Lentiviral Vectors for ASPP2 Isoform Expression

Generation of SIN.LV.SF-GFP-T2A-puro, SIN.LV.SF-T2A-puro (Empty Vector Fig. 6A) and SIN.LV.SF-tMYPT1-T2A-puro was previously described (14 ). t-ASPP2 was isolated with Age1-Sal1 or BamH1-Age1 overhangs and a 5’ FLAG tag from the Trp53bp2^ex13-18 pBABE puro vector previously described (14 ) using Phusion Flash High-Fidelity DNA Polymerase (Thermo scientific). The cDNA fragments were then inserted into SIN.LV.SF (34 (link)) or SIN.LV.SF-T2A-puro to generate SIN.LV.SF-t-ASPP2 and SIN.LV.SF-tASPP2-T2A-puro. T2-ASPP2 was isolated with BamH1-Age1 or BamH1-Sal1 overhangs and a 5’ FLAG tag from SIN.LV.SF tASPP2 using Phusion Flash High-Fidelity DNA Polymerase. The cDNA fragments were then inserted into SIN.LV.SF or SIN.LV.SF-T2A-puro to generate SIN.LV.SF t2-ASPP2 and SIN.LV.SF-t2-ASPP2-T2A-puro. SIN.LV.SF-t-ASPP2^ΔYAP1-T2A-puro and SIN.LV.SF-t-ASPP2^ΔPP1-T2A-puro were generated by site directed mutagenesis using the QuikChange Lightning site directed mutagenesis kit (Agilent). All primers are listed in Supplementary Table 1. Every vector was validated by Sanger sequencing. Concentrated lentiviral stocks were produced by transient co-transfection of four plasmids in 293T cells as previously described (33 (link)). Viral titers were determined using the qPCR lentivirus titration kit from Abm (LV900).