INIBIOLP   05426
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE LA PLATA "PROF. DR. RODOLFO R. BRENNER"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
A regulatable vector harboring the four Yamanaka genes for implementing rejuvenation by cell reprogramming
Autor/es:
CHIAVELLINI P; CANATELLI-MALLAT M; GOYA RG; LEHMANN M; CARRI, NG
Lugar:
Madrid
Reunión:
Conferencia; International Longevity and Cryopreservation Summit; 2017
Resumen:
In the last few years a growing body of evidence is emerging showing that animal and human cell rejuvenation can be achieved by cell reprogramming. Last December, it was 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 (known as the Yamanaka genes) in old progeric mice, transgenic for the Yamanaka genes. In order to implement cell and animal rejuvenation in old rodents we have constructed two plasmid vectors expressing the 4 Yamanaka genes under the control of a regulatable bidirectional promoter which also controls the expression of a reporter gene coding for green fluorescent protein (GFP). In both plasmids the 4 Yamanaka genes are arranged as a bicistronic expression tandem (the STEMCCA cassette, generously donated to us by Gustavo Mostoslavsky, Boston University). One of the plasmids harbors a Tet-Off regulatable system which activates the STEMCCA cassette in the absence of the antibiotic doxycycline (DOX), but silences it when DOX is present. The second plasmid is identical to the previous one except that it harbors a Tet-On regulatory gene which keeps the STEMCCA cassette inactive in the absence of DOX but turns on the Yamanaka genes when the antibiotic is present. We transfected each plasmid in HEK293 cells and characterized the regulatability of both systems by performing dose?response curves, measuring GFP fluorescence versus varying DOX concentrations and found the expected responses which we report here. The transfected cells were submitted to immunocytochemistry in order to verify the expression of the 4 Yamanaka genes and found that both plasmids, when activated, express the 4 genes. A proposed protocol for rejuvenating skin fibroblasts from old mice by means of in vitro cyclic partial cell reprogramming with our STEMCCA system will be described. The discovery of cell rejuvenation and whole animal rejuvenation by cell reprogramming is, in our opinion, opening a new horizon for the application of regenerative medicine to life extension.