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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/loxP controlled conditional shRNA vectors. (A) Scheme for the modification of an shRNA vector into a Cre/loxP controlled version by insertion of a loxP flanked stop cassette into the shRNA loop region. The conditional shRNA vector can be activated by excision of the stop cassette through Cre mediated deletion. A single loxP site remains within the shRNA loop. (B) Transient transfection of β-Galactosidase (lacZ, blue columns) or F-Luciferase (Luc, orange columns) specific conditional shRNA vectors before (U6-lox-lox-shLuc; U6-lox-lox-shLacZ) and after excision of the loxP flanked stop cassette (U6-loxP5-shLuc; U6-loxP5-shLacZ) into ES cells. For F-Luciferase shRNA vectors values are expressed as ratio of Luciferase to β-Galactosidase activity (left Y axis) and for β-Galactosidase shRNA vectors as ratio of β-Galactosidase to Luciferase activity (right Y axis), in comparison to the appropriate positive control. Results are expressed as mean values from duplicate samples with SD. U6-shLuc, U6-shLacZ: parental shRNA vectors; RLU: relative light units.
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Figure 2: Cre/loxP controlled conditional shRNA vectors. (A) Scheme for the modification of an shRNA vector into a Cre/loxP controlled version by insertion of a loxP flanked stop cassette into the shRNA loop region. The conditional shRNA vector can be activated by excision of the stop cassette through Cre mediated deletion. A single loxP site remains within the shRNA loop. (B) Transient transfection of β-Galactosidase (lacZ, blue columns) or F-Luciferase (Luc, orange columns) specific conditional shRNA vectors before (U6-lox-lox-shLuc; U6-lox-lox-shLacZ) and after excision of the loxP flanked stop cassette (U6-loxP5-shLuc; U6-loxP5-shLacZ) into ES cells. For F-Luciferase shRNA vectors values are expressed as ratio of Luciferase to β-Galactosidase activity (left Y axis) and for β-Galactosidase shRNA vectors as ratio of β-Galactosidase to Luciferase activity (right Y axis), in comparison to the appropriate positive control. Results are expressed as mean values from duplicate samples with SD. U6-shLuc, U6-shLacZ: parental shRNA vectors; RLU: relative light units.

Mentions: According to this design we inserted a loxP-flanked 270 bp DNA segment, which includes termination sequences and serves as transcriptional stop element, in our parental U6 driven shRNA vector. The presence of the stop element in the ‘off ’ configuration of the conditional shRNA vector allows only the transcription of the sense region that does not induce RNA interference, provided that the shRNA construct follows the order 5′-sense-loop-antisense-3′ (Figure 2A). Upon Cre mediated deletion of the stop cassette, the shRNA vector acquires the configuration of loxP5 with the remaining loxP site in the loop sequence. To assess the functionality of the stop element, it was first inserted into the loop region of U6-shRNA vectors against the β-Galactosidase or F-Luciferase reporter genes (Figure 2A). Upon transient cotransfection with reporter gene expression plasmids into mouse ES cells, the efficiency of the stop cassette in the ‘off ’ state and RNAi efficiency in the ‘on’ state were analyzed and compared to the non-modified parental vectors U6-shLuc and U6-shLacZ (Figure 2B). The measurement of F-Luciferase and β-Galactosidase activity from transfected cells showed that the ‘off ’ versions did not induce gene silencing whereas the ‘on’ versions induced the same level of RNAi as the parental, non-conditional vectors (Figure 2B). These results show that the stop element and our loxP5 configuration provide a way to control shRNA expression through Cre mediated recombination.Figure 2.


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

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

Cre/loxP controlled conditional shRNA vectors. (A) Scheme for the modification of an shRNA vector into a Cre/loxP controlled version by insertion of a loxP flanked stop cassette into the shRNA loop region. The conditional shRNA vector can be activated by excision of the stop cassette through Cre mediated deletion. A single loxP site remains within the shRNA loop. (B) Transient transfection of β-Galactosidase (lacZ, blue columns) or F-Luciferase (Luc, orange columns) specific conditional shRNA vectors before (U6-lox-lox-shLuc; U6-lox-lox-shLacZ) and after excision of the loxP flanked stop cassette (U6-loxP5-shLuc; U6-loxP5-shLacZ) into ES cells. For F-Luciferase shRNA vectors values are expressed as ratio of Luciferase to β-Galactosidase activity (left Y axis) and for β-Galactosidase shRNA vectors as ratio of β-Galactosidase to Luciferase activity (right Y axis), in comparison to the appropriate positive control. Results are expressed as mean values from duplicate samples with SD. U6-shLuc, U6-shLacZ: parental shRNA vectors; RLU: relative light units.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Cre/loxP controlled conditional shRNA vectors. (A) Scheme for the modification of an shRNA vector into a Cre/loxP controlled version by insertion of a loxP flanked stop cassette into the shRNA loop region. The conditional shRNA vector can be activated by excision of the stop cassette through Cre mediated deletion. A single loxP site remains within the shRNA loop. (B) Transient transfection of β-Galactosidase (lacZ, blue columns) or F-Luciferase (Luc, orange columns) specific conditional shRNA vectors before (U6-lox-lox-shLuc; U6-lox-lox-shLacZ) and after excision of the loxP flanked stop cassette (U6-loxP5-shLuc; U6-loxP5-shLacZ) into ES cells. For F-Luciferase shRNA vectors values are expressed as ratio of Luciferase to β-Galactosidase activity (left Y axis) and for β-Galactosidase shRNA vectors as ratio of β-Galactosidase to Luciferase activity (right Y axis), in comparison to the appropriate positive control. Results are expressed as mean values from duplicate samples with SD. U6-shLuc, U6-shLacZ: parental shRNA vectors; RLU: relative light units.
Mentions: According to this design we inserted a loxP-flanked 270 bp DNA segment, which includes termination sequences and serves as transcriptional stop element, in our parental U6 driven shRNA vector. The presence of the stop element in the ‘off ’ configuration of the conditional shRNA vector allows only the transcription of the sense region that does not induce RNA interference, provided that the shRNA construct follows the order 5′-sense-loop-antisense-3′ (Figure 2A). Upon Cre mediated deletion of the stop cassette, the shRNA vector acquires the configuration of loxP5 with the remaining loxP site in the loop sequence. To assess the functionality of the stop element, it was first inserted into the loop region of U6-shRNA vectors against the β-Galactosidase or F-Luciferase reporter genes (Figure 2A). Upon transient cotransfection with reporter gene expression plasmids into mouse ES cells, the efficiency of the stop cassette in the ‘off ’ state and RNAi efficiency in the ‘on’ state were analyzed and compared to the non-modified parental vectors U6-shLuc and U6-shLacZ (Figure 2B). The measurement of F-Luciferase and β-Galactosidase activity from transfected cells showed that the ‘off ’ versions did not induce gene silencing whereas the ‘on’ versions induced the same level of RNAi as the parental, non-conditional vectors (Figure 2B). These results show that the stop element and our loxP5 configuration provide a way to control shRNA expression through Cre mediated recombination.Figure 2.

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