RT-PCR analysis of the transgene and the X-linked sequences. Total RNA from liver, kidney, brain, muscle, and heart tissues of the C57 or homozygous fad2 females was analysed using the different sets of primers. (A) The X-linked Hprt gene (normalisation control) showed normal expression in all samples from the fad2 and C57 mice. (B) Transgene specific primers revealed that the fad2 gene was expressed only in the transgenic mouse, not in the C57 mouse. (C-F) Transcripts of the region surrounding the X insertion site (from -1123 of the 5' integration to +3069 of the 3' integration site) were not detected in any C57 tissue. In fad2 mice, the examined upstream sequences (-1123 to -977) did not amplify (C), whereas RT-PCR fragments (D-E) of the downstream regions from +859 to +2299 were amplified successfully. The distal downstream region (from +1952 to +3069) was not detected in any of the examined transgenic tissues (F). Amplification without reverse transcriptase in each samples showed no contamination.