Guo Xiao-Ling, Niu Zhi-Yun, Zhang Gai-Ling, Yin Feng-Lei, Yang Ling, Ren Jin-Hai, Dong Zuo-Ren, Zhu Ping, Pan Ling
Department of Hematology, Key Laboratory of Heibei Institute of Hematology, 2nd Hospital of Hebei Medical University, Shijiazhuang 050000, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2012 Jul;43(4):498-502.
To construct the eukaryotic expression vector for Max interacting protein 1 (Mxi1).
The full length cDNA of Mxi1 gene obtained from fetal lymphocyte and KG1 cells were inserted into plasmid pDs-red2-N1 respectively to generate pDs-red2-N1/Mri1 (wild/mutation type). Then the recombinant vector was transfected into Cos-7 cells via liposome. 48 hours post transfection, mRNA of Mri1 gene was detected by RT-PCR and Mxi1 protein expression was detected by flow cytometry and fluorescence microscope in the Cos-7 cells.
The eukaryotic expression vector of Mxi1 was constructed and transfected into eukaryotic cells successfully. The expression of red fluorescence protein in the transfected Cos-7 cells was observed under fluorescence microscope which implied the expression of Mxi1. The transfect efficiency of both wild and mutation type were in a high level in 3 days after transfected, which lasted to 6 d. RT-PCR amplified the total RNA extracted from the transfected Cos-7 cells could find increased mRNA level of Mxi1 gene.
We successfully constructed the eukaryote expression vector for Mri1 gene; Cos-7 cells transfected with the vector via liposome could express Mxi1 protein. These could be useful for the further study of the Myc gene modulation.
构建Max相互作用蛋白1（Mxi1）的真核表达载体。
从胎儿淋巴细胞和KG1细胞中获取的Mxi1基因全长cDNA分别插入质粒pDs-red2-N1中，构建成pDs-red2-N1/Mri1（野生型/突变型）。然后通过脂质体将重组载体转染至Cos-7细胞。转染后48小时，采用RT-PCR检测Mri1基因的mRNA，并用流式细胞术和荧光显微镜检测Cos-7细胞中Mxi1蛋白的表达。
成功构建了Mxi1的真核表达载体并将其转染至真核细胞。在荧光显微镜下观察到转染的Cos-7细胞中有红色荧光蛋白表达，提示Mxi1表达。野生型和突变型转染效率在转染后3天内均处于较高水平，并持续至6天。对转染的Cos-7细胞提取的总RNA进行RT-PCR扩增，发现Mxi1基因的mRNA水平升高。
成功构建了Mri1基因的真核表达载体；通过脂质体转染该载体的Cos-7细胞能够表达Mxi1蛋白。这些结果对进一步研究Myc基因调控具有重要意义。
