Suppression of transforming growth factor β1 in lung alveolar epithelium-derived cells using adeno-associated virus type 2/5 vectors to carry short hairpin RNA.
Since the discovery of RNA interference, short interfering RNA (siRNA) has become a standard research tool. However, expression of siRNA in lung alveolar epithelial cells has remained a problem. Adeno-associated virus (AAV) vectors are known to have low toxicity, and AAV type 5 vectors transduce these cells efficiently. In this study, LacZ expression was higher using AAV2/5-LacZ and LA-4 cells compared with transfection of plasmid or transduction to 3T12-3 cells. The authors designed 10 different siRNAs against mouse transforming growth factor β1 (Tgfβ1), selected one with the highest knockdown efficiency, and transduced the AAV vectors carrying the short hairpin RNA (shRNA) to target cells. The AAV vectors transduced LA-4 cells 50 times more efficiently than 3T12-3 cells, and suppression of Tgfβ1 protein expression was similar, at approximately 50%. Knockdown of mRNA was only seen in LA-4 cells. Inhibition of Tgfβ1 resulted in higher number of LA-4 cells, lower number of 3T12-3 cells, and decreased procollagen expression in LA-4 cells. Higher transduction was seen in H23 cells than in H1975 cells, and low transduction was seen MH-S cells. This study shows that AAV2/5 can be used to carry shRNA and suppress gene function in lung alveolar epithelium-derived cells.
Department of Respiratory Medicine, University of Tokyo, Tokyo, Japan.
Hidenori
H
Kage
Keiki
K
Sugimoto
Atsushi
A
Sano
Hiroshi
H
Kitagawa
Takahide
T
Nagase
Nobuya
N
Ohishi
Daiya
D
Takai
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D017931:DNA Primers / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D055785:Gene Knockdown Techniques / Q000379:methods; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007763:Lac Operon; D016676:Macrophages, Alveolar / Q000378:metabolism; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011347:Procollagen / Q000235:genetics; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection / Q000379:methods; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors* / Q000235:genetics*
D017931:DNA Primers; D011347:Procollagen; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1