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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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Analysis of RNA interference in shBraf+/flox/CamKII-cre mice. (A) Tissue-specific activation of shRNA in the brain with CamKII-cre. Southern blot analysis of BamHI digested genomic DNA from different brain regions of adult shBraf+/flox/CamKII-cre mice. Lane 1: DNA from whole brains of shBraf+/flox control mice; lane 2–10: DNA from indicated regions of shBraf+/flox/CamKII-cre mice. The wild type Rosa26 allele (wt) gives a 5.8 kb band and the band from the shRNA allele is shifted from 5.4 kb with the stop cassette (flox) to 8.6 kb after Cre recombination (del). (B) Expression of shRNA against Braf in adult forebrain. On a Northern blot with small RNAs against the sequence of shBraf the 21 nt band of the processed siRNA against Braf is only detectable with the control oligonucleotide and in mutant mice but not in control mice. (C) BRAF protein reduction in forebrain regions of shBraf+/flox/CamKII-cre mice. On a Western blot with protein from the indicated brain regions from adult mouse brain, knockdown of BRAF protein in forebrain regions of mutant mice is shown in comparison to the protein level from control mice. β-ACTIN was used as a loading control. OB: olfactory bulb, HC: hippocampus, St: Striatum, fCx: frontal cortex, pCx: posterior cortex, Th: Thalamus, MB: midbrain, CB: cerebellum, BS: brainstem, +/flox: shBraf+/flox and +/Δ: shBraf+/flox/CamKII-cre.
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Figure 5: Analysis of RNA interference in shBraf+/flox/CamKII-cre mice. (A) Tissue-specific activation of shRNA in the brain with CamKII-cre. Southern blot analysis of BamHI digested genomic DNA from different brain regions of adult shBraf+/flox/CamKII-cre mice. Lane 1: DNA from whole brains of shBraf+/flox control mice; lane 2–10: DNA from indicated regions of shBraf+/flox/CamKII-cre mice. The wild type Rosa26 allele (wt) gives a 5.8 kb band and the band from the shRNA allele is shifted from 5.4 kb with the stop cassette (flox) to 8.6 kb after Cre recombination (del). (B) Expression of shRNA against Braf in adult forebrain. On a Northern blot with small RNAs against the sequence of shBraf the 21 nt band of the processed siRNA against Braf is only detectable with the control oligonucleotide and in mutant mice but not in control mice. (C) BRAF protein reduction in forebrain regions of shBraf+/flox/CamKII-cre mice. On a Western blot with protein from the indicated brain regions from adult mouse brain, knockdown of BRAF protein in forebrain regions of mutant mice is shown in comparison to the protein level from control mice. β-ACTIN was used as a loading control. OB: olfactory bulb, HC: hippocampus, St: Striatum, fCx: frontal cortex, pCx: posterior cortex, Th: Thalamus, MB: midbrain, CB: cerebellum, BS: brainstem, +/flox: shBraf+/flox and +/Δ: shBraf+/flox/CamKII-cre.

Mentions: Cre recombinase under the CamKII promoter activates the shRNA only in neurons of the adult forebrain (20). To show the deletion of the stop cassette in the U6-shBraf-flox element, a Southern blot analysis with genomic DNA from different brain regions was performed. A shift of the 5.4 kb band with the stop cassette to an 8.6 kb band after Cre recombination indicates activation of RNAi (Figure 5A). This larger band is visible only in forebrain regions of mutant mice, like the olfactory bulb, the hippocampus, striatum, cortex and very weak in the thalamus, but not in more posterior parts of the brain of these mice or in control mice. As expected, the 5.4 kb band from the inactive shRNA allele is still visible in all the forebrain regions of mutant mice, resulting from non-neuronal cells in the forebrain expressing no Cre recombinase.Figure 5.


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

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

Analysis of RNA interference in shBraf+/flox/CamKII-cre mice. (A) Tissue-specific activation of shRNA in the brain with CamKII-cre. Southern blot analysis of BamHI digested genomic DNA from different brain regions of adult shBraf+/flox/CamKII-cre mice. Lane 1: DNA from whole brains of shBraf+/flox control mice; lane 2–10: DNA from indicated regions of shBraf+/flox/CamKII-cre mice. The wild type Rosa26 allele (wt) gives a 5.8 kb band and the band from the shRNA allele is shifted from 5.4 kb with the stop cassette (flox) to 8.6 kb after Cre recombination (del). (B) Expression of shRNA against Braf in adult forebrain. On a Northern blot with small RNAs against the sequence of shBraf the 21 nt band of the processed siRNA against Braf is only detectable with the control oligonucleotide and in mutant mice but not in control mice. (C) BRAF protein reduction in forebrain regions of shBraf+/flox/CamKII-cre mice. On a Western blot with protein from the indicated brain regions from adult mouse brain, knockdown of BRAF protein in forebrain regions of mutant mice is shown in comparison to the protein level from control mice. β-ACTIN was used as a loading control. OB: olfactory bulb, HC: hippocampus, St: Striatum, fCx: frontal cortex, pCx: posterior cortex, Th: Thalamus, MB: midbrain, CB: cerebellum, BS: brainstem, +/flox: shBraf+/flox and +/Δ: shBraf+/flox/CamKII-cre.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Analysis of RNA interference in shBraf+/flox/CamKII-cre mice. (A) Tissue-specific activation of shRNA in the brain with CamKII-cre. Southern blot analysis of BamHI digested genomic DNA from different brain regions of adult shBraf+/flox/CamKII-cre mice. Lane 1: DNA from whole brains of shBraf+/flox control mice; lane 2–10: DNA from indicated regions of shBraf+/flox/CamKII-cre mice. The wild type Rosa26 allele (wt) gives a 5.8 kb band and the band from the shRNA allele is shifted from 5.4 kb with the stop cassette (flox) to 8.6 kb after Cre recombination (del). (B) Expression of shRNA against Braf in adult forebrain. On a Northern blot with small RNAs against the sequence of shBraf the 21 nt band of the processed siRNA against Braf is only detectable with the control oligonucleotide and in mutant mice but not in control mice. (C) BRAF protein reduction in forebrain regions of shBraf+/flox/CamKII-cre mice. On a Western blot with protein from the indicated brain regions from adult mouse brain, knockdown of BRAF protein in forebrain regions of mutant mice is shown in comparison to the protein level from control mice. β-ACTIN was used as a loading control. OB: olfactory bulb, HC: hippocampus, St: Striatum, fCx: frontal cortex, pCx: posterior cortex, Th: Thalamus, MB: midbrain, CB: cerebellum, BS: brainstem, +/flox: shBraf+/flox and +/Δ: shBraf+/flox/CamKII-cre.
Mentions: Cre recombinase under the CamKII promoter activates the shRNA only in neurons of the adult forebrain (20). To show the deletion of the stop cassette in the U6-shBraf-flox element, a Southern blot analysis with genomic DNA from different brain regions was performed. A shift of the 5.4 kb band with the stop cassette to an 8.6 kb band after Cre recombination indicates activation of RNAi (Figure 5A). This larger band is visible only in forebrain regions of mutant mice, like the olfactory bulb, the hippocampus, striatum, cortex and very weak in the thalamus, but not in more posterior parts of the brain of these mice or in control mice. As expected, the 5.4 kb band from the inactive shRNA allele is still visible in all the forebrain regions of mutant mice, resulting from non-neuronal cells in the forebrain expressing no Cre recombinase.Figure 5.

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