Protection from acute cellular injury using Sleeping Beauty mediated telomerase gene transfer.
We developed a Sleeping Beauty (SB) transposon mediated hTERT gene delivery system for in vitro use. We have constructed telomerase or luciferase gene expressing SB-transposons with a SV40 enhancer (pT3.hTERT.Con and pT3.Con, respectively) or without an enhancer (pT3.Pro). Using the SB transposon system in vitro hTERT gene overexpression has protective effects from acute cellular injury by tert-butyl hydroperoxide (t-BH), carbon tetrachloride (CCl(4)), and d-galactosamine (d-GalN) in normal human cells IMR-90. pT3.hTERT.Con vector and helper plasmid co-transfection resulted in a approximately 3-fold increase in telomerase activity which was maintained for 14 days. Trypan blue and Cell Death Detection Assays showed the protective effects of the telomerase gene against toxic agents. Fourteen days after co-transfection with pT3.hTERT.Con vector and helper plasmid, IMR-90 cells were incubated with 1.2mM t-BH for 50 min, 5mM CCl(4) for 1.5h or 30 mM d-GalN for 24h. Cell viability of SB-mediated telomerase overexpressing cells significantly increased by 48% (t-BH), 43% (CCl(4)), and 25% (d-GalN) in comparison to mock treated cells. Cell Death Detection ELISA showed a decrease in the rate of apoptosis by 47%. In summary, SB transposon mediated telomerase gene transfer may have a protective effect against t-BH, CCl(4), or d-GalN induced acute cellular injury, and this results suggested SB-mediated telomerase therapy for tissue engineering.
Song
Joon Seok
JS
University of Pittsburgh Medical Center, Division of Transplantation Pathology, Pittsburgh, PA 15213, USA.
Murase
Noriko
N
Demetris
Anthony J
AJ
Michalopoulos
George K
GK
Ochoa
Erin R
ER
D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D019610:Cytoprotection / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology*; D018014:Gene Transfer Techniques; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D019098:Telomerase / Q000235:genetics* / Q000378:metabolism; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
D019895:Transposases; C494471:sleeping beauty transposase, human; D019098:Telomerase