Differences in integration sites and transcription of human F8 cDNA between sublines E and G. (A) Schematic pictures of 9 overlapping primer pairs spanning the whole human F8 cDNA and of a primer pair designed for long-range PCR. (B) Identification of the integration sites of human F8 cDNA by FISH analysis: chromosomes for FISH analysis were prepared from spleen cells activated with concanavalin A. F8-specific probes were prepared using the 9 overlapping primer pairs shown in panel A and labeled with 2 different fluorochromes: dioxigenin-dUTP and biotin-dUTP. The hybridization signals of the F8-specific probes and the corresponding chromosome mapping for samples obtained from mice of sublines E and G are demonstrated. (C) Detection and sizing of all PCR products obtained from genomic DNA of sublines E and G with the 9 overlapping primer pairs shown in panel A using an Agilent 2100 Bioanalyzer. The molecular size is indicated in the first lane in kilobases. (D) Confirmation of the integration of the full-length human F8 cDNA into the genome of mice of sublines E and G (EM is a male mouse from subline E; EF is a female mouse from subline E; GM is a male mouse from subline G; and GF is a female mouse from subline G) by long-range PCR on genomic DNA using an Agilent 2100 Bioanalyzer. The molecular size is indicated in the first lane in kilobases. (E) Detection and sizing of all PCR products obtained from RNA after reverse transcription into cDNA using an Agilent 2100 Bioanalyzer. The molecular size is indicated in the first lane in kilobases. RNA was prepared from liver samples obtained from mice of sublines E and G.