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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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Vector construction and RMCE for the generation of shBraf-flox and shMek-flox mice. (A) The conditional shRNA expression cassette from the vector pbs-U6-shRNA-flox contains the U6 promoter in front of the sense (s) sequence of the shRNA, the loxP (lox) flanked stop cassette in the loop region and the antisense (as) shRNA sequence. The shRNA expression cassette is cloned into the RMCE donor vector pRMCE behind the promoterless neomycin selection marker (neo-bpA) so that the two attB sites from pRMCE flank the selection marker as well as the shRNA expression cassette. (B) Acceptor ES cells for RMCE harbor a Rosa26 allele where in intron 1 a splice acceptor (SA) is inserted followed by a pgk promoter (pgk) driving a hygromycin selection marker (hygro-bpA) which is flanked by attP sites. (C) Upon RMCE with C31Int, the attP flanked cassette in the acceptor ES cells from B is replaced by the attB flanked cassette from the donor vector in A. FRT (f) sites allow to excise the pgk promoter and the neomycin marker in recombined ES cells or mice. E: EcoRV; B: BamHI; probe: 5′-Rosa26 probe and E1: exon 1.
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Figure 4: Vector construction and RMCE for the generation of shBraf-flox and shMek-flox mice. (A) The conditional shRNA expression cassette from the vector pbs-U6-shRNA-flox contains the U6 promoter in front of the sense (s) sequence of the shRNA, the loxP (lox) flanked stop cassette in the loop region and the antisense (as) shRNA sequence. The shRNA expression cassette is cloned into the RMCE donor vector pRMCE behind the promoterless neomycin selection marker (neo-bpA) so that the two attB sites from pRMCE flank the selection marker as well as the shRNA expression cassette. (B) Acceptor ES cells for RMCE harbor a Rosa26 allele where in intron 1 a splice acceptor (SA) is inserted followed by a pgk promoter (pgk) driving a hygromycin selection marker (hygro-bpA) which is flanked by attP sites. (C) Upon RMCE with C31Int, the attP flanked cassette in the acceptor ES cells from B is replaced by the attB flanked cassette from the donor vector in A. FRT (f) sites allow to excise the pgk promoter and the neomycin marker in recombined ES cells or mice. E: EcoRV; B: BamHI; probe: 5′-Rosa26 probe and E1: exon 1.

Mentions: To facilitate the insertion of shRNA vectors into the Rosa26 locus of ES cells for the fast generation of multiple knockdown mouse lines, we developed an approach for recombinase mediated cassette exchange (RMCE) using the integrase of phage φC31 (C31Int). For this purpose, ES cells with a Rosa26 acceptor allele were generated. These cells harbor a pgk promoter driven hygromycin resistance gene, of which the coding and polyA region is flanked by attP recognition sites, in the Rosa26 locus (Figure 4B). The acceptor allele is designed such that the attP-flanked segment can be replaced by an attB-flanked construct from a donor plasmid (pRMCE) upon transfection of ES cells together with a C31Int expression plasmid (Figure 4A). The donor vector contains a promoterless neomycin resistance coding and polyA region and the shRNA expression cassette between the attB recognition sites. The shift from hygromycin to neomycin resistance in ES cells selects for RMCE events (Figure 4C), such that correct cassette exchange occurs at a frequency of 40–60% among the neomycin resistant ES cell colonies (data not shown).Figure 4.


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

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

Vector construction and RMCE for the generation of shBraf-flox and shMek-flox mice. (A) The conditional shRNA expression cassette from the vector pbs-U6-shRNA-flox contains the U6 promoter in front of the sense (s) sequence of the shRNA, the loxP (lox) flanked stop cassette in the loop region and the antisense (as) shRNA sequence. The shRNA expression cassette is cloned into the RMCE donor vector pRMCE behind the promoterless neomycin selection marker (neo-bpA) so that the two attB sites from pRMCE flank the selection marker as well as the shRNA expression cassette. (B) Acceptor ES cells for RMCE harbor a Rosa26 allele where in intron 1 a splice acceptor (SA) is inserted followed by a pgk promoter (pgk) driving a hygromycin selection marker (hygro-bpA) which is flanked by attP sites. (C) Upon RMCE with C31Int, the attP flanked cassette in the acceptor ES cells from B is replaced by the attB flanked cassette from the donor vector in A. FRT (f) sites allow to excise the pgk promoter and the neomycin marker in recombined ES cells or mice. E: EcoRV; B: BamHI; probe: 5′-Rosa26 probe and E1: exon 1.
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Figure 4: Vector construction and RMCE for the generation of shBraf-flox and shMek-flox mice. (A) The conditional shRNA expression cassette from the vector pbs-U6-shRNA-flox contains the U6 promoter in front of the sense (s) sequence of the shRNA, the loxP (lox) flanked stop cassette in the loop region and the antisense (as) shRNA sequence. The shRNA expression cassette is cloned into the RMCE donor vector pRMCE behind the promoterless neomycin selection marker (neo-bpA) so that the two attB sites from pRMCE flank the selection marker as well as the shRNA expression cassette. (B) Acceptor ES cells for RMCE harbor a Rosa26 allele where in intron 1 a splice acceptor (SA) is inserted followed by a pgk promoter (pgk) driving a hygromycin selection marker (hygro-bpA) which is flanked by attP sites. (C) Upon RMCE with C31Int, the attP flanked cassette in the acceptor ES cells from B is replaced by the attB flanked cassette from the donor vector in A. FRT (f) sites allow to excise the pgk promoter and the neomycin marker in recombined ES cells or mice. E: EcoRV; B: BamHI; probe: 5′-Rosa26 probe and E1: exon 1.
Mentions: To facilitate the insertion of shRNA vectors into the Rosa26 locus of ES cells for the fast generation of multiple knockdown mouse lines, we developed an approach for recombinase mediated cassette exchange (RMCE) using the integrase of phage φC31 (C31Int). For this purpose, ES cells with a Rosa26 acceptor allele were generated. These cells harbor a pgk promoter driven hygromycin resistance gene, of which the coding and polyA region is flanked by attP recognition sites, in the Rosa26 locus (Figure 4B). The acceptor allele is designed such that the attP-flanked segment can be replaced by an attB-flanked construct from a donor plasmid (pRMCE) upon transfection of ES cells together with a C31Int expression plasmid (Figure 4A). The donor vector contains a promoterless neomycin resistance coding and polyA region and the shRNA expression cassette between the attB recognition sites. The shift from hygromycin to neomycin resistance in ES cells selects for RMCE events (Figure 4C), such that correct cassette exchange occurs at a frequency of 40–60% among the neomycin resistant ES cell colonies (data not shown).Figure 4.

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