Summary
A large amount of research activity worldwide is currently directed towards developing lipid- or polymer-based, non-viral gene vectors for therapeutic applications. This strong interest is motivated by their low toxicity, ease of production, ability to transfer large pieces of DNA into cells, and lack of immunogenic protein components. Cationic liposomes (CLs) are one of the most powerful non-viral vectors. In fact, CL-based vectors are among the prevalent synthetic carriers of nucleic acids currently used in human clinical gene therapy trials as well as in cell transfection applications for biological research. Our understanding of the mechanisms of action of CL–DNA complexes is still in its infancy. However, the relevance of a few crucial parameters, such as the lipid/DNA charge ratio (\(\rho_{\rm chg}\)) and the membrane charge density of lamellar complexes (\(\sigma_{\rm M}\)), is well established. To arrive at true comparisons of lipid performance, one must optimize both these parameters using a reproducible, reliable transfection assay. In this chapter, we aim to provide the reader with detailed procedures for liposome formation and transfection. It is our hope that the use of such optimized protocols will improve the comparability of transfection data obtained with novel lipids.
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Acknowledgment
Our work is supported by the National Institutes of Health und grant number GM-59288. Our laboratory is indebted to Prof. C. E. Samuel and Dr. C. X. George for the introduction to and continued support regarding cell culture and biological protocols. We also thank past and present graduate students who have helped to develop the presented protocols: Dr. Nelle L. Slack, Dr. Alison J. Lin, and Alexandra Zidovska.
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Ewert, K.K., Ahmad, A., Bouxsein, N.F., Evans, H.M., Safinya, C.R. (2008). Non-Viral Gene Delivery with Cationic Liposome–DNA Complexes. In: Gene Therapy Protocols. Methods in Molecular Biology™, vol 433. Humana Press. https://doi.org/10.1007/978-1-59745-237-3_10
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DOI: https://doi.org/10.1007/978-1-59745-237-3_10
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