Simple and efficient method for generation of induced pluripotent stem cells using piggyBac transposition of doxycycline-inducible factors and an EOS reporter system

Abstract

PiggyBac (PB) transposition of reprogramming factors (Oct3 ⁄ 4 (O), Sox2 (S), Klf4 (K) and c-Myc) is a safe, nonviral method for generating induced pluripotent stem cells (iPSCs). However, compared with retroviral methods, the reprogramming efficiency of the PB-mediated methods is relatively low. In this study, we describe a simple and efficient system for generating high-quality iPSCs by a single transfection of multiple plasmids that does not require the use of a virus, special instruments or skilled techniques. To improve reprogramming efficiency, we modified the components of the polycistronic 2A vectors used in this study and also investigated the combination of another reprogramming-related factor (L-Myc). By simultaneous transposition of multiple PB vectors containing an EOS (early transposon promoter and Oct3 ⁄ 4 and Sox2 enhancers) reporter and modified polycistronic doxycycline (Dox)-inducible factors, we reprogrammed mouse somatic cells with an efficiency higher than is usually obtained with retroviral methods and we established some iPSC lines that contributed highly to chimeras. By using the Dox-inducible system, we also showed that the appropriate elimination of exogenous-factor expression at appropriate time accelerated the induction of Oct3 ⁄ 4 when a combination of OKS and c-Myc vectors were used.

A Comprehensive System for Generation and Evaluation of Induced Pluripotent Stem Cells Using piggyBac Transposition

“…To generate PB-TRE3G-OKS and PB-TRE3G-c-Myc, pTRE3G-IRES (Clontech) was amplified by PCR and cloned into PB-hCMV*1-cHApA [27], [28] to yield PB-TRE3G-cHApA, and then inserts from PB-TET-OKS [8], [29] and PB-TET-c-Myc were introduced into PB-TRE3G-cHApA. To generate PB-(CAG-Tet3G; EOS-C(3+)-EGFP-IRES-puro), inserts from pCMV-Tet3G (Clontech) and PB-EOS-C(3+)-EiP [8], [9] were cloned into PB-CAG-rtTA_Adv [8], [29].…”

“…Instead, the PB system requires only a simple procedure consisting of a single transfection with multiple plasmids. Previously, we described a simple and efficient method for generating iPSCs by piggyBac transposition [8]. In this study, we modified our previous method to design a comprehensive and non-invasive system for generation and evaluation of iPSCs using simultaneous piggyBac transpositions of reprogramming factors and multiple fluorescent reporters.…”

“…In this study, we modified our previous method to design a comprehensive and non-invasive system for generation and evaluation of iPSCs using simultaneous piggyBac transpositions of reprogramming factors and multiple fluorescent reporters. The multiple-reporter system has three components: an EOS ( e arly transposon promoter and O ct3/4 and S ox2 enhancers)-EGFP reporter [8], [9] to detect pluripotent stem cells, a constitutively active CAG-TagRFP reporter [8], [10] to detect iPSC-derived cells in chimeric animals, and an acrosin reporter [11] to detect iPSC-derived spermatid and sperm in chimeric animals. Acrosin is expressed specifically in late spermatogenesis, and accumulates in acrosomes in spermatids and sperm [12].…”

Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools

“…Engineered transposable elements have been used to generate stably transfected CHO-and human embryonic kidney (HEK293)-derived cell lines for protein production (Alattia et al, 2013;Li et al, 2013;Matasci et al, 2011), and they have also served as gene transfer vectors for applications in gene therapy and gene discovery (Ding et al, 2005;Ivics et al, 1997;Mossine et al, 2013;Tsukiyama et al, 2011;Wu et al, 2006). piggyBac (PB), a class II transposable element originally derived from the cabbage looper moth, is one of the several transposons modified to function in mammalian cells (Fraser et al, 1996;Meir et al, 2011;Wilson et al, 2007).…”

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors

“…Cells were then transfected using Lipofectamine LTX (Invitrogen). Briefly, equal amounts (0.4 μg) of hyPBase vectors (pCAG-hyPBase) [32], PB vectors with reprogramming factors (PB-TET-OKS and pPB-TET-c-Myc) [33–35], the rtTA PB vector (PB-CAG-rtTA Adv, Addgene), and/or the TagRFP PB vector (pPBCAG-TagRFP-IH) [35], 2 μL of Plus reagent (Invitrogen) and 10 μL of Lipofectamine LTX transfection reagent were diluted and mixed in 400 μL Opti-MEM medium (Invitrogen). The DNA-lipid complexes were then added to the culture dish.…”