Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells

Abstract

The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase. The efficiency of iPS cell derivation with SB transposon system was in the range of that obtained with retroviral vectors. Co-expression of the miRNA302/367 cluster together with OSKM significantly improved reprogramming efficiency and accelerated the temporal kinetics of reprogramming. The iPS cells displayed a stable karyotype, and hallmarks of pluripotency including expression of stem cell markers and the ability to differentiate into embryoid bodies in vitro. We demonstrate Cre recombinase-mediated exchange allowing simultaneous removal of the reprogramming cassette and targeted knock-in of an expression cassette of interest into the transposon-tagged locus in mouse iPS cells. This strategy would allow correction of a genetic defect by site-specific insertion of a therapeutic gene construct into ‘safe harbor’ sites in the genomes of autologous, patient-derived iPS cells.

Reprogramming Mouse Fibroblasts with piggyBac Transposons

“…The efficiency of the two systems is comparable, and single vectors allowing the expression of the entire set of reprogramming factors have been developed for both. Furthermore, an additional improvement was to build a recombinase- mediated cassette exchange system into the reprogramming single transgene, which permits their replacement in any other transgene once the expression of the reprogramming factors is no longer needed (Grabundzija et al 2013;Haenebalcke et al 2013 Monothioglycerol is less volatile than b-ME and can be used as a substitute typically at a final concentration of 0.15 mM.…”

Sleeping Beautytransposition: from biology to applications

“…The list includes somatic or germ cells, differentiated or stem cells essentially in all vertebrate species ( Figure 5). SB is suitable for genetic modification of both overexpressing and knocking down (Hu et al, 2011) transgene expression, and can be combined with other recombination techniques (Grabundzija et al, 2013) or delivery approaches (non-viral/viral). Indeed, in the last decade a whole technology platform, a Figure 6.…”

“…The characterized expression cassette can be exchanged relatively simply when the SB cassette is combined with other molecular engineering tools (Grabundzija et al, 2013;Petrakis et al, 2012). A powerful strategy could be used to express a series of expression cassettes from the same genomic locus by cassette exchange using Cre or FLP recombinases (Garrels et al, 2016a;Grabundzija et al, 2013).…”

Myocardial Reprogramming Medicine: The Development, Application, and Challenge of Induced Pluripotent Stem Cells

“…In PB protocols, a PB transposon vector containing reprogramming factors is transfected into MEFs and then removed without trace using PB transposase to generate stable integration-and mutation-free iPSCs with equivalent efficiencies to retroviral transduction [76,77]. Compared to the PB transposon system, SB transposon demonstrated several advantages, including higher transfer/integration efficiency and safety [78]. SB transposon containing OSKM can be cotransfected with hyperactive SB transposase to generate iPSC lines and then the integrations completely removed by transient transfection of excision SB transposase [79,80].…”

“…The exogenous genes integrated by other methods such as retroviral or lentivial vectors can also be removed using a Cre-loxP system to decrease the risk of potential insertional mutagenesis [81][82][83]. Interestingly, SB transposons combined with the Cre-loxP system can also be utilized to create integration-free iPSC lines [78]. In this protocol, the OSKM cassette is flanked by compatible loxP recombination sites in a SB transposon vector and then excised using Cre recombinase after iPSC generation (Figure 2(c)).…”