Pcdh cmv mcs ef1 gfp t2a puro vector

The sequence validated I.M.A.G.E clone IRATp970E0579D corresponding to the BC053594 clone accession number was purchased from Source Bioscience, Nottingham, UK. This plasmid contains the wtPRRT2 cDNA sequence. Primers 5’ GAT CGA ATT CGT TTG CCG CTG TCT CT 3’ (Fw) and 5’ GAT CGC GGC CGC TCA CTT ATA CAC GCC 3’ (Rv) were used to amplify the wtPRRT2 cDNA and subsequently clone the fragment into the pCDH-CMV-MCS-EF1-GFP-T2A-Puro vector (System Biosciences). Primers 5’ GAT CGA ATT CGT TTG CCG CTG TCT CT 3’ (Fw) and 5’ GAT CGC GGC CGC CCC TTC TCA TTC GAT 3’ (Rv) were used to generate a truncated PRRT2 cDNA fragment. This fragment was integrated into the pCDH-CMV-MCS-EF1-GFP-T2A-Puro vector (System Biosciences) for expression of ΔPRRT2 protein. The amplified fragments were ligated to the vector using the Rapid DNA Ligation Kit according to the manufacturer's protocol (Roche). JM109 competent bacteria (Promega) were transformed with the ligation products using the heat shock method and plated in Ampicillin LB agar plates. DNA isolation was performed on selected colonies (Quiagen) and restriction enzyme reactions (BamH^I and Not^I ) confirmed the proper ligation of fragment to vector.

To generate the Rb1 (retinoblastoma-related gene 1) luciferase reporter plasmid (pGL-Rb1), the 3′ untranslated region (3′-UTR) encompassing the miR-155 binding site was cloned between the KpnI and XhoI restriction sites of the pGL-3 basic vector (Promega, Madison, WI, USA) using a Polymerase Chain Reaction (PCR)-generated fragment. The mutation in the miR-155 binding site of the human Rb1 3′-UTR was generated using overlapping PCR. For the ectopic expression of Rb1, the coding region of human Rb1 was cloned into the pCDH-CMV-MCS-EF1-GFP-T2A-Puro vector (System Biosciences, California, USA). Subsequently, virus packaging and infection were performed according to manufacturer’s instructions. The contents of all of these constructs were confirmed by sequencing. The primers used are shown in Additional file 3.