Fig. 2

BAC constructs used to generate transgenic reporter mice. The RP11-571I2 BAC contains the human transthyretin (TTR) coding sequence as well as 42 kb of additional DNA in the 3′-direction and 126 kb of DNA in the 5′-direction (A). The TTR coding sequence from the start codon to stop codon, including introns, was replaced with reporter cDNA, but the 3′- and 5′-untranslated regions of the TTR mRNA remain (B). Sequences distributed throughout the reporter BAC are present in genomic DNA of transgenic mice (C). Founder lines are shown on the left. Sequences of primers are given in Table 1, and their locations within the BAC are designated by black circles in A. Each PCR reaction involved the use of two primer pairs, one amplifying a control sequence present in all mice and one amplifying sequences either within the reporter cDNA or representing human sequences in the BAC that are far removed from the insert in either the 5′- or 3′-direction. Amplicons were separated by electrophoresis in 1% agarose gels containing GreenGlo fluorescent DNA dye. Lanes labeled “a” represent reactions involving experimental transgenic mouse DNA; lanes “b” are negative controls in which water was substituted for DNA; lanes “c” are positive controls involving intact tdTomato BAC; lanes “d” are negative controls involving DNA from a wild type mouse; lanes “e” are positive controls involving DNA from a different, previously genotyped transgenic mouse; and lanes “f” are positive controls involving linearized luc2 BAC DNA