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. 2006 Mar 2;25(6):1344–1352. doi: 10.1038/sj.emboj.7601011

Figure 6.

Figure 6

Profiling gene expression in adult human pancreatic duct cells following ectopic expression of Ngn3 and IA1. (A) Duct cells were transduced with either AdHANgn3, AdIA1 or AdGFP and processed for RT–PCR analysis at 7 days following transduction. Exogenous HANgn3, IA1 and Ngn3-induced endogenous IA1 are readily detected in AdIA1 and AdHANgn3 samples, respectively, but not in the AdGFP control. Viral Ngn3 contains a HA-tag and is detected in the AdHANgn3 samples only. Ectopic IA1 expression does not induce endogenous NeuroD1, Pax4 or Nkx2.2 transcription factor expression, in contrast to Ngn3. Induction of Delta-Notch signaling components DLL1, DLL4 and Hes6 by Ngn3 is not recapitulated by IA1, but remains at expression levels comparable to the GFP control sample. IA1 does not influence Hes1 mRNA abundance. The endocrine marker genes encoding SYP, CHGA, prohormone convertase 1/3 (PC1/3) and, albeit to a lesser extent, insulin (INS) are induced by ectopic Ngn3 expression but remain similar to control levels in IA1-expressing cells. Control RT–PCRs detect transcripts for GFP and GAPD. (B) Cotransduction of Ngn3 and IA1 enhances Ngn3-mediated transdifferentiation in adult human duct cells. Cells were transduced with AdHANgn3 and AdGFP, AdHANgn3 and AdIA1 or AdGFP only at a constant total MOI of 110, and processed for RT–PCR analysis at 3 days following transduction. Exogenous HANgn3, IA1 and Ngn3-induced endogenous IA1 are readily detected. Expression of the Ngn3 target genes NeuroD1, Pax4 and Nkx2.2 is enhanced in the AdHANgn3 and AdIA1 cotransduced samples as compared to the AdHANgn3 sample. RT–PCR results also indicate higher expression of the Delta-Notch component Hes6 and the endocrine marker SYP in Ngn3 and IA1 cotransduced cells. A control GAPD RT–PCR indicates comparable amounts of cDNA input. (C) The level of transcripts shown in (B) was quantified by real-time RT–PCR using specific Taqman probes as described in ‘Materials and methods' and revealed a 2–6-fold increase in target mRNA following cotransduction of adult duct cells with Ngn3 and IA1 as compared to transduction with Ngn3 alone (n=3). Statistical significance of the data was determined by unpaired, two-tailed Student's t-test.