mouse ppard targeting crispr plasmid  (Addgene inc)

Journal: Nature Communications

Article Title: Rapid acceleration of KRAS- mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ

doi: 10.1038/s41467-022-30392-7

Figure Lengend Snippet: a – c Pancreatic tissues from KC or KC/Pd mice fed the HFD or Ctrl diet; and KC/Pd mice fed the GW or Ctrl diet for 3 days ( n = 3–4 biologically independent samples). Venn diagram of differentially expressed chemokines ( a ). Quantitative results of Ccl2 protein levels for KC/Pd mice on the GW diet ( b ) and KC or KC/Pd mice on the HFD ( c ). d Representative images of Ccl2 RNAscope in situ hybridization for normal pancreatic tissues of KC/Pd mice fed the GW or Ctrl diet for 3 days ( n = 3 biologically independent samples). e Venn diagram of differentially expressed chemokines for mouse tdTomato RFP–sorted pancreatic epithelial cells of KC/tdPd mice on GW or control diet for 3 days and for the cell culture media from mouse NB490 KPC cells with WT or Ppard KO. f , g Quantitative results of Ccl2 protein levels for tdTomato-RFP + cells ( f , n = 4 biologically independent samples) and for the cell culture media from mouse NB490 KPC cells from 4 independent experiments ( g ). h , i Ccl2 mRNA expression levels in tdTomato-RFP + pancreatic epithelial cells ( h , n = 4 biologically independent samples) and in mouse NB490 KPC cells from 4 independent experiments ( i ). j Ccl2 mRNA expression levels in mouse KC and KC/Pd PDAC cells with or without GW treatment from 4 independent experiments. k , l PPARδ ( k ) and CCL2 ( l ) mRNA expression levels in human PDAC cells transfected with PPARδ siRNAs (siPPARD) or control siRNA (Ctrl) from 3 independent experiments. m The PPARδ binding to the four predicted PPARδ binding sites (pPDBS) in the mCcl2 promoter in mouse KC PDAC cells stably transduced with mouse DDK-tagged PPARδ expressing lentivirus and treated with 1 µM GW or solvent (DMSO) from 3 independent experiments. Data are mean ± SEM. For ( b ), ( f ), ( h ), unpaired two-tailed Student’s t test, for ( c ), ( k – m ), multiple t -tests, for ( g ), ( i ), one-way ANOVA with Bonferroni correction, and for ( j ), two-way ANOVA with Bonferroni correction. * P < .05, ** P < .01, *** P < .001, and **** P < .0001. Source data are provided as a Source Data file.

Article Snippet: We first constructed a mouse Ppard –targeting CRISPR plasmid by subcloning the following pairs of DNA oligos: 5ʹ-(phos) caccgGAGGAAGTGGCCATGGGTGA-3ʹ (sense) and 5ʹ-(phos) aaacTCACCCATGGCCACTTCCTCc-3ʹ (antisense) into pSpCas9(BB)-2A-Puro (PX459) plasmid (Addgene, #48139) between two BbsI restriction enzyme sites.

Techniques: RNAscope, In Situ Hybridization, Control, Cell Culture, Expressing, Transfection, Binding Assay, Stable Transfection, Transduction, Solvent, Two Tailed Test

Journal: Nature Communications

Article Title: Rapid acceleration of KRAS- mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ

doi: 10.1038/s41467-022-30392-7

Figure Lengend Snippet: a Representative image of co-staining for Ccl2 RNAscope in situ hybridization with Ccr2 IF (top) or with F4/80 IHC (bottom) in pancreatic normal, acinar-to-ductal metaplasia (ADM), and PanIN tissues in the KC/Pd mice fed the GW diet for 3 days ( n = 5 per group). b KC/Pd mice at 6–8 weeks on the GW diet (50 mg/kg) for 0, 3, or 9 days were euthanized, and then pancreatic tissues were harvested for further analyses ( n = 4–6 per group). Representative images of co-IF staining of Ccr2 with F4/80 (TAMs) (top) or with Gr1 (MDSCs) (bottom). c – h Ccr2 inhibitor PF4136309 (PF) or control solvent (corn oil) was administered via subcutaneous injection at 80 mg/kg twice daily to the KC/Pd mice for 4 days ( n = 5 mice per group), and the mice were fed the GW diet (50 mg/kg) for the last 3 days, and then pancreatic tissues were harvested for further analyses. c Timeline for the mice with PF4136309 and GW diet treatment. d , e Representative images of H&E staining of pancreata ( d ) and percentage of pancreatic neoplastic area per mouse ( e ) for GW-fed KC/Pd mice treated with PF4136309 or Ctrl. f – h Representative images of co-IF staining of Ccr2 with F4/80 (TAMs) ( f , top) or with Gr1 (MDSCs) ( f , bottom), and quantitative co-IF staining results of double-positive cells per 40× field for Ccr2 + /F4/80 + cells ( g ) and Ccr2 + /Gr1 + cells ( h ) for the indicated mice. i Schematic flow showing PPARδ hyperactivation by either the HFD or the GW diet upregulates CCL2, which chemoattracts macrophages and MDSCs into pancreata via the CCL2/CCR2 axis, leading to an inflammatory and immunosuppressive TME (e.g., IL6/STAT3) and subsequent progression of KRAS mu -initiated pancreatic tumorigenesis to PDAC, while PPARD -genetic KO and a CCR2 inhibitor (PF4136309) suppress those tumorigenic effects. Data are mean ± SEM. Unpaired two-tailed Student’s t test. ** P < .01, *** P < .001, and **** P < .0001. Source data are provided as a Source Data file.

Article Snippet: We first constructed a mouse Ppard –targeting CRISPR plasmid by subcloning the following pairs of DNA oligos: 5ʹ-(phos) caccgGAGGAAGTGGCCATGGGTGA-3ʹ (sense) and 5ʹ-(phos) aaacTCACCCATGGCCACTTCCTCc-3ʹ (antisense) into pSpCas9(BB)-2A-Puro (PX459) plasmid (Addgene, #48139) between two BbsI restriction enzyme sites.

Techniques: Staining, RNAscope, In Situ Hybridization, Control, Solvent, Injection, Two Tailed Test