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, SeungHyeok Han, TaeHyun Yoo, Shin-Wook Kang

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)F1060-F1069
JournalAmerican Journal of Physiology - Renal Physiology
Volume309
Issue number12
DOIs
Publication statusPublished - 2015 Dec 15

Fingerprint

Aquaporin 3
Lentivirus
Kidney
Small Interfering RNA
Genetic Recombination
Injections
Collecting Kidney Tubules
Gene Transfer Techniques
Genes
enhanced green fluorescent protein
Proteins

All Science Journal Classification (ASJC) codes

  • Physiology
  • Urology

Cite this

Nam, Bo Young ; Kim, Dong Ki ; Park, Jung Tak ; Kang, Hye Young ; Paeng, Jisun ; Kim, Seonghun ; Park, Jimin ; Um, Jae Eun ; Oh, Hyung Jung ; Han, SeungHyeok ; Yoo, TaeHyun ; Kang, Shin-Wook. / Double transduction of a Cre/LoxP lentiviral vector : A simple method to generate kidney cell-specific knockdown mice. In: American Journal of Physiology - Renal Physiology. 2015 ; Vol. 309, No. 12. pp. F1060-F1069.
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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.",
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Double transduction of a Cre/LoxP lentiviral vector : A simple method to generate kidney cell-specific knockdown mice. / Nam, Bo Young; Kim, Dong Ki; Park, Jung Tak; Kang, Hye Young; Paeng, Jisun; Kim, Seonghun; Park, Jimin; Um, Jae Eun; Oh, Hyung Jung; Han, SeungHyeok; Yoo, TaeHyun; Kang, Shin-Wook.

In: American Journal of Physiology - Renal Physiology, Vol. 309, No. 12, 15.12.2015, p. F1060-F1069.

Research output: Contribution to journalArticle

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T1 - Double transduction of a Cre/LoxP lentiviral vector

T2 - A simple method to generate kidney cell-specific knockdown mice

AU - Nam, Bo Young

AU - Kim, Dong Ki

AU - Park, Jung Tak

AU - Kang, Hye Young

AU - Paeng, Jisun

AU - Kim, Seonghun

AU - Park, Jimin

AU - Um, Jae Eun

AU - Oh, Hyung Jung

AU - Han, SeungHyeok

AU - Yoo, TaeHyun

AU - Kang, Shin-Wook

PY - 2015/12/15

Y1 - 2015/12/15

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