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Am J Physiol Renal Physiol. 2015 Dec 15;309(12):F1060-9. doi: 10.1152/ajprenal.00251.2015. Epub 2015 Sep 16.

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

Author information

1
Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and.
2
Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.
3
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.

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.

KEYWORDS:

Cre/LoxP; cell-specific knockdown; lentiviral vector

PMID:
26377795
DOI:
10.1152/ajprenal.00251.2015
[Indexed for MEDLINE]
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