title: Pre-selection of integration sites imparts repeatable transgene expression.
abstract: Variable gene expression amongst transgenic lines occurs due to copy number and to random associations of incoming DNA with chromosomal elements at the site of integration. Here we describe a method of identifying sites permissive for transgene expression and their use for efficient introduction of single copy transgenes by homologous recombination. ES clones were selected in HAT medium for expression of a randomly integrated HPRT marker lying 5' to an Oct4/ lacZ transgene. 794 clones were assessed in vitro for appropriate down-regulation of lacZ following differentiation. Two clones were chosen for further analysis which displayed appropriate and inappropriate gene regulation (clones 710 and 91, respectively). Three developmental promoters (thyroglobulin, Hox2.6 and Myf5) were then sequentially introduced into the original insertion sites in each clone (710 and 91) by homologous recombination, to drive expression of lacZ. Transgenic embryos were assessed for their ability to direct lacZ expression to tissues in which the respective promoter sequences are normally active. The site which appropriately down-regulated lacZ in vitro (710) also showed appropriate in vivo regulation of lacZ from the three developmental promoters. Site 91, however, directed an additional pattern of ectopic expression, which was common to all four promoters. Pre-selection of genomic sites for the introduction of transgenes by gene targeting improves the repeatability of transgene expression and provides an efficient means of single copy transgene introduction by homologous recombination.
authors.affiliation: Department of Molecular Biology, Roslin Institute, Roslin, Midlothian EH29 5PS, UK.
authors.forename: H
authors.initials: H
authors.lastname: Wallace
authors.forename: R
authors.initials: R
authors.lastname: Ansell
authors.forename: J
authors.initials: J
authors.lastname: Clark
authors.forename: J
authors.initials: J
authors.lastname: McWhir
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002999:Clone Cells / Q000166:cytology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005801:Genes, Homeobox / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009124:Muscle Proteins / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000235:genetics; D051787:Myogenic Regulatory Factor 5; D050814:Octamer Transcription Factor-3; D011401:Promoter Regions, Genetic / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D013954:Thyroglobulin / Q000235:genetics; D015534:Trans-Activators; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D009124:Muscle Proteins; C497418:Myf5 protein, mouse; D051787:Myogenic Regulatory Factor 5; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D013954:Thyroglobulin