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

 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 
 American Journal of Physiology - Renal Physiology, Vol.309(12) : F1060-F1069, 2015 
Journal Title
 American Journal of Physiology - Renal Physiology 
Issue Date
Animals ; Gene Knockdown Techniques ; Gene Transfer Techniques ; Genetic Vectors* ; Green Fluorescent Proteins/genetics ; Integrases/genetics* ; Kidney/cytology* ; Lentivirus ; Mice, Transgenic ; Promoter Regions, Genetic/genetics
Cre/LoxP ; cell-specific knockdown ; lentiviral vector
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.
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1. College of Medicine (의과대학) > Dept. of Internal Medicine (내과학교실) > 1. Journal Papers
1. College of Medicine (의과대학) > Yonsei Biomedical Research Center (연세의생명연구원) > 1. Journal Papers
1. College of Medicine (의과대학) > BioMedical Science Institute (의생명과학부) > 1. Journal Papers
Yonsei Authors
Kang, Shin Wook(강신욱) ORCID logo https://orcid.org/0000-0002-5677-4756
Kang, Hye Young(강혜영)
Kim, Seonghun(김성훈)
Nam, Bo Young(남보영)
Park, Jung Tak(박정탁) ORCID logo https://orcid.org/0000-0002-2325-8982
Park, Jimin(박지민)
Um, Jae Eun(엄재은)
Oh, Hyung Jung(오형중)
Yoo, Tae Hyun(유태현) ORCID logo https://orcid.org/0000-0002-9183-4507
Paeng, Ji Sun(팽지선)
Han, Seung Hyeok(한승혁) ORCID logo https://orcid.org/0000-0001-7923-5635
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