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.

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