Pcmv myc mammalian expression vector

KCC2 cDNAs inserted in pCR-Blunt II-TOPO were mutagenized to introduce a unique SfiI site at position –5 (relative to start codon A¹TG) necessary for further sub-cloning/tagging. Mutagenesis was performed using the following sense/antisense primers: 5′-GTG CGA TCC CGC GGC CCC GGA GGC CAT GAG CCG C-3′ and 5′-GCG GCT CAT GGC CTC CGG GGC CGC GGG ATC GCA C-3′ (the SfiI site is underlined, first codon in bold). Mutagenized inserts were cloned into SfiI/XhoI sites of the pCMV-myc mammalian expression vector (Clontech, Mountain View, CA). After sequencing confirmation, pCMV-myc plasmids were transfected into COS7 or MIN6 β-cells to verify expression.

We have utilized mHypoA 2/28 cell line in this study, as this cell line was originally generated from the hypothalamus, and therefore, includes a broad library of hypothalamic neuronal phenotypes (44 (link), 45 (link)). mHypoA 2/28 cells (CELLutions Biosystems Inc. Ontario, Canada) were maintained in high-glucose Dulbecco’s Modified Eagle Medium (Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin (Hyclone) in a humidified atmosphere with 5% CO₂ at 37°C. When the cells had grown to 70% confluence, they were transiently transfected with expression vectors or small interfering RNA (siRNA) using jetPRIME^® reagent (Polyplus, New York, NY, USA): expression vector pCMV-myc containing the TonEBP coding region (a gift from Dr. H.M. Kwon, 500 ng) or control pCMV-myc; TonEBP siRNA (50 nM, sense sequence, 5’-GAC CAU GGU CCA AAU GCA A-3’; antisense sequence, 5’-UUG CAU UUG GAC CAU GGU C-3’) or control RNA (50 nM) with a scrambled sequence. pCMV-myc mammalian expression vector was originally developed from Clontech, and general information for this vector is available at http://www.takara.co.kr/file/manual/pdf/PT3282-5.pdf. To determine the effect of osmotic stress on TonEBP and AVP expression, the cells were treated with NaCl (50 or 100 mM) for 24 h, which did not induce cell death.