Pre-selection of integration sites imparts repeatable transgene expression

Variable gene expression amongst transgenic lines occurs due to copy number and to random associations of incoming DNA with chromosomal elements at th e site of integration. Here we describe a method of identifying sites permi ssive 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 appropria te down-regulation of lacZ following differentiation. Two clones were chose n for further analysis which displayed appropriate and inappropriate gene r egulation (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 recombin ation, to drive expression of lacZ, Transgenic embryos were assessed for th eir ability to direct lacZ expression to tissues in which the respective pr omoter sequences are normally active. The site which appropriately down-reg ulated lacZ in vitro (710) also showed appropriate in vivo regulation of la cZ from the three developmental promoters. Site 91, however, directed an ad ditional pattern of ectopic expression, which was common to all four promot ers. Pre-selection of genomic sites for the introduction of transgenes by g ene targeting improves the repeatability of transgene expression and provid es an efficient means of single copy transgene introduction by homologous r ecombination.