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Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference.

Hitz C, Wurst W, Kühn R - Nucleic Acids Res. (2007)

Bottom Line: In the last years, RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner.By placing conditional RNAi constructs into the defined genomic Rosa26 locus and by using recombinase mediated cassette exchange (RMCE) instead of laborious homologous recombination, we developed a fast, easy and reproducible approach to assess gene function in adult mice.We applied this technique to three genes of the MAPK signaling pathway-Braf, Mek1 and Mek2-and demonstrate here the potential of this new tool in mouse mutagenesis.

View Article: PubMed Central - PubMed

Affiliation: GSF National Research Center for Environment and Health, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.

ABSTRACT
In the last years, RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of this technique in developing or adult mice, short hairpin (sh)RNA vectors expressed stably from the genome are a faster alternative to conventional knockout approaches. Here we describe an advanced strategy for conditional gene knockdown in mice, where we used the Cre/loxP system to activate RNAi in a time and tissue dependent manner in the adult mouse brain. By placing conditional RNAi constructs into the defined genomic Rosa26 locus and by using recombinase mediated cassette exchange (RMCE) instead of laborious homologous recombination, we developed a fast, easy and reproducible approach to assess gene function in adult mice. We applied this technique to three genes of the MAPK signaling pathway-Braf, Mek1 and Mek2-and demonstrate here the potential of this new tool in mouse mutagenesis.

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Cre mediated activation of a single copy conditional shRNA vector within the Rosa26 locus of murine ES cells. (A) One Rosa26 allele of ES cells was modified by a gene-targeting vector (R26.5) that introduced a splice acceptor-lacZ cassette and a hygromycin resistance gene such that β-Galactosidase (lacZ) is expressed from the endogenous Rosa26 promoter. R26.5 ES cells were further modified with a gene targeting vector (R26.9) that introduced a neomycin resistance gene and the conditional shRNA vector U6-lox-lox-shLacZ. The R26.5/R26.9 ES cells were transiently transfected with a Cre expression vector and subclones that recombined the R26.9 allele (R26.9Δ) were isolated. (B) X-Gal staining of fixed R26.5/R26.9 ES cells in comparison to a R26.5/R26.9Δ clone shows highly reduced β-Galactosidase activity in the latter cells (magnification 20×). (C) Comparison of β-Galactosidase activity in lysates of R26.5/R26.9 ES cells (mean of three non-deleted subclones, blue column) in comparison to two deleted subclones (R26.9Δ-1, R26.9Δ-2, red columns) and wild type ES cells (WT). Values are shown as β-Galactosidase activity in RLU per micrograms protein of the lysates in comparison to the non-deleted clones and are expressed as mean values with SD.
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Figure 3: Cre mediated activation of a single copy conditional shRNA vector within the Rosa26 locus of murine ES cells. (A) One Rosa26 allele of ES cells was modified by a gene-targeting vector (R26.5) that introduced a splice acceptor-lacZ cassette and a hygromycin resistance gene such that β-Galactosidase (lacZ) is expressed from the endogenous Rosa26 promoter. R26.5 ES cells were further modified with a gene targeting vector (R26.9) that introduced a neomycin resistance gene and the conditional shRNA vector U6-lox-lox-shLacZ. The R26.5/R26.9 ES cells were transiently transfected with a Cre expression vector and subclones that recombined the R26.9 allele (R26.9Δ) were isolated. (B) X-Gal staining of fixed R26.5/R26.9 ES cells in comparison to a R26.5/R26.9Δ clone shows highly reduced β-Galactosidase activity in the latter cells (magnification 20×). (C) Comparison of β-Galactosidase activity in lysates of R26.5/R26.9 ES cells (mean of three non-deleted subclones, blue column) in comparison to two deleted subclones (R26.9Δ-1, R26.9Δ-2, red columns) and wild type ES cells (WT). Values are shown as β-Galactosidase activity in RLU per micrograms protein of the lysates in comparison to the non-deleted clones and are expressed as mean values with SD.

Mentions: For the genomic integration of conditional shRNA vectors, a single-copy approach is preferable since multi copy integrations could undergo unpredictable and non-functional rearrangements upon Cre mediated recombination. We tested the efficiency of a single shRNA vector copy in the Rosa26 locus (15) of murine ES cells with β-Galactosidase as reporter gene. We first inserted a splice acceptor sequence and the β-Galactosidase coding region by gene targeting downstream of the first exon of one Rosa26 allele of murine ES cells. These modified R26.5 ES cells express β-Galactosidase from the endogenous, ubiquitously active Rosa26 promoter. Next, the second Rosa26 allele of R26.5 ES cells was targeted with a single copy of the conditional shRNA vector U6-lox-lox-shLacZ giving rise to R26.5/R26.9 ES cells. In the double-targeted R26.5/R26.9 cells, the shRNA vector stop cassette was deleted by transient transfection with a Cre expression plasmid (Figure 3A). Three ES cell clones harboring the non-activated and two clones carrying the recombined shRNA construct (R26.5/R26.9Δ cells) were isolated and analyzed for β-Galactosidase activity by histochemical staining (X-Gal) and by a chemiluminescence assay using cell lysates. X-Gal staining revealed a strong reduction of β-Galactosidase activity in R26.5/R26.9Δ ES cells as compared to non-recombined R26.5/R26.9 cells (Figure 3B). The quantitative analysis revealed that the β-Galactosidase activity in R26.5/R26.9Δ cells is reduced by 90% (Figure 3C). Thus, a single genomic shRNA vector copy within the Rosa26 locus is sufficient to induce RNAi in ES cells and the extent of gene silencing can reach a similar level as obtained by transient transfections (compare to Figure 2B).Figure 3.


Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference.

Hitz C, Wurst W, Kühn R - Nucleic Acids Res. (2007)

Cre mediated activation of a single copy conditional shRNA vector within the Rosa26 locus of murine ES cells. (A) One Rosa26 allele of ES cells was modified by a gene-targeting vector (R26.5) that introduced a splice acceptor-lacZ cassette and a hygromycin resistance gene such that β-Galactosidase (lacZ) is expressed from the endogenous Rosa26 promoter. R26.5 ES cells were further modified with a gene targeting vector (R26.9) that introduced a neomycin resistance gene and the conditional shRNA vector U6-lox-lox-shLacZ. The R26.5/R26.9 ES cells were transiently transfected with a Cre expression vector and subclones that recombined the R26.9 allele (R26.9Δ) were isolated. (B) X-Gal staining of fixed R26.5/R26.9 ES cells in comparison to a R26.5/R26.9Δ clone shows highly reduced β-Galactosidase activity in the latter cells (magnification 20×). (C) Comparison of β-Galactosidase activity in lysates of R26.5/R26.9 ES cells (mean of three non-deleted subclones, blue column) in comparison to two deleted subclones (R26.9Δ-1, R26.9Δ-2, red columns) and wild type ES cells (WT). Values are shown as β-Galactosidase activity in RLU per micrograms protein of the lysates in comparison to the non-deleted clones and are expressed as mean values with SD.
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Figure 3: Cre mediated activation of a single copy conditional shRNA vector within the Rosa26 locus of murine ES cells. (A) One Rosa26 allele of ES cells was modified by a gene-targeting vector (R26.5) that introduced a splice acceptor-lacZ cassette and a hygromycin resistance gene such that β-Galactosidase (lacZ) is expressed from the endogenous Rosa26 promoter. R26.5 ES cells were further modified with a gene targeting vector (R26.9) that introduced a neomycin resistance gene and the conditional shRNA vector U6-lox-lox-shLacZ. The R26.5/R26.9 ES cells were transiently transfected with a Cre expression vector and subclones that recombined the R26.9 allele (R26.9Δ) were isolated. (B) X-Gal staining of fixed R26.5/R26.9 ES cells in comparison to a R26.5/R26.9Δ clone shows highly reduced β-Galactosidase activity in the latter cells (magnification 20×). (C) Comparison of β-Galactosidase activity in lysates of R26.5/R26.9 ES cells (mean of three non-deleted subclones, blue column) in comparison to two deleted subclones (R26.9Δ-1, R26.9Δ-2, red columns) and wild type ES cells (WT). Values are shown as β-Galactosidase activity in RLU per micrograms protein of the lysates in comparison to the non-deleted clones and are expressed as mean values with SD.
Mentions: For the genomic integration of conditional shRNA vectors, a single-copy approach is preferable since multi copy integrations could undergo unpredictable and non-functional rearrangements upon Cre mediated recombination. We tested the efficiency of a single shRNA vector copy in the Rosa26 locus (15) of murine ES cells with β-Galactosidase as reporter gene. We first inserted a splice acceptor sequence and the β-Galactosidase coding region by gene targeting downstream of the first exon of one Rosa26 allele of murine ES cells. These modified R26.5 ES cells express β-Galactosidase from the endogenous, ubiquitously active Rosa26 promoter. Next, the second Rosa26 allele of R26.5 ES cells was targeted with a single copy of the conditional shRNA vector U6-lox-lox-shLacZ giving rise to R26.5/R26.9 ES cells. In the double-targeted R26.5/R26.9 cells, the shRNA vector stop cassette was deleted by transient transfection with a Cre expression plasmid (Figure 3A). Three ES cell clones harboring the non-activated and two clones carrying the recombined shRNA construct (R26.5/R26.9Δ cells) were isolated and analyzed for β-Galactosidase activity by histochemical staining (X-Gal) and by a chemiluminescence assay using cell lysates. X-Gal staining revealed a strong reduction of β-Galactosidase activity in R26.5/R26.9Δ ES cells as compared to non-recombined R26.5/R26.9 cells (Figure 3B). The quantitative analysis revealed that the β-Galactosidase activity in R26.5/R26.9Δ cells is reduced by 90% (Figure 3C). Thus, a single genomic shRNA vector copy within the Rosa26 locus is sufficient to induce RNAi in ES cells and the extent of gene silencing can reach a similar level as obtained by transient transfections (compare to Figure 2B).Figure 3.

Bottom Line: In the last years, RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner.By placing conditional RNAi constructs into the defined genomic Rosa26 locus and by using recombinase mediated cassette exchange (RMCE) instead of laborious homologous recombination, we developed a fast, easy and reproducible approach to assess gene function in adult mice.We applied this technique to three genes of the MAPK signaling pathway-Braf, Mek1 and Mek2-and demonstrate here the potential of this new tool in mouse mutagenesis.

View Article: PubMed Central - PubMed

Affiliation: GSF National Research Center for Environment and Health, Institute of Developmental Genetics, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany.

ABSTRACT
In the last years, RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of this technique in developing or adult mice, short hairpin (sh)RNA vectors expressed stably from the genome are a faster alternative to conventional knockout approaches. Here we describe an advanced strategy for conditional gene knockdown in mice, where we used the Cre/loxP system to activate RNAi in a time and tissue dependent manner in the adult mouse brain. By placing conditional RNAi constructs into the defined genomic Rosa26 locus and by using recombinase mediated cassette exchange (RMCE) instead of laborious homologous recombination, we developed a fast, easy and reproducible approach to assess gene function in adult mice. We applied this technique to three genes of the MAPK signaling pathway-Braf, Mek1 and Mek2-and demonstrate here the potential of this new tool in mouse mutagenesis.

Show MeSH
Related in: MedlinePlus