KLF4 N-terminal variance modulates induced reprogramming to pluripotency.
Bottom Line: Yet, subtle differences in methodology confound comparative studies of reprogramming mechanisms.Strikingly, global gene expression patterns elicited by published polycistronic cassettes diverged according to each KLF4 variant.Our data expose a Klf4 reference cDNA variation that alters polycistronic factor stoichiometry, predicts reprogramming hallmarks, and guides comparison of compatible public data sets.
Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8507, Japan.Show MeSH
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Mentions: Reprogramming studies in mouse have made use of unique polycistronic factor arrangements and delivery vectors. For uniform evaluation of factor-order effects, we employed a fundamental reprogramming scheme based on factor transposition in MEFs (Woltjen et al., 2009). The PB transposon vector (PB-TAC) employs doxycycline (dox)-responsive reprogramming cassette expression co-incidentally with a mCherry reporter (Figure 1A). ROSA-rtTA; Nanog-GFP MEFs combine the m2-rtTA transactivator (Ohnishi et al., 2014) with a Nanog-GFP reporter (Okita et al., 2007). Thus, dox-responsive, PB-TAC-transgenic cells can be monitored throughout reprogramming initiation and maturation (day 2 [d2]–14) by mCherry fluorescence, while stabilization of pluripotency (d14–18) is indicated by activation of Nanog-GFP. Gain of factor independence through autonomous transgene silencing despite continued dox treatment is an established hallmark of the stabilization phase (Golipour et al., 2012), signaled here by a decrease in mCherry expression. For all polycistronic reprogramming cassettes tested, we routinely passaged populations on d8 and d18 using equal cell numbers without fractionation for extended culture and fluorescence-activated cell sorting (FACS) analysis. Dox-independent maintenance of iPSCs was verified by culture until d24, after which pluripotency was assayed by gene expression array and chimera contribution.
Affiliation: Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8507, Japan.