756240.fig.002a
(a)
756240.fig.002b
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756240.fig.002c
(c)
Figure 2: The inducible transgene and excisable system for iPSC generation. (a) The inducible transgene system conta ins two main components, reverse-tetracycline-dependent transactivator (rtTA) and the inducible promoter controlling the expression of reprogramming genes (OSKM). In the absence of doxycycline (Dox) induction, the specific promoter lacks binding sites for endogenous TFs, so it is virtually silent. In the presence Dox, the transactivator binds tightly and specifically to the promoter and activates transcription of OSKM genes. (b) The primary somatic cells (such as fibroblasts) were infected with inducible lentivirus encoding OSKM to generate “primary” iPSCs (1-iPSCs) with Dox. These were then injected into blastocysts to create chimeras or formed into embryoid bodies for the generation of “secondary” somatic cells. These then carry the inducible reprogramming factors, which were isolated and used to generate secondary iPSCs (2-iPSCs) using Dox induction. (c) The schematic of integration-free iPSCs generated by the transposon and Cre-loxP excisable system. The OSKM cassette was flanked by compatible loxP-loxP257 sites and inserted between the specific inverted terminal repeat (ITR) sequences of transposon vector. The insertion was excised (“cut”) by hyperactive transposase from ITR to integrate (“paste”) into the TTAA sites of chromosomal DNA in transfected somatic cells. After integration-iPSC generation, the reporter tracking gene (such as RFP) can be introduced by Cre recombinase which can specifically recognize loxP sites, resulting in the removal of OSKM. Finally, the integrated fragment can be re-excised by excision transponsase to generate integration-free iPSCs.