We previously generated transgenic mice carrying a large P1-artificial chromosome (PAC160) encompassing a 160Kb segment containing the human renin gene, two upstream genes and one downstream gene. We also previously generated mutant PAC160 constructs lacking the distal enhancer and concluded it is required to maintain baseline expression of human renin, but is not required for tissue-specific, cell-specific and regulated expression of renin in vivo. We now report two additional transgenic lines carrying random truncations of PAC160 upstream of the renin gene. Southern and PCR mapping studies indicate that the truncation break points in the two lines are located approximately 10.4 Kb and 2.5 Kb upstream of the renin gene causing a deletion of all DNA upstream of the break. We tested the hypothesis that large scale deletion of DNA upstream of the human renin gene including the enhancer would cause dysregulation of human renin expression. Phenotypically, these truncations cause a severe dysregulation of human renin expression, but remarkably, a preservation of the normal tissue-specific expression of the human ethanolamine kinase 2 (ETNK2) gene which lies immediately downstream of renin. Several functional binding sites for CTCF, a mammalian insulator protein, were identified in and around the renin and ETNK2 loci by gel shift and chromatin immunoprecipitation. We conclude that there are sequences in and around the renin and ETNK2 loci which act as boundaries between neighboring genes which insulate them from each other. The study illustrates the value of taking a much wider genomic perspective when studying mechanisms regulating gene expression.