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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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Diagram of retroviral gene trap constructs. (A) Schematic of how gene traps create mutations after integration. As an example, integration of a proviral gene trap between exons 1 and 2 leads to loss of exons 2 and 3. (B) Diagrams of PGGV2-PGGV7 are depicted. Grey bars in PGGV5-7 represent unique insertion in LTR. The titer for each construct is shown to the right. SA, splice acceptor; LacZ, beta-galactosidase; Neo, neomycin resistance marker; PolA, polyadenylation.
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Figure 2: Diagram of retroviral gene trap constructs. (A) Schematic of how gene traps create mutations after integration. As an example, integration of a proviral gene trap between exons 1 and 2 leads to loss of exons 2 and 3. (B) Diagrams of PGGV2-PGGV7 are depicted. Grey bars in PGGV5-7 represent unique insertion in LTR. The titer for each construct is shown to the right. SA, splice acceptor; LacZ, beta-galactosidase; Neo, neomycin resistance marker; PolA, polyadenylation.

Mentions: To screen for RNAi mutant cells, we used a recombinant retroviral gene trap strategy to mutagenize the reporter cell lines. Retroviral gene trap mutagenesis has been successfully used in recessive genetic screens performed in Blm-deficient ES cells (22,23). The major advantage of this approach is the ability to use the gene trap provirus as a molecular tag to identify the integration site. Integration within a gene results in splicing of upstream exons to the gene trap, and a polyadenylation signal in the gene trap prematurely terminates the mRNA leading to a loss of downstream exons (Figure 2A). The gene trap contains a neomycin resistance marker to allow for Geneticin (G418) selection of the stably integrated and expressed gene traps. After integration, the gene trap contains two loxP sites, and Cre-expression removes the gene trap leaving behind a single long terminal repeat (LTR) in the locus. In some cases, a gene trap event can be reverted after Cre excision provided that the remaining viral LTR does not interfere with the endogenous locus.Figure 2.


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)

Diagram of retroviral gene trap constructs. (A) Schematic of how gene traps create mutations after integration. As an example, integration of a proviral gene trap between exons 1 and 2 leads to loss of exons 2 and 3. (B) Diagrams of PGGV2-PGGV7 are depicted. Grey bars in PGGV5-7 represent unique insertion in LTR. The titer for each construct is shown to the right. SA, splice acceptor; LacZ, beta-galactosidase; Neo, neomycin resistance marker; PolA, polyadenylation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Diagram of retroviral gene trap constructs. (A) Schematic of how gene traps create mutations after integration. As an example, integration of a proviral gene trap between exons 1 and 2 leads to loss of exons 2 and 3. (B) Diagrams of PGGV2-PGGV7 are depicted. Grey bars in PGGV5-7 represent unique insertion in LTR. The titer for each construct is shown to the right. SA, splice acceptor; LacZ, beta-galactosidase; Neo, neomycin resistance marker; PolA, polyadenylation.
Mentions: To screen for RNAi mutant cells, we used a recombinant retroviral gene trap strategy to mutagenize the reporter cell lines. Retroviral gene trap mutagenesis has been successfully used in recessive genetic screens performed in Blm-deficient ES cells (22,23). The major advantage of this approach is the ability to use the gene trap provirus as a molecular tag to identify the integration site. Integration within a gene results in splicing of upstream exons to the gene trap, and a polyadenylation signal in the gene trap prematurely terminates the mRNA leading to a loss of downstream exons (Figure 2A). The gene trap contains a neomycin resistance marker to allow for Geneticin (G418) selection of the stably integrated and expressed gene traps. After integration, the gene trap contains two loxP sites, and Cre-expression removes the gene trap leaving behind a single long terminal repeat (LTR) in the locus. In some cases, a gene trap event can be reverted after Cre excision provided that the remaining viral LTR does not interfere with the endogenous locus.Figure 2.

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