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

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.

Functional-genomic analysis reveals intraspecies diversification of antiviral receptor transporter proteins in Xenopus laevis

“…In addition to identifying RNA viruses that naturally infect amphibians and could therefore serve as models for RTP knockout studies, an important focus of future work will be the development of tissue culture-based models that permit the study of Xenopus RTPs and other antiviral effectors in a native cellular background. Of note, ectopic gene expression in A6 cells is inefficient [ 32 ], so it would be helpful to test other Xenopus cell lines as possible models.…”

Transgenic Xenopus laevis Line for In Vivo Labeling of Nephrons within the Kidney

“…Previously, an immortal kidney cell line (A6) was generated from spontaneous kidney tumor in X. laevis [ 41 ]. Although stable transgenic cell lines have been generated using A6 cells, this process is frequently difficult, because A6 cells grow slowly and transfection rates are low [ 42 , 43 , 44 ]. Additionally, these cells are a cancerous line that requires growth factors to survive [ 43 ].…”

Still Finding Ways to Augment the Existing Management of Acute and Chronic Kidney Diseases with Targeted Gene and Cell Therapies: Opportunities and Hurdles

“…Several reports have indicated inconsistent outcomes regarding the effectiveness of existing gene and cell transfer techniques. Studies in the kidney have illustrated this variability [153,154,[156][157][158][159][160][161][162]. In general, an in vivo gene and cell transfer system's success relies on various factors.…”