Pcdh ef1 mcs t2a puro vector

As described in our previous study (12 (link)), full-length human NPM1-ALK cDNA (a gift from Dr. Toshiki Watanabe, The University of Tokyo, Japan) was amplified by PCR. The PCR product was then subcloned between XbaI and NheI restriction sites into the lentiviral plasmid pCDH-EF1-MCS-T2A-Puro vector (purchased from System Biosciences, Palo Alto, CA, USA). The clones were confirmed through Sanger DNA sequencing. The lentiviral plasmids, either empty vector pCDH-EF1-MCS-T2A-Puro or recombinant plasmid with FLAG (FG) tag, pCDH-EF1-FG-NPM1-ALK were transfected along with packaging and envelop plasmids psPAX2 and pMD2.G into HEK293T cells. The empty vector or pCDH-EF1-FG-NPM1-ALK lentiviral particles were transduced into Ba/F3 cells and stable clones were selected with puromycin. The Ba/F3-pCDH-vector cell growth was IL3-dependent while the transformed Ba/F3-FG-NPM1-ALK exhibited IL3-independent growth. The NPM1-ALK fusion gene mRNA expression levels were confirmed by RT-qPCR and protein levels were determined by Western blot using FLAG and NPM1-ALK antibodies.