Figure 2

TAIL-PCR analysis of the 3' integration site. (A) Schematic diagram of the transgenic construct indicating the positions of three transgene primers (tgp) used, along with an arbitrary degenerate primer. (B) Gel analysis of TAIL-PCR products amplified from the fad2 mice. Total DNA from three fad2 mice (nos. 3, 5, and 15) was used for TAIL-PCR, and the specific amplified fragments (*) in each sample from the tertiary amplification were sequenced directly. (C) Nucleotide sequence of transgenic forward primers (underlined) tgp1 (-627 to -610), tgp2 (-381 to -360), and tgp3 (-206 to -187) and the 3' integration region in fad2 mice. The sequence from -172 to the 3' end was determined by direct sequencing. The sequencing results indicate that nucleotides from -172 to +7 were 100% identical to the transgene sequences and that the nucleotides from +1 to the 3' end were the same as the downstream sequence of the 87,507,732nd nucleotide of the XC1 region of the X chromosome. Seven nucleotides, from +1 to +7 (5'-TTAATAG-3'), were shared by the transgene and the X chromosome as a very short homologous arm. Furthermore, the sequences from -172 to -22 and -22 to +7, corresponding to the 3' end and the 5' initial transgene sequences, indicate that the foreign DNA molecules integrated as a head-to-tail array.