title: Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice.
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.
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Bo Young
authors.initials: BY
authors.lastname: Nam
authors.affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
authors.forename: Dong Ki
authors.initials: DK
authors.lastname: Kim
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Jung Tak
authors.initials: JT
authors.lastname: Park
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Hye-Young
authors.initials: HY
authors.lastname: Kang
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Jisun
authors.initials: J
authors.lastname: Paeng
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Seonghun
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Jimin
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Jae Eun
authors.initials: JE
authors.lastname: Um
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Hyung Jung
authors.initials: HJ
authors.lastname: Oh
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Seung Hyeok
authors.initials: SH
authors.lastname: Han
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
authors.forename: Tae-Hyun
authors.initials: TH
authors.lastname: Yoo
authors.affiliation: Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and kswkidney@yuhs.ac.
authors.forename: Shin-Wook
authors.initials: SW
authors.lastname: Kang
mesh_terms: D000818:Animals; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D007668:Kidney / Q000166:cytology*; D016086:Lentivirus; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases
keywords: Cre/LoxP; cell-specific knockdown; lentiviral vector