pG-miR-7-Sponge转基因小鼠载体的构建及鉴定

Identification and construction of vector of pG- miR-7- Sponge transgenic mice

目的构建pG-miR-7-Sponge转基因小鼠的真核载体并检测其表达活性,为转基因小鼠的建立提供前期实验基础。方法利用miRNA sponge(海绵体)技术原理,合成miR-7-Sponge序列,酶切连接入真核表达载体eGFP-C2并转化Stbl3感受态细胞,克隆PCR、酶切和测序鉴定重组载体eGFP-C2-miR-7-Sponge(简称pG-miR-7-Sp);将重组载体pG-miR-7-Sp体外瞬时转染人肺癌95D细胞,荧光显微镜下观察细胞的GFP表达情况;Real-Time PCR检测细胞中miRNA-7的表达;MTT法检测细胞的增殖变化。结果经克隆PCR、酶切鉴定及测序分析,成功构建了真核表达载体pGmiR-7-Sp;pG-miR-7-Sp瞬时转染95D细胞48 h后,与对照组细胞相比,miR-7表达水平明显降低(P<0.05);同时细胞的生长明显加快(P<0.05)。结论成功构建pG-miR-7-Sp真核表达载体。

Objective To construct the vector of pG -miR -7 -Sponge transgenic mice model and detect its expression. Methods According to miRNA sponge technological rule, the chemically synthesized miR - 7 - Sponge sequence was linked to the eukaryotic expression vector eGFP - C2. The recombinant vector （termed as pG - miR -7 -Sp） was transformed into the competent cells Stbl3 and analyzed by colon PCR, restriction enzyme di- gestion and DNA sequencing. Subsequently, pG - miR - 7 - Sp was transiently transfected into human lung cancer 95D cells and then the GFP expression was observed by fluorescence microscopy. The miR-7 expression was detected by real - time PCR assay. The cell proliferation was determined by MTT assay. Results The recom- binant eukaryotic expression plasmid pG - miR - 7 - Sponge was confirmed through DNA recombinant technique, enzyme cleavage and DNA sequencing. Forty -eight h later, the miR- 7 expression was significantly decreased in pG- miR-7- Sp transfected group compared with in the control group （P 〈 0.05）. Conversely, the cell proliferation was markedly induced in vitro （ P 〈 0. 05 ）. Conclusion The recombinant eukaryotic expression vec- tor pG - miR - 7 - Sponge is successfully constructed, which provides the fundamental basis for the successive research work related to transgenic mice.