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[Experimental Research of Circular RNA hsa_circ_0000254 in Acute Myeloid Leukemia]
1Ningbo Central Blood Station, Ningbo 315000, Zhejiang Province, China.
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.
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