Construction of mouse wide-type and mutant dynactin-1 vectors and their expression in mouse podocytes / 第二军医大学学报

ABSTRACT

Objective To construct mouse wide-type and mutant dynactin-1 expression vectors and investigate their expression in mouse podocytes. Methods Mouse cDNA was synthesized from mouse total RNA and was used as a template for PCR amplification to obtain full length dynactin-1 cDNA. The DNA fragment was then cloned into pcDNA3. l(+)-FLAG and pEGFP-Nl vector to produce wide-type dynactin-1 vector. The mutant dynactin-1 was obtained by site-direct mutagenesis kit. All the constructs were verified by restriction enzyme digestion, sequenced, and then transfected into mouse podocyte clone 5 (MPC5). Western blotting analysis and immunofluorescence microscopy were employed to determine dynactin-1 protein expression. Results The amplified mouse dynactin-1 cDNA fragment was analyzed by agarose gel electrophoresis and a single discrete band of the correct size (3. 8 kb) was observed. The vectors containing mouse dynactin-1 were subjected to restriction enzyme digestion and two vector fragments (pcDNA3. l[+]-FLAG(5. 4 kb) and pEGFP-Nl[4. 7 kb] individually) and the 3. 8 kb insert fragment were observed by electrophoresis. The result of sequencing showed that the sequence of cloned dynactin-1 was identical to that reported in Genbank. Dynactin-1 protein band with the correct relative molecular weight was detected by Western blotting analysis, and immunofluorescence microscopy showed dynactin-1 protein expression in the cytoplasm of the mouse podocytes. Conclusion We have successfully constructed wide type and mutant dynactin-1 vectors expressed them in mouse podocytes.