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Testis-specific fankl gene in knockdown mice produces oligospermia via apoptosis

Asian Journal of Andrology 2014年第1期16卷 124-130页

作者： Wan-Wei Dong Hua-Liang Huang Wei Yang Jia Liu Yang Yu Sheng-Lai Zhou Wei Wang Xiang-Chuan Lv Zhao-Yang LP Mei-Ying Zhang Zhi-Hong Zheng Wei Yan Laboratory Animal Centre China Medical University Shenyang China Department of Clinical Laboratory The Third Affiliated Hospital of Inner Mongonia MedicalUniversity Baogang Hospital Baotou China Department of Pathology and Pathophysiology Research China Medical University Shenyang China and Department ofPhysiology and Cell Biology University of Nevada School of Medicine Reno Nevada USA.

fankl is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of fankl by establishing a fankl-knockdown transgenic mouse model. The apoptotic statuses of the testes of the transgenic mice were tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） method. The FANK1 consensus DNA-binding sequence was identified using cyclic amplification of sequence target （CAST） analysis. Differentially expressed genes were examined using microarray analysis. A reduction in sperm number and an increase in apoptotic spermatocytes were observed in fankl-knockdown mice, and the apoptotic cells were found to be primarily spermatogonia and spermatocytes. The CAST results demonstrated that the consensus DNA-binding sequence was AAAAAG, in which the percentage occurrence of each base at each position ranged from 55 to 86%. This sequence was present in the promoter regions of 10 differentially expressed genes that were examined using microarray analysis. In total, 17 genes were differentially expressed with changes in their expression levels greater than twofold. The abnormal expression of fankl target genes that were regulated directly or indirectly by fankl reduced the number of sperm in the knockdown mice. Thus, FANK1 may Dlav a pivotal role in sDermato^enesis as a transcription *** is exclusively expressed in the testis from the meiosis phase to the haploid phase of spermatogenesis. In this study, we examined the function of fankl by establishing a fankl-knockdown transgenic mouse model. The apoptotic statuses of the testes of the transgenic mice were tested using the terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） method. The FANK1 consensus DNA-binding sequence was identified using cyclic amplification of sequence target （CAST） analysis. Differentially expressed genes were examined using microarray analysis, A reduction in sperm number an

关键词： fankl knockdown mice spermatogenesis

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人源Fank1蛋白质FN3结构域多肽的原核表达，纯化及晶体筛选

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医学分子生物学杂志 2009年第3期6卷 193-196页

作者：黄海涛宋伟缪时英王琳芳中国医学科学院基础医学研究所医学分子生物学国家重点实验室北京市100005

目的纯化人源Fank1（fibronectin type Ⅲ and ankyrin repeat domain1）蛋白质N端FN3（fibronectin typeⅢ，Ⅲ型纤黏连蛋白）结构域蛋白，用于晶体生长的三维结构分析。方法将FN3结构域基因片段克隆至原核表达载体pGEX-6P-1中，将菌落... 详细信息

目的纯化人源Fank1（fibronectin type Ⅲ and ankyrin repeat domain1）蛋白质N端FN3（fibronectin typeⅢ，Ⅲ型纤黏连蛋白）结构域蛋白，用于晶体生长的三维结构分析。方法将FN3结构域基因片段克隆至原核表达载体pGEX-6P-1中，将菌落PCR和测序鉴定正确的重组质粒转化*** BL21（DE3）后获得表达菌株。该菌株经IPTG诱导高效表达出带有GST标签的可溶性的融合蛋白，经过Glutathione Sepha-rose^TM 4B亲和层析、Hiload16／60 superdex200分子筛层析纯化后，蛋白纯度达到95％以上。结果纯化蛋白采用悬滴气相扩散法得到棒状晶体。结论成功制备了高纯度FN3蛋白，获得FN3蛋白质晶体，为进一步的三维结构解析及Fank1功能研究奠定了基础。

关键词： fankl FN3结构域表达蛋白质纯化晶体生长

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