Construction of molecular tools for implementing in vivo rejuvenation by cell reprograming.

LEHMANN M; CHIAVELLINI P; CANATELLI-MALLAT M; MULET AP; SCHLAPP G; MEIKLE MN; CRISPO M; COMINI M; REGGIANI PC; GOYA RG

Buenos Aires

Congreso; Segunda Reunión Conjunta de Sociedades de BioCiencias.; 2017

Sociedad Argentina de Investigación Clínica

It was recently reported that animal rejuvenation and life extension can be achieved by partial cell reprogramming using repeated cycles of expression and silencing of the four pluripotency genes Sox2, Oct4, Klf4 and c-Myc (the Yamanaka genes) in old mice, transgenic for the Yamanaka genes. We have constructed an expression vector harboring the 4 Yamanaka genes (the STEMCCA cassette) under the control of a bidirectional Tet-On or Tet-Off regulatable promoter. With the future aim to study the redox biology of cell reprogramming (collaboration with Institut Pasteur de Montevideo), a redox-sensitive GFP fused to a glutaredoxin (hGrx-roGFP2) was included into the STEMCCA plasmid. The redox biosensor is highly sensitive to detect small changes in the intracellular glutathione-dependent redox homeostasis. The regulatability of the plasmids by doxycycline (DOX) was characterized in HEK293 cells expressing the four Yamanaka genes as assessed by ICC. Subsequently, a plasmid harboring the final STEMCCA cassette flanked by two homology arms 1,200 and 3,200 bp long will be microinjected in one-cell embryos along with RNA encoding for the enzyme Cas9 and a guide RNA targeted to the Rosa26 locus. The embryos will be cultivated in M16+50ìM SCR7 until they reach a two-cell stage and further transferred to pseudopregnant surrogate females. Once the STEMCCA transgenic mice are generated, we will investigate in vivo rejuvenation by subjecting them to several cycles of partial reprogramming according to the procedure described by Ocampo et al (In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming Cell 167: 1719-1733 (2016)). In vivo rejuvenation by cell reprogramming is an emerging technology that is revolutionizing the field of the biology of aging.