Publication Date:
2011-06-17
Description:
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572410/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572410/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Skarnes, William C -- Rosen, Barry -- West, Anthony P -- Koutsourakis, Manousos -- Bushell, Wendy -- Iyer, Vivek -- Mujica, Alejandro O -- Thomas, Mark -- Harrow, Jennifer -- Cox, Tony -- Jackson, David -- Severin, Jessica -- Biggs, Patrick -- Fu, Jun -- Nefedov, Michael -- de Jong, Pieter J -- Stewart, A Francis -- Bradley, Allan -- 077188/Wellcome Trust/United Kingdom -- U01-HG004080/HG/NHGRI NIH HHS/ -- Wellcome Trust/United Kingdom -- England -- Nature. 2011 Jun 15;474(7351):337-42. doi: 10.1038/nature10163.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. skarnes@sanger.ac.uk〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21677750" target="_blank"〉PubMed〈/a〉
Keywords:
Alleles
;
Animals
;
Computational Biology
;
Embryonic Stem Cells/cytology/metabolism
;
*Gene Deletion
;
Gene Knockout Techniques/*methods
;
Genes/*genetics
;
Genes, Lethal/genetics
;
Genetic Association Studies/*methods
;
Genetic Vectors/genetics
;
Genome/*genetics
;
Genomics
;
Genotype
;
Humans
;
Mice
;
Mice, Inbred C57BL
;
Mice, Knockout/*genetics
;
Mutagenesis, Insertional/methods
;
Phenotype
;
Polymerase Chain Reaction
;
Rats
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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