(A) Diagram of the imprinted domain on distal Chr7 showing the location of the insertion upstream of Ins2 (arrowhead), relative to the two imprinting centers (IC). Maternally and paternally expressed genes are in white and grey, non-imprinted genes in black; arrows show transcriptional orientation. Structure of the targeted I2loxP allele, with its promoterless loxP-neo-polyA (loxP-neopA) cassette inserted 2.6 kb telomeric to Ins2 (below). (B) Proposed mechanism to generate a circular intermediate in I2loxP/+ ES cells. The linear targeting vector is postulated to have multimerized, then a circular intermediate was resolved by Cre from this array. This provided a substrate for Cre-mediated insertion at I2loxP. (C) Resulting structure of the loxP-flanked Tel7KI allele showing the telomeric repeats (tel), the ubiquitous EGFP reporter (pCX-EGFP), and active Pgk-loxP-neopA marker.