Benchmarks Gene Transfer into Retinoblastoma Cells

Abstract

Retinoblastoma cells have been widely used in retinal research for in vitro models of photoreceptors. Although attributes of “retinoblastoma cells” are still controversial (3,6), investigators use them in careful ways and under defined conditions of growth and induction such that they possess phenotypes of different retinal cells, e.g., cone and rod photoreceptors, ganglion cells and pigmented epithelial cells (3,6). Two widely studied, representative retinoblastoma cell lines are Y79 (7) and WERI (4). Transfection of retinoblastoma cells with reporter constructs and expression vectors is becoming increasingly important, allowing for studies involving, e.g., promoter identification with the goal of understanding retinal specific gene expression or the molecular biology of photoreceptor transformation. Common approaches for gene delivery are the use of liposomes (1,5,8) or viral vectors (Reference 2 and references therein). Viral-mediated gene transfer is efficient; however, it requires production of recombinant viruses using cell packaging and screening techniques. Transfecting retinoblastoma cells using liposomes is simpler, but current protocols are generally regarded as inefficient. It is therefore important to develop improved procedures for DNA transfer into these cells. Here, we assessed electroporation and use of liposomes for retinoblastoma cell transfection. We varied critical parameters after considering manufacturers’ recommendations, and compared their effects by measuring reporter gene products and cell viabilities after transfection. Electroporation was carried out using well-established conditions in 4-mm cuvettes (Cuvettes Plus; BTX, San Diego, CA, USA), in a volume of 300 μL of cells/DNA mixture using the Electroporator II (Invitrogen, Carlsbad, CA, USA), at a constant capacitance of 1000 μF and infinite load resistance. We tested the two most important variables: (i) the choice of electroporation buffer (Dulbecco’s phosphate-buffered saline (PBS), RPMI 1640 or Ham’s F-12; Life Technologies, Gaithersburg, MD, USA) and (ii) the voltage (200 or 300 V). After each electroporation cells were resuspended in 6 mL complete media and incubated for another 44 h at 30°C. For liposome-mediated transfection, three types of reagents were assessed

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Cite this paper

@inproceedings{Chau1999BenchmarksGT, title={Benchmarks Gene Transfer into Retinoblastoma Cells}, author={Kai - Yin Chau and Santa Jeremy Ono}, year={1999} }