ABSTRACT Primary cells derived from human or animal tissues provide physiologically relevant models in which to study regulatory elements in genes and the function of the transcription factors associated with them. Unfortunately, most primary cells are notoriously difficult to transfect, with transfection efficiencies often below 1% using common transfection methods. Thus, techniques such as RNA interference (RNAi) and transient chromatin immunoprecipitation assays are not feasible with these valuable experimental models. Here, we report the optimization of the Amaxa NucleofectorTM technology for the primary tubular gland cells of the chicken oviduct. Nucleofection involves the use of a modified electroporation procedure and a proprietary reagent to directly transfer DNA into the nucleus rather than into the cytoplasm. The transfection efficiency of the oviduct cells was increased from less than 1% to an amazing 60-70%. Interestingly, protein expression was observed within 4 hours of transfection. Furthermore, a small interfering RNA (siRNA) designed against a GFP expression plasmid (pmaxGFP) was successful in silencing GFP by about 85%. Thus, by using nucleofection the transfection efficiency of primary oviduct cells was increased to such an extent that a highly expressed mRNA was successfully targeted and silenced. This technical advance ensures that the chick oviduct model system will continue to be an important paradigm for investigating the mechanism of action of estrogen and opens the way for comparable experiments in other reproductive tissues.
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