Figure 1: Oncogenic KRAS expression and tumour suppressor inactivation immortalizes purified primary human duct cells. | Nature Communications

Figure 1: Oncogenic KRAS expression and tumour suppressor inactivation immortalizes purified primary human duct cells.

From: Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells

Figure 1

(a) Schematic diagram summarizing experimental procedures. (b) FACS histogram of the dissociated human adult pancreas stained with antibody specific for CD133. Results are representative of three independent experiments. (c) Schematics of lentiviral constructs used and sgRNA sequences for the construction of lentiCRISPRv2. (d) Representative images of spheres cultured for 27 days from CD133+ ductal cells infected with combinations of lentiviruses (CTRL-NeoR, Control-NeoR alone; KRAS-NeoR, KRAS-NeoR alone; CTRLmix, Control-NeoR and lentiCRISPRv2-Control; KCST, KRAS-NeoR plus lentiCRISPRv2 against CDKN2A#1, SMAD4#1 and TP53#2). (e) Genomic DNA PCR for confirming the presence of lentiviral transgenes in uninfected (S1; Supplementary Table 1) and infected (S1 KCST) spheres. bp=base pair. (f) Relative mRNA expression level of oncogenic KRAS transgene (left) and the transgene plus endogenous KRAS (right); n=2. (g) Indel efficiency of each indicated genomic locus assessed by TIDE analysis, (h) Quantification of the total cell number in each cell passage of CD133+ cells infected with indicated combinations of lentiviruses. Data are presented as fold increase over day 1. (i) Representative stereoscopic and haematoxylin and eosin (H&E) staining images of S1 KCST spheres after 52 days in culture. Error bars=s.d., scale bars, 200 μm.

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