AML1-MTG16融合基因的构建及其致瘤性的研究

Construction and tumorigenic study on a novel fusion gene AML1-MTG16

目的 研究用重叠延伸剪接术 (splicing overlaping extension,SOE)方法获取少见融合基因的转录本。了解 AML1- MTG16融合基因的致瘤性。方法 通过 SOE重组 ,将 AML1、MTG16基因的两个片段经 PCR融合起来 ,形成 AML 1- MTG16融合基因的编码部分。将 MTG16 ,AML 1- MTG16融合基因及切除 AML1- MTG16融合基因 、 两个功能域 (motif)的基因片段 (AML1- MTG16 - S )分别插入p EGFP- C1,p Ds Red- N1载体中 ,利用脂质体转染 NIH3T3细胞 ,4 8小时后激光共聚焦显微镜观察。后G4 185 0 0 μg/ ml筛选 1月 ,获稳定转染细胞后绘制生长曲线 ;做软琼脂克隆形成实验及裸鼠致瘤实验。结果 测序结果表明 ,利用 SOE重组获得的 DNA片段含有 AML 1- MTG16融合基因完整的 CDS部分。比较p EGFP- C1、p EGFP- C1- AML1- MTG16质粒稳定转染的 NIH3T3细胞的生长曲线 ,后者增殖明显。软琼脂克隆形成试验显示 ,转染了 p EGFP- C1的 NIH3T3细胞在 6孔板内未形成克隆 ;转染了 p EGFP- C1-AML1- MTG16的 NIH3T3细胞在 6孔板内每孔平均形成 (47.7± 2 .7)个克隆 ,且使裸鼠致瘤。通过激光共聚焦显微镜观察 ,融合有 MTG16、AML 1- MTG16、AML 1- MTG16 - S 基因片段的绿荧光蛋白或红荧光蛋白在胞核表达 ,MTG16与 AML1- MTG16、AML1-

Objective To test whether splicing overlapping extension(SOE) method can be a tool for obtaining rare fusion gene's transcripts and to study the tumorigenic capacity of a novel fusion gene AML1 MTG16. Methods SOE method was used to obtain AML1 MTG16 fusion gene's transcripts. MTG16 , AML1 MTG16 and AML1 MTG16 without Ⅲ,Ⅵ conserved domains of MTG16 segment were inserted into pEGFP C1,pDsRed N1 vector respectively,then transfected NIH3T3 cell line by lipofection. Forty eight hours later, the transfected cells were examined by laser scanning confocal microscopy. Stable transfected cells were obtained by G418 500μg/μl selection for one month. Growth curve, soft agar colonies formation, tumorigenesis in nude mice were done to compare the difference between stable transfected cells. Results Recombined AML1 MTG16 by SOE contained its CDS. NIH3T3 expressing AML1 MTG16 had a faster proliferation in medium,colony growth in soft agar. AML1 MTG16 expression cells also induced tumors formation following injection into nude mouse. MTG16,AML1 MTG16 and AML1 MTG16 without Ⅲ,Ⅵ conserved domains of MTG16 were colocalized in the nucleus of cotransfected NIH3T3 cells under the examination of laser scanning confocal microscope. Conclusion SOE is an effective method to get rare fusion gene's transcripts. AML1 MTG16 plays an important role in leukemogenesis. MTG16 may also have a carcinogenic property within the AML1 MTG16 fusion gene.Carcinogenic property of AML1 MTG16 is restricted to its localization in the nuclear matrix.N terminal of MTG16 may play an important part in the carcinogenic activity of AML1 MTG16.