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[Experimental Research of Circular RNA hsa_circ_0000254 in Acute Myeloid Leukemia].

OBJECTIVE
To clone the circular RNA hsa_circ_0000254 and construct its lentiviral over-expression vector.


METHODS
The sequence of hsa_circ_0000254 (a total of 524 bp long) was synthesized and cloned by using pGH vector. The vector was cut by EcoR I and BamH I, and artificial hsa_circ_0000254 was obtained, then inserted in pLCDH-ciR to construct the recombinant expression vector pLCDH-circ254(C254), which was confirmed by DNA sequencing. The lentiviral expression vectors pLCDH-circ254(C254) and NC(pLCDH-ciR) were cotransfected into 293T cells by lipofectamineTM 2000(lipo2000). After transfection for 40 hours, the cells were collected and verified by PCR and sequencing.


RESULTS
Restriction analysis and DNA sequencing demonstrated that the lentiviral vector pLCDH-circ254(C254) was constructed successfully, the expression efficiency increased 10000 times after transfection of cells.


CONCLUSION
The successful construction of the lentiviral expression vector pLCDH-circ254(C254) results in the production of high-titer lentivirus, so as to facilitate further study of the molecular functions of hsa_circ_0000254.