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A genetic screen for components of the mammalian RNA interference pathway in Bloom-deficient mouse embryonic stem cells.

Trombly MI, Su H, Wang X - Nucleic Acids Res. (2009)

Bottom Line: This result demonstrates that true RNAi components can be isolated by this screening strategy.Furthermore, Ago2 homozygous mutant ES cells provide a genetic background to perform mutational analyses of the Ago2 protein.Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the isolation of putative homozygous RNAi mutant cells. Candidate clones were confirmed by an independent RNAi-based reporter assay and the causative gene trap integration site was identified using molecular techniques. Our screen identified multiple mutant cell lines of Argonaute 2 (Ago2), a known essential component of the RNAi pathway. This result demonstrates that true RNAi components can be isolated by this screening strategy. Furthermore, Ago2 homozygous mutant ES cells provide a genetic background to perform mutational analyses of the Ago2 protein. Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.

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Isolation of Ago2 mutants. (A) All Ago2 gene-trap mutants were identified in the first intron of the Ago2 locus, six are depicted in the diagram with the gene trap construct that was used in the screen shown above the arrowheads. A western blot for Ago2 shows that endogenous Ago2 was below detection in the mutant cells. (B) To confirm that the isolated Ago2 mutant was homozygous for the gene trap, a Southern blot was performed on EcoRI digested DNA from the reporter cell line and one of the Ago2 mutants, Ago2# 13. A probe for a region flanking the Ago2 gene trap was used to detect the wild-type locus as a 3.1 kb fragment and the gene trap locus as a 5.7 kb fragment. The Ago2 mutant is homozygous for the gene trap as shown by the detection of only the 5.7 kb fragment.
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Figure 4: Isolation of Ago2 mutants. (A) All Ago2 gene-trap mutants were identified in the first intron of the Ago2 locus, six are depicted in the diagram with the gene trap construct that was used in the screen shown above the arrowheads. A western blot for Ago2 shows that endogenous Ago2 was below detection in the mutant cells. (B) To confirm that the isolated Ago2 mutant was homozygous for the gene trap, a Southern blot was performed on EcoRI digested DNA from the reporter cell line and one of the Ago2 mutants, Ago2# 13. A probe for a region flanking the Ago2 gene trap was used to detect the wild-type locus as a 3.1 kb fragment and the gene trap locus as a 5.7 kb fragment. The Ago2 mutant is homozygous for the gene trap as shown by the detection of only the 5.7 kb fragment.

Mentions: After confirmation of the RNAi mutant phenotype by luciferase assay, we performed Splinkerette PCR to identify the gene trap integration site. Six independent clones had gene trap integrations within the 38-kb intron 1 of Ago2 (Figure 4A). Loss of Ago2 was confirmed by western blot on several of the mutants (Figure 4A). Other clones, which were isolated once, included VIG (by PGGV6), and CA150 (by PGGV7). To confirm that the Ago2 clones were indeed homozygous for the gene trap, we performed Southern blot analysis. For Ago2 clone #13, genomic DNA was digested with EcoRI and hybridized with a probe detecting a region flanking the gene trap. Southern blot analysis showed that the Ago2 mutant cells are homozygous for the gene trap (Figure 4B). However, two other mutant clones (VIG and CA150) were found to be heterozygous for the gene trap (data not shown). The significance of these heterozygous gene trap clones remains to be determined.Figure 4.


A genetic screen for components of the mammalian RNA interference pathway in Bloom-deficient mouse embryonic stem cells.

Trombly MI, Su H, Wang X - Nucleic Acids Res. (2009)

Isolation of Ago2 mutants. (A) All Ago2 gene-trap mutants were identified in the first intron of the Ago2 locus, six are depicted in the diagram with the gene trap construct that was used in the screen shown above the arrowheads. A western blot for Ago2 shows that endogenous Ago2 was below detection in the mutant cells. (B) To confirm that the isolated Ago2 mutant was homozygous for the gene trap, a Southern blot was performed on EcoRI digested DNA from the reporter cell line and one of the Ago2 mutants, Ago2# 13. A probe for a region flanking the Ago2 gene trap was used to detect the wild-type locus as a 3.1 kb fragment and the gene trap locus as a 5.7 kb fragment. The Ago2 mutant is homozygous for the gene trap as shown by the detection of only the 5.7 kb fragment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651804&req=5

Figure 4: Isolation of Ago2 mutants. (A) All Ago2 gene-trap mutants were identified in the first intron of the Ago2 locus, six are depicted in the diagram with the gene trap construct that was used in the screen shown above the arrowheads. A western blot for Ago2 shows that endogenous Ago2 was below detection in the mutant cells. (B) To confirm that the isolated Ago2 mutant was homozygous for the gene trap, a Southern blot was performed on EcoRI digested DNA from the reporter cell line and one of the Ago2 mutants, Ago2# 13. A probe for a region flanking the Ago2 gene trap was used to detect the wild-type locus as a 3.1 kb fragment and the gene trap locus as a 5.7 kb fragment. The Ago2 mutant is homozygous for the gene trap as shown by the detection of only the 5.7 kb fragment.
Mentions: After confirmation of the RNAi mutant phenotype by luciferase assay, we performed Splinkerette PCR to identify the gene trap integration site. Six independent clones had gene trap integrations within the 38-kb intron 1 of Ago2 (Figure 4A). Loss of Ago2 was confirmed by western blot on several of the mutants (Figure 4A). Other clones, which were isolated once, included VIG (by PGGV6), and CA150 (by PGGV7). To confirm that the Ago2 clones were indeed homozygous for the gene trap, we performed Southern blot analysis. For Ago2 clone #13, genomic DNA was digested with EcoRI and hybridized with a probe detecting a region flanking the gene trap. Southern blot analysis showed that the Ago2 mutant cells are homozygous for the gene trap (Figure 4B). However, two other mutant clones (VIG and CA150) were found to be heterozygous for the gene trap (data not shown). The significance of these heterozygous gene trap clones remains to be determined.Figure 4.

Bottom Line: This result demonstrates that true RNAi components can be isolated by this screening strategy.Furthermore, Ago2 homozygous mutant ES cells provide a genetic background to perform mutational analyses of the Ago2 protein.Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the isolation of putative homozygous RNAi mutant cells. Candidate clones were confirmed by an independent RNAi-based reporter assay and the causative gene trap integration site was identified using molecular techniques. Our screen identified multiple mutant cell lines of Argonaute 2 (Ago2), a known essential component of the RNAi pathway. This result demonstrates that true RNAi components can be isolated by this screening strategy. Furthermore, Ago2 homozygous mutant ES cells provide a genetic background to perform mutational analyses of the Ago2 protein. Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.

Show MeSH
Related in: MedlinePlus