【摘要】 目的研究制备地高辛(DIG)标记的Tex13(testis expressed gene 13)基因非放射性原位杂交探针,用于检测小鼠性腺Tex13基因的表达。方法采用RT-PCR方法扩增Tex13靶序列,将其插入带SP6和T7 RNA聚合酶启动子的pGEM-T Easy质粒,转入大肠杆菌DH10B,经蓝白斑筛选获得重组质粒,用T7和SP6引物对重组质粒进行PCR扩增,以此PCR产物为模板,以DIG RNA Labeling Mix为底物,经SP6 RNA聚合酶和T7 RNA聚合酶体外转录,分别制备DIG标记的反义链和正义链RNA探针。结果应用正常小鼠睾丸组织非放射性原位杂交实验,证实制备的探针具有较高的特异性和敏感性。结论 DIG标记Tex13基因杂交探针的成功制备,为进一步研究Tex13基因在小鼠性腺中的表达定位和发育规律以及它在减数分裂过程中的作用提供了实验基础。更多还原

【Abstract】 Objective To prepare digoxigenin(DIG)-labeled non-radioactive in situ hybridization cRNA probes of testis expressed gene 13(Tex13 gene) in order to determine Tex13 gene expression in the mouse gonad.Methods RT-PCR was performed for amplification of Tex13 gene,and the PCR fragments were cloned into the polylinker site of the pGEM-T Easy vector which contained a promoter for SP6 and T7 RNA polymerase.DH10B competent cells were used for transformations and the recombinant clones were identified by blue/white screening.The recombinant plasmids served as templates for generation of PCR products of Tex13 gene with the SP6 and T7 primer.We used recovered PCR products to synthesize DIG-labeled anti-sense and sense RNA probes by in vitro transcription with SP6 and T7 RNA polymerases and DIG RNA Labeling Mix.Results Non-radioactive in situ hybridization studies on the expression of Tex13 gene in the normal mouse testis showed that the probes had relatively high specificity and sensitivity.Conclusion The successful preparation of DIG-labeled probes of Tex13 gene lays an experimental foundation for detecting the developmental expression pattern of Tex13 gene in the mouse gonad and studying its role in the meiosis.更多还原