AN INTERFERING RNA PROTOCOL FOR PRIMARY PORCINE ALVEOLAR MACROPHAGES

ABSTRACT

RNA interference (RNAi) is a cellular process of post-transcriptional gene silencing in which a short interfering dsRNA (siRNA, 21–23 nt) targets a homologous mRNA for degradation by ribonuclease. RNAi has been used successfully to inhibit targeted gene expression and viral replication in mammalian cells. In this study we established an RNAi transfection protocol for primary porcine alveolar macrophages and evaluated potential off-target effects of siRNA introduction into these cells. Porcine alveolar macrophages were transfected using a fluorescence-labeled siRNA to compare transfection reagents from different suppliers. Under optimized transfection conditions, up to 95% of macrophages were fluorescent at 12 and 24 h post-transfection using an amine-based transfection reagent. An siRNA targeting GAPDH suppressed macrophage endogenous GAPDH transcript levels as much as 60% through 24 h. Further, we did not detect a significant interferon response following siRNA transfection. These data suggest that RNAi will be an efficient and convenient approach for studying loss of gene function in primary porcine alveolar macrophages.

Keywords:

• SiRNA
• Macrophage
• Protein kinase R

ACKNOWLEDGMENTS

This work was supported in part by a grant from USDA-NRICGP, #2002-35204-12665. We thank Dr. Mitchell S. Abrahamsen for critical reading of this manuscript.