Figure 1

Analysis of the transgene structure in the BAC (ASS-EGFP) transgenic mice. (A) Overall view of BAC (ASS-EGFP) constructs showing relative positions of the EGFP transgene and the human ASS exons. Insertions of the EGFP gene with a polyadenylation signal into exon 3 (EGFP-pA) or an IRES-EGFP sequence into exon 16 of the ASS gene of BAC clone RP11-52B13 created the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) constructs, respectively. The lengths of the 5′ and 3′ human genomic sequences included in the BAC construct and the ASS structural gene are shown. Wavy line at the end represents the vector sequence. (B) Determination of transgene copy number. Genomic DNAs from offspring of the second generation transgenic mice (F2) was digested with EcoRI and Southern blot analysis was performed using an EGFP probe. The horizontal grey arrows are schematic representation of the transgene; the green bars represent the EGFP sequence; downward arrows indicate the EcoRI sites shown with expected sizes of the hybridized EcoRI fragments. The signal of a BAC (ASS-Ex3-EGFP) DNA preparation with predetermined copy number was used to estimate the transgene copy number in the mouse lines. The abbreviated transgenic line designations 3G, 3 J and 16E, 16H were two different lines each from the BAC (ASS-Ex3-EGFP) and BAC (ASS-Ex16-EGFP) transgenes, respectively; the abbreviated designations are also used in other figures. (C) Confirmation of head-to-tail transgene integration. The PstI sites (downward arrows) and the expected 1,044-bp head-to-tail hybridized fragments are shown. The size of junction fragment may be greater than 939 bp (>939 bp) which is determined by position of the next PstI site in the mouse genome. (D) Monitoring of ASS-EGFP transgene transmission by fluorescence signals of tail samples. Pedigree analysis of the transgenic line Tg (ASS-Ex16-EGFP) 16 F is shown. Upper and lower rows were observed by dissecting microscopy under fluorescent light or white light, respectively.