Evaluation of RNA interference in developing porcine granulosa cells using fluorescence reporter genes.
Gene silencing using small interfering RNA (siRNA) may be useful for functional analyses of unidentified genes expressed during cell differentiation. The present study was performed to evaluate RNA interference (RNAi) in porcine granulosa cells stimulated with bovine FSH, by using two fluorescence reporter genes: a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescence microscopy and analyzed by flow cytometry. Strong fluorescence was observed after introduction of both plasmids into cells. The intensity of green fluorescence generated by GFP was greatly suppressed by introduction of GFP-siRNA, showing an approximate 70% decrease in the ratio of green to red fluorescence. Consequently, we concluded that gene silencing by siRNA can be used to analyze the functions of genes of interest during differentiation of porcine granulosa cells.
Hirano
Takuro
T
Laboratory of Reproductive Physiology and Biotechnology, Department of Animal and Marine Bioresource Sciences, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki, Fukuoka, Japan.
Yamauchi
Nobuhiko
N
Sato
Fuminori
F
Soh
Tomoki
T
Hattori
Masa-aki
MA
D000818:Animals; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D006107:Granulosa Cells / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D034622:RNA Interference; D034421:Sus scrofa; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein