23 Jul Construction and application method of efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector
Abstract
The invention relates to construction of an efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector, belonging to the technical fields of animal gene engineering and animal molecule breeding engineering. The construction of the efficient double promoter PLEGFP-N1-spMyoD1 green fluorescence retrovirus vector is characterized by artificially synthesizing a skeletal muscle specific promoter sequence, using a RT-PCR method to amplify a gene overall length sequence from a skeletal muscle tissue of a pig, designing a fusion primer, using an overlapped PCR amplification method to obtain the promoter and a fusion gene sequence, cloning T/A to a pMD18-T vector, constructing the efficient expression vector of the PLEGFP-N1-spMyoD1 double promoter, and identifying with restriction enzyme and sequencing, packaging with a liposome method and amplifying, and monitoring virus titer and efficiency of infection. The invention has the beneficial effects: 1, Primer Premier 5.0, DNA Club and Oligo 6.0 are utilized to self-design the primer; 2, the restriction that an objective gene can not be subject to specific tissue expression in the past is overcome; 3, pertinency and accuracy of study on the objective gene functions are simultaneously improved; and 4, detection is fast and convenient, and has strong reliability.
Year: 2011
Country: CN
Doc No: 101993895