| Autor: |
Kok CY; Centre for Heart Research, The Westmead Institute for Medical Research, Westmead, NSW 2145, Australia.; Westmead Clinical School, The University of Sydney, Westmead, NSW 2145, Australia., MacLean LM; Centre for Heart Research, The Westmead Institute for Medical Research, Westmead, NSW 2145, Australia., Rao R; Centre for Heart Research, The Westmead Institute for Medical Research, Westmead, NSW 2145, Australia., Tsurusaki S; Centre for Heart Research, The Westmead Institute for Medical Research, Westmead, NSW 2145, Australia.; Westmead Clinical School, The University of Sydney, Westmead, NSW 2145, Australia., Kizana E; Centre for Heart Research, The Westmead Institute for Medical Research, Westmead, NSW 2145, Australia.; Westmead Clinical School, The University of Sydney, Westmead, NSW 2145, Australia.; Department of Cardiology, Westmead Hospital, Westmead, NSW 2145, Australia. |
| Abstrakt: |
Lentiviral vectors are a robust gene delivery tool for inducing transgene expression in a variety of cells. They are well suited to facilitate the testing of therapeutic candidate genes in vitro, due to relative ease of packaging and ability to transduce dividing and non-dividing cells. Our goal was to identify a gene that could be delivered to the heart to protect against cancer-therapy-induced cardiotoxicity. We sought to generate a lentivirus construct with a ubiquitous CMV promoter driving expression of B-cell lymphocyte/leukemia 2 gene ( Bcl-2 ), a potent anti-apoptotic gene. Contrary to our aim, overexpression of Bcl-2 induced cell death in the producer HEK293T cells, resulting in failure to produce usable vector titre. This was circumvented by exchanging the CMV promoter to the cardiac-specific NCX1 promoter, leading to the successful production of a lentiviral vector which could induce cardioprotective expression of Bcl-2. In conclusion, reduced expression of Bcl-2 driven by a weaker promoter improved vector yield, and led to the production of functional cardioprotective Bcl-2 in primary cardiomyocytes. |
