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Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
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Biologie
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Journal JoVE Biologie
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

DOI:
10.3791/2468-v

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08:53 min

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February 17, 2011

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, ,
1Department of Biomedical Informatics,The Ohio State University, 2Departments of Molecular Immunology and Clinical Oncology,Tohoku University

Chapitres

  • 00:05Titre
  • 02:01Preparation of Cell Line with an Integrated Recombination Substrate
  • 03:04Transfection to Deplete Endogenous BRCA1 and add back Mutant BRCA1
  • 05:19Analysis of Transfectants
  • 06:07Analysis of the Effects of BRCA1 Mutations on Homologous Recombination via GFP-Expression
  • 07:54Conclusion

Summary

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We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Tags

BRCA1 MutationsHomologous RecombinationEndogenous BRCA1Missense MutationsFunctional AnalysisCell LinesRNAi TargetingBRCA1 MRNABRCA1 VariantAcute SilencingSecondary MutationsCompensation For Loss Of FunctionHeLa-derived Cell LineHomologous Recombination AssayRecombination SubstrateI-SceI Restriction Enzyme SiteGFP Allele

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