Novel Gene Expression Cassette Design

The newly constructed expression cassettes are shown in Fig. 1a. The promoters, RU5′, BGH (bovine growth hormone) polyadenylation (polyA) signal, and a sequence for multiple cloning sites, were synthesized by IDT Inc. (Coralville, IA) and inserted into pDNR-1r promoter-less vector (Clontech, Mountain View, CA) or pIDT-SMART promoter-less vector (IDT Inc.). The RU5′ sequence (269 bp: Accession No. J02029 (374–642)) is derived from the R segment and a part of the U5 sequence of HTLV Type 1 long terminal repeat and used to enhance transcription efficiency [9 (link)]. Sequences of the promoter elements were as follows: hTERT (189 bp: Accession No. DQ264729 (1618–1806)), SV40 (319 bp: Accession No. AY864928 (2156–2474)), and CMV (479 bp: Accession No. AJ318513 (159–637)). The CAG promoter was obtained from the pCAGGS vector (a kind gift from Dr. Jun-ichi Miyazaki; Osaka University, Japan). pTracer-EF/V5-His-A and pEF6/Myc-His-A were purchased from Invitrogen. Full-length cDNAs of human S100A11, REIC/Dkk-3, CD133, LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5), telomerase, erythropoietin (EPO), and green fluorescence protein (GFP) were amplified by RT-PCR.Fig. 1

Schematic diagram of modified gene expression systems and their capabilities for gene expressions. a A series of indicated plasmids were constructed on the basis of the promoter-less pDNR-1r vector. b Expression of KLF16 protein was assessed by Western blot analysis after transfecting the indicated plasmids carrying KLF16 cDNA in HEK293, MCF7, PC-3, HeLa, and HepG2 cells. c Plasmid vectors carrying various cDNAs were constructed using the same series of vectors as those shown in (A). The vectors were transfected to HEK293 cells, and the level of each protein was determined by Western blot analysis. Lane numbers in b and c correspond to the vector numbers shown in (a)