[Establishment of BV2 cell line with steady knockdown of Mg 2+ /Mn 2+ -dependent protein phosphatase 1A(PPM1A)].

Objective To construct a short hairpin RNA (shRNA) lentiviral of Mg2+ /Mn2+ -dependent protein phosphatase 1A (PPM1A) gene and establish mouse brain microglia cell line(BV2) which PPM1A was knocked down stably. Methods According to the Coding sequence (CDS) region of PPM1A gene, two pairs of short hairpin RNA (shRNA) sequences (shRNA1, shRNA2) were designed and cloned into GV493 vector, then it was transformed into competent Escherichia coli DH5α strain and cultured. The positive clones were picked for sequencing. Finally, lentiviral packaging and titer determination were performed. Two groups of lentiviruses were transfected into BV2 cells and screened with puromycin. Fluorescence microscopy and flow cytometry were used to observe the expression of green fluorescent protein (GFP), and the expression of PPM1A were evaluated by RT-qPCR and Western blot analysis. Results The PPM1A shRNA lentiviral expression vector was successfully constructed. The expression of GFP was more than 80%. Compared with the control group, the mRNA and protein levels of PPM1A were significantly decreased in shRNA1 and shRNA2 knockdown groups. Conclusion The PPM1A knockdown BV2 cell line was successfully constructed using the lentiviral shRNA.