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Generation of Viable Systemic Chimeras with Rabbit Induced Pluripotent Stem Cells Reprogrammed with KLF2, ERAS and PRMT6
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International audience. Embryo-derived and induced pluripotent stem cells (ESCs and iPSCs, respectively) exist in two different states, designated naive and primed. Naïve and primed states differ by the signalling pathways, transcription factors and epigenetic regulators that hold the cells in one of either state. In rabbits, ESCs and iPSCs only exist in the primed state of pluripotency.We developed a strategy that led to identify factors capable of reprogramming rabbit iPSCs to a naïve-like, embryo-colonization competent state. We conducted an unbiased screening of a cDNA library encoding a panel of 36 factors, including transcription factors, epigenetic regulators and signalling molecules associated with naïve-state pluripotency in rodents and primates. All the factors were randomly introduced into rabbit primed iPSCs by means of lentivector infection, followed by clonogenic growth in a FGF2/KOSR-deprived culture media containing LIF, activin A, PKC and WNT inhibitors. The three transgenes most frequently detected were PRMT6 that encodes an arginine methyl transferase, ERAS that encodes a GTPase, and KLF2 that encodes Krüppel-like factor 2.To investigate the transgene action, KLF2, ERAS and PRMT6 were overexpressed, either separately, in pairwise combinations, or all three together, in rabbit iPSC cells, either constitutively or with induction. KLF2 was necessary and sufficient to sustain self-renewal in a naïve-like state. However, the addition of both ERAS and PRMT6 led to the appearance of a subset of cells expressing the naïve state-specific marker CD75 at a high level. These CD75high cell population underwent epigenetic reconfiguration typical of naïve-state pluripotency including reactivation of the 2nd X-chromosome.To investigate the ability of KLF2/ERAS/PRMT6 reprogrammed iPSCs to generate chimeras, we developed iPSC line that express the three factors in a reversible manner, using FKBP12 degron. Remarkably, CD75high cells displayed the ability to produce chimeric foetuses, newborns, and viable adults with almost 100% efficiency. The injected cells showed a high contribution in all major organs of the analysed foetuses and newborns.Our results describe systemic chimeras in rabbits for the first time. They pave the way for the creation of knock-out rabbits to study development in lagomorphs, and for the creation of rabbit models of human genetic diseases.
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