Pires2 dsred2 vector

A humanized IgG₁ monoclonal antibody MAb0014 was chosen as the model antibody in this work. HC (gamma 1) and LC (kappa) cDNAs were commercially synthesized (Genscript) and cloned into separate expression plasmid vectors under the control of the same human cytomegalovirus (CMV) immediate-early enhancer and promoter. HC cDNA sequence was cloned into the pTracer-CMV2 vector (Invirtogen) which contained the zeocin resistance gene for selection of transfected cells. LC cDNA sequence was cloned into the pIRES2-DsRed2 vector (Clontech) with neomycin resistance gene. Heavy and light chain expression vectors were termed pH and pL, respectively. To construct UCOE containing vectors, synthesized 2.8 kb A2UCOE sequence (Genscript) was inserted into the upstream of CMV promoter of pH and pL vectors. These UCOE containing vectors were named pUH and pUL, respectively. The schematic maps of plasmid vectors used in this study are showed in Fig. 1. All vectors were constructed based on standard cloning methods [24 ]. The cloned sequences were confirmed by DNA sequencing.Fig. 1

The schematic structure of plasmid vectors constructed and used in the studies. a Heavy chain coding plasmid vectors; pTracer-CMV2-HC (pH) and pTracer-CMV2-UCOE-HC (pUH). b Light chain coding plasmid vectors; pIRES2-DsRed2-LC (pL) and pIRES2-DsRed2-UCOE-LC (pUL)

The pIRES-Cx43 construct was generated as previously reported [19 (link)] by ligating a PCR-amplified fragment encoding the rat Cx43 sequence (NM_012567) into the XhoI-BamHI sites of the bicistronic pIRES2-DsRed2 vector (Clontech, Palo Alto, CA, USA). The Cx43Y247F/Y265F mutation was introduced into pIRES-Cx43 by site-directed mutagenesis [19 (link)]. C6-Ires, C6-Cx43 and C6-Cx43 Y247F/Y265F clones were generated and characterized as previously described [19 (link)]. Unless otherwise specified, the C6 glioma cells were stably transfected with the empty vector (C6-Ires), the construct containing Cx43 (C6-Cx43) or the construct containing the mutant Cx43 (C6-Cx43Y247F/Y265F) using Lipofectamine 2000 (Life Technologies). For stable transfection cells were selected with 0.5 mg/ml G418 (Promega, Madison, WI, USA) in DMEM supplemented with 10% (v/v) FCS.
The pSG5L, pSG5L HA-PTEN, pCMV FLAG WT-PTEN and pCMV FLAG ΔPDZ-PTEN (1–400) plasmids were a gift from William Sellers and Hong Wu and were obtained from Addgene (Addgene plasmid 10737, 10750, 22231 and 22232, respectively) [58 (link), 59 (link)]. Cells were transiently transfected using Lipofectamine 2000, as described above.

The 6.74-kb pCMV-hFTSJ2-IRES2-DsRed plasmid was constructed by inserting the full-length human FTSJ2 (hFTSJ2) protein coding sequence into the EcoRI restriction site of the pIRES2-DsRed2 vector (Clontech Laboratories Inc., Mountain View, CA, USA). The TE671 and HepG2 cell lines were transfected with this plasmid via electroporation with a BTX ECM2001 system (BTX, Holliston, MA, USA). Briefly, 6×10⁶ TE671 or 2×10⁷ HepG2 cells were suspended in 400 µL of DMEM, which contained 5 µg or 50 µg of plasmid DNA, respectively, and then, the cells were subjected to electroporation at 200 V for 4 msec or 100 V for 30 msec, respectively. After electroporation, the cells were grown in a culture medium containing 400 µg/mL of the antibiotic G418 for the selection of cells that were stably expressing hFTSJ2.