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Transfection of urothelial cells using methyl-β-cyclodextrin solubilized cholesterol and Dotap

Gene Therapy volume 8, pages 760–768 (2001)Cite this article

Abstract

The murine urothelial cell line, mb49 was transfected with the reporter gene pcmvlacz using a number of commercial transfection agents. the transfection efficiency of these agents, as determined by β-galactosidase activity, is in the order of dotap>superfect>Fugene. The addition of methyl-β-cyclodextrin solubilized cholesterol (MBC) to Dotap and Superfect further improved their transfection efficiency by 3.8-fold and 2.6-fold, respectively. β-Galactosidase activity was detectable within 1 h of transfection and peaked at 48 h. Nuclear and cytoplasmic separation showed that with Dotap + methyl-β-cyclodextrin solubilized cholesterol (DMBC), the DNA plasmid complex was found in both the nucleus and the cytoplasm. In vivo, murine bladders were transfected with an intravesical instillation of DMBC + DNA for 2 h. Two days later the bladder, lungs, liver, spleen and heart were assayed for the presence of the β-galactosidase gene by staining and PCR. Expression of the gene was confined to the bladder. Both in vitro and in vivo expression was observed after as little as a 15 min exposure to DMBC:DNA. Expression of the marker gene was present up to 30 days after transfection in vivo. From our data it appears that DMBC is the best nonviral agent for the transfection of urothelial cells in vitro and in vivo.

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Acknowledgements

We would like to thank Associate Prof Hui Kam Man and Dr Gao Hui of the National Cancer Centre for the synthesis of Dotap:cholesterol (1:1) and Associate Prof Bay Boon Huat of the Department of Anatomy, National University of Singapore for reviewing the electron micrographs of transfected bladder epithelial cells. We are grateful to Prof Leaf Huang (University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania) for helpful discussions. We would like to thank Associate Prof Eric Yap and Mr Jimmy Goh of the Defence Military Institute for their assistance and the use of their real time PCR machine. This work was supported by grants from the Singapore Cancer Society, National University of Singapore (GR6655) and the National Medical Research Council (NMRC/0170/1996) to Associate Prof Kesavan Esuvaranathan.

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  1. Department of Surgery, National University of Singapore, 10 Kent Ridge Crescent, 119260, Singapore

    C Lawrencia, R Mahendran & K Esuvaranathan

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  1. C Lawrencia
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  2. R Mahendran
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  3. K Esuvaranathan
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Lawrencia, C., Mahendran, R. & Esuvaranathan, K. Transfection of urothelial cells using methyl-β-cyclodextrin solubilized cholesterol and Dotap. Gene Ther 8, 760–768 (2001). https://doi.org/10.1038/sj.gt.3301462

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