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Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5
Published on: November 13, 2015
Foreign gene transfer to cardiomyocyte using a replication-defective recombinant coxsackievirus B3 without
Byung-Kwan Lim1, Soo-Hyeon Yun, Chae-Ok Gil
1Division of Cardiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Background:
Replication-competent coxsackievirus B3 (CVB3) has been used as a gene transfer vector for cultured cardiomyocytes and hearts in vivo. However, CVB3 induces cell lysis when it replicates in infected cells. In this study, we investigated whether a replication-defective rCVB3 vector could be generated and used as a noncytotoxic gene transfer vector for cardiomyocytes.
Methods:
We generated a replication-defective luciferase-expressing CVB3 plasmid. This recombinant cDNA and pCMV-P1 plasmids were amplified and cotransfected into Hek293 cells using transfection reagents. Replication-defective rLuCVB3 virus was recovered from the cells and cell culture supernatants for 3 days after transfection. The generated rLuCVB3 viruses were concentrated on a 30% sucrose cushion and semiquantified using a luciferase assay. In addition, foreign gene delivery by the rLuCVB3 was tested in cultured cardiomyocytes and intact mouse hearts after rLuCVB3 infection.
Results:
Luciferase was expressed in Hek293, HeLa cells and cardiomyocytes after rLuCVB3 infection. In addition, these cells did not show a significant cytopathic effect after 72 h. Luciferase protein expression or activity were detected for 3 days in the myocardium of rLuCVB3-infected mouse hearts without producing cytotoxicity or inflammation.
Conclusion:
As a proof-of-concept, these data indicate that a replication-defective rCVB3 vector can be generated and used as a novel gene transfer system to transfect exogenous genes into cardiomyocytes without generating cytotoxicity.
