Chloroplast vectors and PCR confirmation of transgene integration. A) Two chloroplast expression cassettes were made, one with the IGF-1n gene and another with the IGF-1s gene (for complete sequence, see figure 1). A) The blue dotted lines show regions of homologous recombination with the chloroplast genome. Regulatory sequences used were from the tobacco chloroplast genome: Prrn: 16S rRNA promoter; 5'UTR: the psbA promoter and 5' UTR; 3'UTR: the psbA 3' UTR. The primers 3P & 3M and 5P & 2M were used to confirm the integration of the IGF-1 gene cassette into the chloroplast genome. The primer (3P, 3M or 5P, 2M) landing sites for PCR reactions are shown in green boxes. Transgenic lines should produce a 1.65 kb PCR product with 3P & 3M primers and 2.5 kbp products with 5P & 2M primers. B) Lanes 1–3: IGF-1s transgenic lines; lane 4: Untransformed control. C) Lanes 1–4: IGF-1n transgenic lines; lane 5: Untransformed control. D) Lanes 1–3: transgenic lines with the 5'UTRZZTEVIGF-1s gene cassette; Lanes 4–6: Transgenic lines with the 5'UTRZZTEVIGF-1n gene cassette. Lane 7: Untransformed control. Lanes marked MW show 1 kbp DNA ladder.