Related Experiment Videos
[Construction of eukaryotic expression plasmid for mouse myogenic regulatory factor MyoD gene]
1Department of Maxillofacial Surgery, Qin-Du Stomatological College, Fourth Military Medical University, Xi'an Shanxi, P. R. China 710032.
Objective:
To construct eukaryotic expression plasmid of mouse myogenic regulatory factor MyoD gene for further study on MyoD gene function in molecular regulatory mechanism in skeletal muscle repair.
Methods:
The plasmids PEMMBC2 beta 5 containing full cDNA length of MyoD inserted in EcoRI restriction site, were first propagated in Escherichia coli DH5a, then extracted and purified with the Wizard Plus Minipreps DNA Purification System (Promega, USA). The coding sequence of MyoD in PEMMBC2 beta 5 was confirmed by agarose gel electrophoresis and DNA sequence analysis. After plasmids PEMMBC2 beta 5 and plasmids pcDNA3-neo were prepared by digestion with EcoRI, the MyoD cDNA fragment was inserted into EcoRI site in pcDNA3-neo eukaryotic expression vector, and pcDNA3/MyoD was formed. The pcDNA3/MyoD, digested with restriction enzymes, was found to contain the MyoD cDNA sequence by agarose gel electrophoresis analysis.
Results:
The extracted and purified PEMMBC2 beta 5 contained the correct nucleotide sequence for the full length of MyoD cDNA fragment. The MyoD cDNA fragment had been inserted into the eukaryotic expression plasmid pcDNA3-neo, which formed the pcDNA3/MyoD.
Conclusion:
The pcDNA3/MyoD, a eukaryotic expression plasmid, for MyoD is constructed successfully.