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Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes

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Abstract

The Xenopus inner ear provides a useful model for studies of hearing and balance because it shares features with the mammalian inner ear, and because amphibians are capable of regenerating damaged mechanosensory hair cells. The structure and function of many proteins necessary for inner ear function have yet to be elucidated and require methods for analysis. To this end, we seek to characterize Xenopus inner ear genes outside of the animal model through heterologous expression in cell lines. As part of this effort, we aimed to optimize physical (electroporation), chemical (lipid-mediated; Lipofectamine™ 2000, Metafectene® Pro), and biological (viral-mediated; BacMam virus Cellular Lights™ Tubulin-RFP) gene delivery methods in amphibian (Xenopus; A6) cells and mammalian (Chinese hamster ovary (CHO)) cells. We successfully introduced the commercially available pEGFP-N3, pmCherry-N1, pEYFP-Tubulin, and Cellular Lights™ Tubulin-RFP fluorescent constructs to cells and evaluated their transfection or transduction efficiencies using the three gene delivery methods. In addition, we analyzed the transfection efficiency of a novel construct synthesized in our laboratory by cloning the Xenopus inner ear calcium-activated potassium channel β1 subunit, then subcloning the subunit into the pmCherry-N1 vector. Every gene delivery method was significantly more effective in CHO cells. Although results for the A6 cell line were not statistically significant, both cell lines illustrate a trend towards more efficient gene delivery using viral-mediated methods; however the cost of viral transduction is also much higher. Our findings demonstrate the need to improve gene delivery methods for amphibian cells and underscore the necessity for a greater understanding of amphibian cell biology.

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Acknowledgments

We would like to acknowledge Karla Almaraz for assistance with cell culture. Funding for this research was provided by Graduate Fellowships from NSF to DRG (New Mexico AMP, HRD-0331446; IGERT, DGE0504304) and NIH grants to EES (R01DC03292 and P50GM068762).

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  1. Department of Biology, New Mexico State University, P.O. Box 30001, MSC 3AF, Las Cruces, NM, 88003, USA

    Daniel Ramirez-Gordillo, Casilda Trujillo-Provencio, V. Bleu Knight & Elba E. Serrano

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  1. Daniel Ramirez-Gordillo
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  2. Casilda Trujillo-Provencio
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  3. V. Bleu Knight
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  4. Elba E. Serrano
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Correspondence to Elba E. Serrano.

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Editor: T. Okamoto

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Ramirez-Gordillo, D., Trujillo-Provencio, C., Knight, V.B. et al. Optimization of gene delivery methods in Xenopus laevis kidney (A6) and Chinese hamster ovary (CHO) cell lines for heterologous expression of Xenopus inner ear genes. In Vitro Cell.Dev.Biol.-Animal 47, 640–652 (2011). https://doi.org/10.1007/s11626-011-9451-2

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