303. Deletion of Pdx1 in Mouse Pancreatic Ducts by CRISPR-Cas9 Mediated Gene Editing

Abstract

Introduction: The homeodomain transcription factor Pdx1 is crucial for pancreas formation. Pdx1-expressing cells are first observed at embryonic day 8.5 (E8.5), prior even to the earliest indication of morphogenesis, in endodermal cells designated to give rise to the pancreas. In adult mouse, Pdx1 protein is transiently expressed when pancreas is injured, such as Langerhans islets damage in alloxan induced diabetic mice, implying Pdx1 may be necessary for the neogenetic formation of β-cells from mature ducts. In order to study the role of pdx1 in the pancreatic duct for pancreatic regeneration, most investigators use the Cre-lox system to generate duct-specific Pdx1 deletions in mice. This method is able to delete Pdx1specifically from tissue or cells with or without a tamoxifen-inducible (cre-ERT2) system; however, several caveats and limitations exist with tamoxifen-induced depletion. In addition, the cre-ERT2 system may be leaky, resulting in constitutive rather than inducible activation in some cells. To overcome these flaws from the Cre-ERT2/lox system, we decided to use a novel CRISPR-Cas9 gene editing technique to delete pdx1 in pancreatic ducts. Methods: We previously used a mouse short Sox9 promoter (468 bp) to generate a recombinant adeno-associated virus (AAV) carrying a reporter GFP (AAV6-Sox9-GFP), and this recombinant virus construct was infused into the pancreatic ducts, through the common bile/pancreatic duct, to specifically tag the duct cells. In order to address pancreatic duct specific deletion of Pdx1 after alloxan ablation of beta-cells, we first to applied the CRISPR-Cas9 gene editing technique on the HEK293-CMV-GFP cell line in vitro (Figure 2Figure 2) and ROSA26 LSL tomato reporter mice ex vivo (Figure 3Figure 3), and then employed AAV6 mediated CRISPR-SaCas9 under the control of the mouse sox9 short promoter, coupled with paired guide RNAs (gRNAs) flanking the Pdx1 exon2 (Figure 4Figure 4) through pancreatic duct infusion into the pancreas. RESULTS AND CONCLUSIONS: These CRISPR-SaCas9 and gRNAs AAV viruses were co-infused into pancreases of C57BL/6J mice 2 days after alloxan treatment. One week after AAV6-SaCas9+gRNAs infusion, it appeared that only the Pdx1 in pancreatic ductal cells were deleted. Comparatively, PBS or AAV6-CMV-GFP viral infused C57BL/6J mice and ROSA26 LSL tomato reporter mice expressed fluorescence in the entire pancreas as a result of the deleted loxP sites that removed the stop cassette, the Pdx1 is not deleted. Thus, we first report here a highly efficient and specific ablation of Pdx1 in adult mouse pancreatic ducts using the CRISPR-Cas9 technique. Figure 1Figure 1. Construction of AAV CRISPR-SaCas9 and gRNA vectors. Figure 2Figure 2. Deletion of ZsGeen in HEK293-CMV-GFP cells using SacCas9 and gRNA in vitro. Figure 3Figure 3. Valiation of SaCas9 and gRNA in vivo deletion of loxP sites in ROSA26-LSL tomato mice pancreas. Figure 4Figure 4. Deletion of Pdx1 exon2 in alloxan treated C57 pancreatic ducts.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide