[Cloning of hsa-miR-148a and construction of its retroviral expression vector].
OBJECTIVE
To clone hsa-miR-148a and construct its retroviral expression vector.


METHODS
The pre-miR-148a amplified by PCR was inserted to pMSCV to construct the recombinant retroviral expression plasmid pMSCV-miR-148a, which was confirmed by restriction endonuclease analysis and DNA sequencing. The retroviral expression vector pMSCV-miR-148a and PIK packaging plasmid were cotransfected into 293FT packaging cells by calcium phosphate-mediated transfection to produce the retrovirus, and the retrovirus titer was measured by infection of NIH3T3 cells.


RESULTS
Restriction enzyme digestion and DNA sequencing demonstrated that the retroviral vector pMSCV-miR-148a was constructed successfully, and the virus titer was 5x10(8) CFU/ml after infection of NIH3T3 cells.


CONCLUSIONS
The successful construction of the retroviral expression vector MSCV-miR-148a allows the production of high-titer retrovirus to facilitate further study of the molecular functions of miR-148a.
Xu
Xue-hu
XH
Department of General Surgery, Third Affiliated Hospital of Guangzhou Medical College, Guangzhou 510150, China. maxtiger@126.com
Quan
Tian-yi
TY
Zeng
Wei-xia
WX
Chen
Xin-jie
XJ
Li
Wei-ming
WM
D003001:Cloning, Molecular; D019175:DNA Methylation; D005822:Genetic Vectors; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
C550570:MIRN148 microRNA, human; D035683:MicroRNAs