PMID: 9548976Jun 13, 1998Paper

Modification of bacterial artificial chromosome clones using Cre recombinase: introduction of selectable markers for expression in eukaryotic cells

Genome Research
S Y KimC A Westbrook

Abstract

Bacterial artificial chromosome clones (BACs) are widely used at present in human genome physical mapping projects. To extend the utility of these clones for functional genomic studies, we have devised a method to modify BACs using Cre recombinase to introduce a gene cassette into the loxP sequence, which is present in the vector portion of the BAC clone. Cre-mediated integration is site specific and thus maintains the integrity of the genomic insert sequences, while eliminating the steps that are involved in restriction digest-based DNA cloning strategies. The success of this method depends on the use of a DNA construct, RETRObac, which contains the reporter marker green fluorescent protein (GFP) and the selectable marker neomycin phosphotransferase (neo), but does not contain a bacterial origin of replication. BAC clones have been modified successfully using this method and the genomic insert shows no signs of deletions or rearrangements. Transfection efficiencies of the modified BACs into human or murine cell lines ranged from 1% to 6%. After culture in media containing G418 for 3 weeks, approximately 0. 1% of cells previously sorted for GFP expression acquired stable antibiotic resistance. Introduction of a human BAC clone ...Continue Reading

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Citations

May 7, 2014·PloS One·Branden S MoriarityDavid A Largaespada
Jul 11, 2008·Genes, Chromosomes & Cancer·Ryan L RaglandThomas W Glover
May 26, 1999·Proceedings of the National Academy of Sciences of the United States of America·M R RondonJ Handelsman
Jun 5, 2003·Genomics·Elena G BochukovaAnthony P Monaco
Dec 20, 2003·Biochemical and Biophysical Research Communications·Rachael L BrakePaul M Watt

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