Multiple shRNA expressions in a single plasmid vector improve RNAi against the XPA gene.
To improve the efficiency of stable knockdown with short hairpin RNA (shRNA), we inserted multiple shRNA expression sequences into a single plasmid vector. In this study, the DNA repair factor XPA was selected as a target gene since it is not essential for cell viability and it is easy to check the functional knockdown of this gene. The efficiency of knockdown was compared among single and triple expression vectors. The single shRNA-expressing vector caused limited knockdown of the target protein in stable transfectants, however, the multiple expression vectors apparently increased the frequency of knockdown transfectants. There were correlations between the knockdown level and marker expression in multiple-expressing transfectants, whereas poorer correlations were observed in single vector transfectants. Multiple-transfectants exhibited reduced efficiency of repair of UV-induced DNA damage and an increased sensitivity to ultraviolet light-irradiation. We propose that multiple shRNA expression vectors might be a useful strategy for establishing knockdown cells.
Nagao
Akihiro
A
Laboratory of Human Molecular Genetics, Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192, Japan.
Zhao
Xia
X
Takegami
Tsutomu
T
Nakagawa
Hideaki
H
Matsui
Shinobu
S
Matsunaga
Tsukasa
T
Ishigaki
Yasuhito
Y
D002470:Cell Survival; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein / Q000037:antagonists & inhibitors* / Q000235:genetics
D034741:RNA, Small Interfering; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein