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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 study the rare fusion gene transcripts obtained by the method of overlap extension splicing (SOE). To understand the tumorigenicity of the AML1-MTG16 fusion gene. Methods Two fragments of AML1 and MTG16 genes were fused by SOE recombination to form the coding part of AML 1-MTG16 fusion gene. The MTG16, AML1-MTG16 fusion gene and the AML1-MTG16 fusion gene and the two gene motifs (AML1-MTG16-S) were inserted into pEGFP-C1 and pDs Red-N1 vector respectively. Lipofectamine was transfected into NIH3T3 cells and observed by laser scanning confocal microscopy after 48 hours. After G4 185 0 0 μg / ml was screened in January, the cells were stably transfected and the growth curve was drawn. The colony formation assay of soft agar and tumorigenicity in nude mice were performed. Results The sequencing results showed that the DNA fragment obtained by SOE recombination contained the complete CDS part of the AML 1-MTG16 fusion gene. The growth curves of NIH3T3 cells stably transfected with p EGFP-C1, p EGFP-C1-AML1-MTG16 plasmids were compared, and the latter showed significant proliferation. Soft agar colony formation assay showed that NIH3T3 cells transfected with p EGFP-C1 did not form a clone in a 6-well plate; NIH3T3 cells transfected with p EGFP-C1-AML1-MTG16 formed averagely in 6-well plates 47.7 ± 2 .7) clones, and tumorigenic nude mice. By confocal laser scanning microscopy, green fluorescent protein or red fluorescent protein fused with MTG16, AML1-MTG16, AML1-MTG16-S gene fragment was expressed in the nucleus, MTG16 and AML1-MTG16, AML1-