Pcdh mcs t2a puro mscv vector

The Sp110 ORF sequence was amplified by PCR using cDNA from C57BL/6 mouse lung. The lentiviral expression vector pCDH-Sp110 was generated by inserting Sp110 ORF sequence into the pCDH-MCS-T2A-Puro-MSCV vector (System Biosciences, Mountain View, CA). To generate lentiviral vector expressing Bmf, the ORF sequences encode isoforms of Bmf were amplified by PCR using different upstream primers and a common downstream primer, and a FLAG tag was fused at the N-terminus, the resulting fragments were cloned into pCDH-MCS-T2A-Puro-MSCV, respectively. To construct the pCDH-miR-125a vector, DNA sequence of the primary miR-125a was amplified and inserted into the pCDH-CMV-MCS-EF1-copGFP vector (System Biosciences). To construct the luciferase reporter plasmids, the 3′ UTR sequence of mouse Bmf mRNA was amplified by RT-PCR and cloned into psiCHECK-2 (Promega, Madison, WI). The mutations in the 3′ UTR of mouse Bmf mRNA was introduced by overlap extension PCR. To generate stably expressed cells, RAW264.7 cells were transduced with viral supernatants collected from the HEK293T cells transfected with lentiviral constructs. After 48 h transduction, puromycin (5 μg/ml) was added to the dishes for additional 5 d to screen stably transfected cells, and expression of target genes was verified by immunoblotting.