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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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RNAi activity of H1 or U6 promoter based shRNA expression vectors modified by loxP sites. (A) Overview of the tested shRNA vectors. The position of the loxP sites (red triangle) in relation to the start of transcription (black arrow) and the shRNA region (blue arrows) are indicated. In the resulting shRNAs loxP derived sequences are shown in red color. All constructs contain identical sense and antisense shRNA sequences with specificity to β-Galactosidase (lacZ). (B) Sequences of the H1 and U6 promoter constructs. The different positions of the loxP and lox2272 sites (red), respectively, within or outside of the promoter are shown. The nucleotide of the transcriptional start is highlighted in gray. In capital letters: TATA box of the promoter; bold: original loop region and in blue and underlined: shRNA sense and antisense regions. (C) Silencing activity of β-Galactosidase specific shRNA vectors upon transient cotransfection with β-Galactosidase and F-Luciferase expression plasmids into ES cells. Values are expressed as ratio of β-Galactosidase to Luciferase activity in RLU in comparison to the positive control. The results obtained with the parental shRNA vectors H1-shLacZ and U6-shLacZ are shown as black columns. Numbers above the columns indicate the efficiency of silencing of β-Galactosidase activity in percent in comparison to the positive control. All results from duplicate samples are expressed as mean values with standard deviation.
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Figure 1: RNAi activity of H1 or U6 promoter based shRNA expression vectors modified by loxP sites. (A) Overview of the tested shRNA vectors. The position of the loxP sites (red triangle) in relation to the start of transcription (black arrow) and the shRNA region (blue arrows) are indicated. In the resulting shRNAs loxP derived sequences are shown in red color. All constructs contain identical sense and antisense shRNA sequences with specificity to β-Galactosidase (lacZ). (B) Sequences of the H1 and U6 promoter constructs. The different positions of the loxP and lox2272 sites (red), respectively, within or outside of the promoter are shown. The nucleotide of the transcriptional start is highlighted in gray. In capital letters: TATA box of the promoter; bold: original loop region and in blue and underlined: shRNA sense and antisense regions. (C) Silencing activity of β-Galactosidase specific shRNA vectors upon transient cotransfection with β-Galactosidase and F-Luciferase expression plasmids into ES cells. Values are expressed as ratio of β-Galactosidase to Luciferase activity in RLU in comparison to the positive control. The results obtained with the parental shRNA vectors H1-shLacZ and U6-shLacZ are shown as black columns. Numbers above the columns indicate the efficiency of silencing of β-Galactosidase activity in percent in comparison to the positive control. All results from duplicate samples are expressed as mean values with standard deviation.

Mentions: To control the activity of shRNA vectors through Cre/loxP mediated recombination, their transcription must be initially aborted by a loxP flanked stop element. The shRNA vector should be activated upon Cre mediated deletion of the stop segment. Since a single 34 bp loxP site remains in the activated vector after Cre recombination, we first tested whether this additional sequence disturbs RNAi efficiency. Therefore, we used vector configurations with different positions for the remaining loxP site, where the shRNA is driven by the human U6 or H1 promoter (Figure 1A and B). We found that the loxP site in all configurations of H1 promoter driven constructs strongly diminished the efficiency of gene silencing whereas the loxP site in all constructs driven by the U6 promoter did not significantly affect RNAi-mediated gene silencing (Figure 1C). Thus, the U6 but not the H1 promoter is of use for Cre mediated control of shRNA expression and we chose the loxP5 configuration (Figure 1A) as one effective configuration that includes the loxP sequence within the loop region.


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

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

RNAi activity of H1 or U6 promoter based shRNA expression vectors modified by loxP sites. (A) Overview of the tested shRNA vectors. The position of the loxP sites (red triangle) in relation to the start of transcription (black arrow) and the shRNA region (blue arrows) are indicated. In the resulting shRNAs loxP derived sequences are shown in red color. All constructs contain identical sense and antisense shRNA sequences with specificity to β-Galactosidase (lacZ). (B) Sequences of the H1 and U6 promoter constructs. The different positions of the loxP and lox2272 sites (red), respectively, within or outside of the promoter are shown. The nucleotide of the transcriptional start is highlighted in gray. In capital letters: TATA box of the promoter; bold: original loop region and in blue and underlined: shRNA sense and antisense regions. (C) Silencing activity of β-Galactosidase specific shRNA vectors upon transient cotransfection with β-Galactosidase and F-Luciferase expression plasmids into ES cells. Values are expressed as ratio of β-Galactosidase to Luciferase activity in RLU in comparison to the positive control. The results obtained with the parental shRNA vectors H1-shLacZ and U6-shLacZ are shown as black columns. Numbers above the columns indicate the efficiency of silencing of β-Galactosidase activity in percent in comparison to the positive control. All results from duplicate samples are expressed as mean values with standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919482&req=5

Figure 1: RNAi activity of H1 or U6 promoter based shRNA expression vectors modified by loxP sites. (A) Overview of the tested shRNA vectors. The position of the loxP sites (red triangle) in relation to the start of transcription (black arrow) and the shRNA region (blue arrows) are indicated. In the resulting shRNAs loxP derived sequences are shown in red color. All constructs contain identical sense and antisense shRNA sequences with specificity to β-Galactosidase (lacZ). (B) Sequences of the H1 and U6 promoter constructs. The different positions of the loxP and lox2272 sites (red), respectively, within or outside of the promoter are shown. The nucleotide of the transcriptional start is highlighted in gray. In capital letters: TATA box of the promoter; bold: original loop region and in blue and underlined: shRNA sense and antisense regions. (C) Silencing activity of β-Galactosidase specific shRNA vectors upon transient cotransfection with β-Galactosidase and F-Luciferase expression plasmids into ES cells. Values are expressed as ratio of β-Galactosidase to Luciferase activity in RLU in comparison to the positive control. The results obtained with the parental shRNA vectors H1-shLacZ and U6-shLacZ are shown as black columns. Numbers above the columns indicate the efficiency of silencing of β-Galactosidase activity in percent in comparison to the positive control. All results from duplicate samples are expressed as mean values with standard deviation.
Mentions: To control the activity of shRNA vectors through Cre/loxP mediated recombination, their transcription must be initially aborted by a loxP flanked stop element. The shRNA vector should be activated upon Cre mediated deletion of the stop segment. Since a single 34 bp loxP site remains in the activated vector after Cre recombination, we first tested whether this additional sequence disturbs RNAi efficiency. Therefore, we used vector configurations with different positions for the remaining loxP site, where the shRNA is driven by the human U6 or H1 promoter (Figure 1A and B). We found that the loxP site in all configurations of H1 promoter driven constructs strongly diminished the efficiency of gene silencing whereas the loxP site in all constructs driven by the U6 promoter did not significantly affect RNAi-mediated gene silencing (Figure 1C). Thus, the U6 but not the H1 promoter is of use for Cre mediated control of shRNA expression and we chose the loxP5 configuration (Figure 1A) as one effective configuration that includes the loxP sequence within the loop region.

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