ObjectiveTo construct mouse wide-type and mutant dynactin-1 expression vectors and investigate their expression in mouse podocytes. MethodsMouse 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 pcDNA3.1(+)-FLAG and pEGFP-N1 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. ResultsThe 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 (pcDNA3.1\[+\]-FLAG(5.4 kb) and pEGFP-N1\[4.7 kb\] 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. ConclusionWe have successfully constructed wide-type and mutant dynactin-1 vectors and expressed them in mouse podocytes, which provides a foundation for future research on dynactin-1.