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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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A recessive genetic screen to isolate RNAi mutants in Blm-deficient ES cells. The screen is diagrammed on the left and the drug resistance phenotype of the cells is shown on the right. Viable ES cells appear dark blue when stained with methylene blue. (A) Blm−/−Hprt+/+ cells are HATR and 6-TGS. (B) After the addition of a puromycin-linked U6-shRNA-Hprt to silence the Hprt gene, cells become HATS and 6-TGR. (C) Gene trap mutagenesis generates mutations in the cells and can be selected with the drug G418. (D) The Blm-deficient gene trapped ES cells accumulate homozygous gene trap mutations. Mutations that inactivate the RNAi pathway lead to Hprt expression and these cells are HATR and PuroR.
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Figure 1: A recessive genetic screen to isolate RNAi mutants in Blm-deficient ES cells. The screen is diagrammed on the left and the drug resistance phenotype of the cells is shown on the right. Viable ES cells appear dark blue when stained with methylene blue. (A) Blm−/−Hprt+/+ cells are HATR and 6-TGS. (B) After the addition of a puromycin-linked U6-shRNA-Hprt to silence the Hprt gene, cells become HATS and 6-TGR. (C) Gene trap mutagenesis generates mutations in the cells and can be selected with the drug G418. (D) The Blm-deficient gene trapped ES cells accumulate homozygous gene trap mutations. Mutations that inactivate the RNAi pathway lead to Hprt expression and these cells are HATR and PuroR.

Mentions: We constructed our selection system using a Blm-deficient ES cell line, PGG5-4, that contains two copies of the Hprt gene (Blm-deficient Hprt+/+ ES cells) (22). This cell line was originally engineered from NM5 ES cells that are Hprt-deficient and Blm-deficient. Two copies of the PGK-Hprt minigene were sequentially targeted at the mouse gdf7 locus in order to maintain homozygosity for the Hprt gene required in an LOH-based recessive genetic screen. The expression of Hprt in the cells was confirmed by drug selection with HAT and 6-TG (Figure 1A). We electroporated the cells with a U6-promoter driven shRNA to silence the Hprt gene through RNAi. The shRNA contained a puromycin (puro) marker (puro::shRNA) to select for cells that stably incorporated the transgene. Blm-deficient cells expressing the puro::shRNA effectively silenced the Hprt gene and the cells became puromycin resistant (puroR), HAT sensitive (HATS) and 6-TGR (Figure 1B). These reporter cell lines were expanded and used as the cell lines for screening. Although our preliminary results showed that one copy of shRNA was sufficient to knock-down Hprt expression to an undetectable level, the use of two independent shRNA transgenes can compensate for the loss of a single transgene through mitotic recombination. Therefore, reporter cell lines were created that contained a second copy of Hprt shRNA, selectable with zeocin (zeo::shRNA) in addition to the puro::shRNA (not depicted in the diagram for simplicity). In the presence of Hprt shRNA, RNAi competent cells were selected with 6-TG and then expanded to establish several cell lines including 59, a8, c9 (containing puro::shRNA) 59Z4 and 59Z12 (containing both puro::shRNA and zeo::shRNA) (Figure 1B).Figure 1.


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)

A recessive genetic screen to isolate RNAi mutants in Blm-deficient ES cells. The screen is diagrammed on the left and the drug resistance phenotype of the cells is shown on the right. Viable ES cells appear dark blue when stained with methylene blue. (A) Blm−/−Hprt+/+ cells are HATR and 6-TGS. (B) After the addition of a puromycin-linked U6-shRNA-Hprt to silence the Hprt gene, cells become HATS and 6-TGR. (C) Gene trap mutagenesis generates mutations in the cells and can be selected with the drug G418. (D) The Blm-deficient gene trapped ES cells accumulate homozygous gene trap mutations. Mutations that inactivate the RNAi pathway lead to Hprt expression and these cells are HATR and PuroR.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: A recessive genetic screen to isolate RNAi mutants in Blm-deficient ES cells. The screen is diagrammed on the left and the drug resistance phenotype of the cells is shown on the right. Viable ES cells appear dark blue when stained with methylene blue. (A) Blm−/−Hprt+/+ cells are HATR and 6-TGS. (B) After the addition of a puromycin-linked U6-shRNA-Hprt to silence the Hprt gene, cells become HATS and 6-TGR. (C) Gene trap mutagenesis generates mutations in the cells and can be selected with the drug G418. (D) The Blm-deficient gene trapped ES cells accumulate homozygous gene trap mutations. Mutations that inactivate the RNAi pathway lead to Hprt expression and these cells are HATR and PuroR.
Mentions: We constructed our selection system using a Blm-deficient ES cell line, PGG5-4, that contains two copies of the Hprt gene (Blm-deficient Hprt+/+ ES cells) (22). This cell line was originally engineered from NM5 ES cells that are Hprt-deficient and Blm-deficient. Two copies of the PGK-Hprt minigene were sequentially targeted at the mouse gdf7 locus in order to maintain homozygosity for the Hprt gene required in an LOH-based recessive genetic screen. The expression of Hprt in the cells was confirmed by drug selection with HAT and 6-TG (Figure 1A). We electroporated the cells with a U6-promoter driven shRNA to silence the Hprt gene through RNAi. The shRNA contained a puromycin (puro) marker (puro::shRNA) to select for cells that stably incorporated the transgene. Blm-deficient cells expressing the puro::shRNA effectively silenced the Hprt gene and the cells became puromycin resistant (puroR), HAT sensitive (HATS) and 6-TGR (Figure 1B). These reporter cell lines were expanded and used as the cell lines for screening. Although our preliminary results showed that one copy of shRNA was sufficient to knock-down Hprt expression to an undetectable level, the use of two independent shRNA transgenes can compensate for the loss of a single transgene through mitotic recombination. Therefore, reporter cell lines were created that contained a second copy of Hprt shRNA, selectable with zeocin (zeo::shRNA) in addition to the puro::shRNA (not depicted in the diagram for simplicity). In the presence of Hprt shRNA, RNAi competent cells were selected with 6-TG and then expanded to establish several cell lines including 59, a8, c9 (containing puro::shRNA) 59Z4 and 59Z12 (containing both puro::shRNA and zeo::shRNA) (Figure 1B).Figure 1.

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