PCR analysis of the 5' integration site. (A) Schematic diagram of the approximate positions of five primer sets used for PCR analysis of the 5' integration site. P1 (-54,088 to -53,884), P2 (-29,872 to -29,553), and P3 (-44,562 to -44,144) primer pairs were designed according to the sequence upstream of nucleotide 87,507,732 (+1), whereas the forward primers of P4 (from -46,680) and P5 (from -45,797) were designed corresponding to the X sequence. The reverse primer (5'-gccaagtgggcagttta-3') corresponded to the CMV enhancer sequence included in the transgene construct. X+ represents the transgenic X. The double-dashed line represents the foreign DNA molecules. (B) Gel analysis of the PCR products amplified from the fad2 or C57 mice. The 205-bp fragments using P1 primers were successfully amplified in X+X+, X+X, X+Y, or C57 samples. The 320-bp fragments using P2 primers and the 419-bp fragments using P3 primers were only amplified in the X+X and C57, not in X+X+ or X+Y, samples. The 1732-bp fragment amplified by P4 primers and the 799-bp fragment amplified using P5 primers were present in all fad2, but not C57, mouse samples, and they were sequenced directly. (C) Nucleotide sequence of the P5 primer pair (underlined) and the 5' integration region in fad2 mice. Sequences from the 5' initial nucleotide to +509 were determined by direct sequencing. The partial sequence from the 5' initial nucleotide to -1 was 100% identical to the region upstream of the 87,462,177th nucleotide of the XC1 region of the X chromosome. The sequence from -3 to +509 showed 100% identity to the transgene sequence. A 5'-TGT-3' sequence (-3 to -1) was shared by the transgene and the X chromosome as a very short homologous arm. Transgenic sequences were divided into two classes based on the presence of five additional nucleotides (TACTG). The sequence from -3 to +324 was 100% identical to the foreign complementary sequence (from 3580 to 3259). The sequence from +330 to the 3' end was 100% identical to the 5' initial sequence of the transgene.