OBJECTIVETo explore an efficient way to knockout microRNA genes in hemapoietic cell lines with a very low transfection efficiency, so as to facilitate the study of microRNA function in hematopoietic malignancies.

METHODSTALE-nucleases was utilized to knockout the microRNA-21 gene in human diffuse large B-cell lymphoma cells (OCI-Ly3). The OCI-Ly3 single cell clones without expression of miR-21 were established through eGFP(+) enrichment, PCR screening, and microRNA quantification. Finally, the miR-21 changes of mutant clones were identified by sequencing.

RESULTSFour miR-21-knockouted OCI-Ly3 single-cell-derived clones were established after 2 round transfection and screening. The miR-21 knockout efficiency was around 10/10(6) original cells. Sequencing the mutant clones indicated that miR-21 expression could be drastically reduced by simply altering sequences immediately adjacent to the microRNA duplex.

CONCLUSIONThis strategy may be applied to knockout any microRNA of interest even in hemapoietic cell lines with very low transfection efficiency.