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Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice

Authors
 Bo Young Nam  ;  Dong Ki Kim  ;  Jung Tak Park  ;  Hye-Young Kang  ;  Jisun Paeng  ;  Seonghun Kim  ;  Jimin Park  ;  Jae Eun Um  ;  Hyung Jung Oh  ;  Seung Hyeok Han  ;  Tae-Hyun Yoo  ;  Shin-Wook Kang 
Citation
 American Journal of Physiology - Renal Physiology, Vol.309(12) : F1060-F1069, 2015 
Journal Title
 American Journal of Physiology - Renal Physiology 
ISSN
 1931-857X 
Issue Date
2015
MeSH
Animals ; Gene Knockdown Techniques ; Gene Transfer Techniques ; Genetic Vectors* ; Green Fluorescent Proteins/genetics ; Integrases/genetics* ; Kidney/cytology* ; Lentivirus ; Mice, Transgenic ; Promoter Regions, Genetic/genetics
Keywords
Cre/LoxP ; cell-specific knockdown ; lentiviral vector
Abstract
In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species.
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/156922
Full Text
http://www.physiology.org/doi/abstract/10.1152/ajprenal.00251.2015
DOI
10.1152/ajprenal.00251.2015
Appears in Collections:
1. Journal Papers (연구논문) > 1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실)
1. Journal Papers (연구논문) > 1. College of Medicine (의과대학) > Yonsei Biomedical Research Center (연세의생명연구원)
1. Journal Papers (연구논문) > 1. College of Medicine (의과대학) > BioMedical Science Institute (의생명과학부)
Yonsei Authors
강신욱(Kang, Shin Wook) ORCID logo https://orcid.org/0000-0002-5677-4756
강혜영(Kang, Hye Young)
김성훈(Kim, Seonghun)
남보영(Nam, Bo Young)
박정탁(Park, Jung Tak)
박지민(Park, Jimin)
엄재은(Um, Jae Eun)
오형중(Oh, Hyung Jung)
유태현(Yoo, Tae Hyun)
팽지선(Paeng, Ji Sun)
한승혁(Han, Seung Hyeok)
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