that in control groups at 3, 7 and 14d after injury, by detecting the mean optical density value of its expression (P＜0.05). At 14 and 29d after injury, the expression level of Integrina5 in experimental group was significant higher than that in control groups (P＜0.05). Conclusion The application of the hVEGF genetically engineering membrane on the full-thickness cutaneous defect upregulated the expression of Fn in experimental group, especially in the early and metaphase of wound healing. Integrina5 expression significantly increased at metaphase and later period of wound healing. These could accelerate the healing process.

Screening of Microsatellite Primers Used for Genetic Mark of Closed Colony in Beagle Dogs

SUN Zhao-zeng，ZENG Lin，ZHAO Tai-yun，KONG De-qiang，HONG Bao-qing，HU Zhong-ming

2009, 29(2):  105-108. 

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Objective To select the effective microsatellite primers and analyze the genetic circumstance of individual Beagle dogs. Methods Twenty-four pair of primers with the heterozygotic degree between 0.4?0.8 were selected from the literature. The primers were authenticated used the mix-genome of 10 Beagle dogs. Results Among the 24 pair primers, 7 pair of primers were polymorphism and reproducibility. The 7 pair of primers used the mix-genome of 10 Beagle dogs of MaSi corporation were also tested，The results showed obviously different from Beagle dogs of our plant. The genetic circumstance of 6 individual Beagle dogs were also analyzed used the same 7 pair primers, four of them showed obviously different. Conclusion The 7 pair primers were suitable for analyzed the genetic circumstance of individual Beagle dogs primarily.

Analysis of Microsatellite DNA Polymorphisms and Genetic Monitoring of Rat

WEI Xiao-feng，XIE Jian-yun，HU Jian-hua，GAO Cheng

2009, 29(2):  109-112. 

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Objective Choose a microsatellite loci with better polymorphism to analyse DNA polymorphism of 6 strains of rat with a view to establishing a reliable and accurate, efficient and feasible genetic monitoring method. Method Fifteen rat microsatellite loci on different chromosomes were used to analyse the microsatellite DNA polymorphism among 6 rat strains by PCR methods. Result The nine microsatellites DNA could be amplified efficiently and displayed polymorphism among the various strains and no polymorphism in the same rat strains. Conclusion With the continuous deepening study of microsatellite DNA polymorphism of rat, the microsatellite DNA markers can replace the biochemical analysis，and be widely used in genetic monitoring.

Analysis of Genetic Stability of on Closed Colony Mice by mtDNA Sequence

GUAN Min-qiang1，CAO Qiong-jie2，CHEN Zhong-yi2，RUAN Dong-fen2，Lou Zhe-feng2，JIN Long-jin2

2009, 29(2):  113-116. 

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Objective To analyse genetic polymorphisms and genetic stability in mitochondrial ATPase6 and D-Loop gene of Closed Colony mice. Methods Twenty male ICR mice from 1000 mice of stochastic colony (offspring of 100 female parent mice) were studied, mitochondria ATPase6 gene and D-Loop gene of them were amplified respectively by using PCR of two pairs primers. The results from DNA sequencing were compared with the standard sequence of mice. Results The sequence of mtATPase6 gene and D-Loop gene was same in twenty ICR mice. Conclusion Polymorphismof mitochondrial DNA was not found. The ICR mice colony showed good genetic stability.

A Effective Heterotopic Heart Transplantation Model in Rat

ZHOU Yun，GUO Kun-liang，YANG Shao-jun，YANG Da-kuan

2009, 29(2):  117-119. 

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Objective To establish a quick and effective heterotopic heart transplantation model in rat. Method Male SD rats served as donors and Wistar as recipients,the ascending aorta of allograft was anastomosed to the recipient abdominal aorta, the recipient underwent a left nephrectomy, and the right pulmonary artery of allograft inserted into the left renal vein of donors. Results The mean ischemic time was (37.8 ±1.8) min in 35 experiments. The successful rate was 91.4%. Complications were anastomotic orificces hemorrhange, thrombosis. Conclusion This method has advantage of simpleness, and requiring no operating microscope. So it is easily to be applied.