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Optimized expression vector for ion channel studies in Xenopus oocytes and mammalian cells using alfalfa mosaic virus

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Abstract

Plasmid vectors used for mammalian expression or for in vitro cRNA translation can differ substantially and are rarely cross-compatible. To make comparisons between mammalian and Xenopus oocyte expression systems, it would be advantageous to use a single vector without the need for shuttle vectors or subcloning. We have designed such a vector, designated pUNIV for universal, with elements that will allow for in vitro or ex vivo expression in multiple cell types. We tested the expression of pUNIV-based cDNA cassettes using enhanced green fluorescent protein and two forms of the type A γ-aminobutyric acid receptor (GABAAR) and compared pUNIV to vectors optimized for expression in either Xenopus oocytes or mammalian cells. In HEK293 cells, radioligand binding was robust, and patch clamp experiments showed that subtle macroscopic GABAAR kinetics were indistinguishable from our previous results. In Xenopus oocytes, agonist median effective concentration measurements matched previous work using a vector optimized for oocyte expression. Furthermore, we found that expression using pUNIV was significantly enhanced in oocytes and was remarkably long-lasting in both systems.

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Acknowledgement

We thank Dr. Cynthia Czajkowski for support and encouragement and Mark Nowak for helpful discussions. This work was funded by National Institute of Health grants MH66406 to C.C./A.B. and NS34727 to C.C.

Jeremy D. Bushman and José L. Mercado contributed equally to this work.

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Authors and Affiliations

  1. Department of Physiology, University of Wisconsin-Madison, 601 Science Drive, Madison, WI, 53711, USA

    Srinivasan P. Venkatachalan, Jeremy D. Bushman & Andrew J. Boileau

  2. Neuroscience Training Program, University of Wisconsin-Madison, 601 Science Drive, Madison, WI, 53711, USA

    José L. Mercado & Feyza Sancar

  3. Molecular and Cellular Pharmacology Program, University of Wisconsin-Madison, 601 Science Drive, Madison, WI, 53711, USA

    Kelly R. Christopherson

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  1. Srinivasan P. Venkatachalan
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  2. Jeremy D. Bushman
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  3. José L. Mercado
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  4. Feyza Sancar
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  5. Kelly R. Christopherson
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  6. Andrew J. Boileau
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Correspondence to Andrew J. Boileau.

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Venkatachalan, S.P., Bushman, J.D., Mercado, J.L. et al. Optimized expression vector for ion channel studies in Xenopus oocytes and mammalian cells using alfalfa mosaic virus. Pflugers Arch - Eur J Physiol 454, 155–163 (2007). https://doi.org/10.1007/s00424-006-0183-1

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