Precise BAC targeting of genetically polymorphic mouse ES cells
| العنوان: | Precise BAC targeting of genetically polymorphic mouse ES cells |
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| المؤلفون: | Tahsin Stefan Barakat, Frank Sleutels, Eveline Rentmeester, J. Anton Grootegoed, Joost Gribnau |
| المساهمون: | Developmental Biology, Cell biology |
| المصدر: | Nucleic Acids Research Nucleic Acids Research, 39(18):e121. Oxford University Press |
| بيانات النشر: | Oxford University Press, 2011. |
| سنة النشر: | 2011 |
| مصطلحات موضوعية: | Genetics, Chromosomes, Artificial, Bacterial, Bacterial artificial chromosome, Transcription, Genetic, Ubiquitin-Protein Ligases, Biology, Cell Line, Mice, Inbred C57BL, Gene Knockout Techniques, Mice, genomic DNA, Open reading frame, Complementary DNA, Methods Online, Animals, Restriction fragment length polymorphism, Homologous recombination, Gene, Embryonic Stem Cells, Polymorphism, Restriction Fragment Length |
| الوصف: | The use of bacterial artificial chromosomes (BACs) provides a consistent and high targeting efficiency of homologous recombination in embryonic stem (ES) cells, facilitated by long stretches of sequence homology. Here, we introduce a BAC targeting method which employs restriction fragment length polymorphisms (RFLPs) in targeted polymorphic C57BL/6/Cast/Ei F1 mouse ES cell lines to identify properly targeted ES cell clones. We demonstrate that knockout alleles can be generated either by targeting of an RFLP located in the open reading frame thereby disrupting the RFLP and ablating gene function, or by introduction of a transcription stop cassette that prematurely stops transcription of an RFLP located downstream of the stop cassette. With both methods we have generated Rnf12 heterozygous knockout ES cells, which were identified by allele specific PCR using genomic DNA or cDNA as a template. Our results indicate that this novel strategy is efficient and precise, by combining a high targeting efficiency with a convenient PCR based readout and reliable detection of correct targeting events. |
| تدمد: | 1362-4962 0305-1048 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::54688384ec6595621567832128baaa06 https://pure.eur.nl/en/publications/c4420b2f-54b6-4547-8c3f-108f4dab34f5 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....54688384ec6595621567832128baaa06 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 13624962 03051048 |
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