Fig. 4

Transgene expression detected by RT-PCR

DNase I-treated RNA was used for all reactions, and the predicted 238 bp band is shown. (A) Low concentrations of RNase reduced the amplified transgene produced by RT-PCR. Absence of reverse transcriptase results in no amplification, indicating that DNase I treatment eliminated all of the DNA and that the amplified product results from RNA-derived cDNA generated by the reverse transcriptase. Samples came from intestine of homozygous transgenic animals 4402 (lanes 1–3), 4302 (lanes 4, 5), and 4503 (lanes 6, 7). Lanes 1, 4, and 6: reaction with reverse transcription; lane 2: reaction with reverse transcription plus pretreatment with low concentration of RNase; lanes 3, 5, and 7: reaction without reverse transcription. (B) Multiple-tissue expression of transgene. Lanes 1–4: brain, intestine, liver, lung RNA from animal 4402; lane 5: liver from animal 4302; lane 6: liver RNA from animal 4503; lane 7: intestinal RNA from animal 4503; lane 8: abdominal and chest RNA in day 1 newborn animal 4301; lane 9: abdominal and chest RNA in day 2 newborn animal 4501. (C) Quantitative RT-PCR from intestinal RNA from animals 5308 and 4603. Lanes 1–5: actin and gli expression in a sick, zinc-induced transgenic animal (5308); lanes 6–10: actin and gli expression in a healthy, Zn⁺⁺-induced transgenic animal (4603). Lanes 1 and 6: 5 ng RNA; lanes 2 and 7: 10 ng RNA; lanes 3 and 8: 20 ng RNA; lanes 4 and 9: 40 ng RNA; lanes 5 and 10: 80 ng of RNA. Actin expression was linear from 5 to 20 ng, and GLI expression was linear from 5 to 40 ng of intestinal RNA. (D) Quantification of relative RNA expression. The ratio of GLI/actin band intensities from gels as those shown in C were compared using NIH Image after digital scanning.