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Figure 1 | BMC Gastroenterology

Figure 1

From: Characterisation of a transgenic mouse expressing R122H human cationic trypsinogen

Figure 1

a. Schematic illustration of the targeting vector: The vector is carrying the minus-205 fragment of the rat elastase promotor, the Kozak consensus, the cDNA of human cationic trypsinogen with the R122H mutation, and the bGH-polyadenylation signal of the bovine growth hormone. The position of the PCR primer for the verification of the transgene on genomic and on mRNA levels (NruI-Ela and Poly-rev) as well as the controls are indicated (TG-forward, TG-reverse, TryUp) b. Verification of the transgene in genomic DNA: Lane 1: 1000 bp marker, lane 2: 100 bp marker. Genomic DNA from transgene animals (lane 4–6), positive control (targeting vector diluted 1:10 000, lane 3), and negative control (water, lane 7) were used as a template with primers spanning the elastase promoter, the cDNA of human cationic trypsinogen, and the polyadenylation signal of the bovine growth hormone (NruI-Ela and Poly-rev). The PCR resulted in a single band of the expected size of 1352 bp. c. Verification of transgene on mRNA level: After mRNA preparation from pancreatic tissue a reverse transcription PCR was performed with either two upstream primers complementary to cationic trypsinogen (pTry) (used in lane 3, 4, 6) or to the elastase promotor (TG-forward) as a control. The downstream primer was complementary to trypsinogen (TG-reverse) (used in lane 5 and 7). Position of the primers see figure 1a. lane 1: 1000 bp marker, lane 2: 100 bp marker, lane 3: water control, lane 4: cDNA from a transgenic animal; PCR product with the expected size of 409 bp, lane 5: cDNA from a transgenic animal; no PCR product, lane 6: positive control for lane 4: targeting vector diluted 1:10 000, lane 7: positive control for lane 5: targeting vector diluted 1:10 000. d. Verification of the transgenic protein: Zymogene preparation from mouse pancreata or pancreatic juice were subjected to isoelectric focussing followed by western blot using a antibody directed against human cationic trypsinogen (lane 1: pancreatic juice; lane 2–5: control animals; lane 6: transgenic animal)

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