pcdna3 1 entry vector  (Addgene inc)

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Schematic of the Organoid Easytag workflow. ssRNP and a circular plasmid repair template are nucleofected into dissociated cells at day 0. By day 3, cells have proliferated to become tiny colonies and are removed from the Matrigel and dissociated for selection by flow cytometry. EGFP + cells are re-plated sparsely (~1000–1500 cells/well of a 24-well plate) and grown until day 15 when organoids reach a sufficient size to be manually picked under a fluorescent microscope. Typically, 10–40 organoid colonies formed per ~1000 cells seeded. Organoids are picked into individual wells and passaged until sufficient cells are obtained for both genotyping and freezing down the line. Cells with red nuclei represent incorrectly targeted cells. Cells with white nuclei denote correctly targeted cells. ( b ) Schematic of repair template design for N terminal fusion mEGFP-ACTB gene targeting and the final product. Arrow shows the position of gRNA. E1, exon 1; E2, exon 2; 5′HA, 5′ homology arm; 3′HA, 3′ homology arm. ( c ) Representative flow cytometry results showing the percentage of EGFP cells 72 hr after nucleofection is performed. ( d ) Representative image showing mEGFP-ACTB organoid. DIC channel on the left and EGFP channel on the right. ( e ) Immunofluorescence of mEGFP-ACTB organoids. Blue: DAPI (nuclei); green: EGFP (ACTB fusion protein); red: SOX9 (lung progenitor marker). ( f ) Schematic of the AAVS1 targeting repair template design and the final product. E1, exon 1, E2, exon 2. Arrow indicates the position of the gRNA. ( g ) Immunofluorescence of AAVS1-EF1a-mTagRFP-T organoids. Blue: DAPI (nuclei); red: mTagRFP-T (membrane localised reporter); white: SOX2 (lung progenitor marker). Scale bars in all panels denote 50 μm.

Article Snippet: The gRNA entry vector was cloned by infusion cloning of a EF1a promoter into pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W vector (a gift from Kosuke Yusa, Addgene plasmid # 67974) using BamHI and EcoRI sites, and then cloned a EGFP-CAAX to swap the EGFP sequence using XhoI and NotI sites.

Techniques: Plasmid Preparation, Selection, Flow Cytometry, Microscopy, Immunofluorescence, Marker, Membrane

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Strategy for targeting transcriptionally silent genes using the Organoid Easytag workflow. First step: knocking-in to the 3’ end of the locus an exogenous EF1a promoter, which is flanked by two Rox sites, driving nuclear-localised Venus expression. A T2A peptide sequence is inserted before the EF1a promoter in order to minimise the influence of the future fluorescent reporter on the gene of interest (GOI). Second step: transient transfection of a plasmid encoding the Dre-recombinase to remove the exogenous EF1a promoter. This results in Venus expression becoming dependent on transcription of the targeted locus. ( b ) Schematic of the SFTPC locus repair template design and final product. E5, exon 5; 5’ HA, 5’ homology arm; 3’ HA, 3’ homology arm. Arrow indicates the position of the gRNA. Red bar indicates the position of the stop codon. ( c ) Representative images showing SFTPC-T2A-Rox-EF1a-Rox-Venus-NLS organoids before and after Dre recombinase expression. Upper panel: nuclear-localised Venus fluorescence can be observed under a standard epifluorescent microscope before Dre recombinase expression; bottom panel: Venus fluorescent signal is lost after Dre recombinase expression. Arrows indicate nuclear-localised Venus signal. ( d ) Schematic of the strategy for testing SFTPC reporter function by NKX2-1 overexpression. SFTPC reporter lines were dissociated and transduced with NKX2-1 conditional overexpression lentivirus. Transduced cells are marked by TagRFP expression which can be enriched via flow cytometry. TagRFP expression is coupled to tet3G which, upon doxycycline (Dox) administration, binds to the tetON promoter and drives NKX2-1 overexpression. ( e ) Representative immunofluorescence showing that the SFTPC reporter is functional. Blue: DAPI (nuclei); green: Venus (SFTPC reporter); white: proSFTPC protein. ( f ) Western blot against Venus in the SFTPC reporter revealed a higher molecular weight (MW) band than expected. The expected MW for proSFTPC and Venus are 21 kDa and 27 kDa, respectively, the size of higher MW band (40–50 kDa) correlated well with the predicted size of a fusion protein (~48 kDa), suggesting an inefficient T2A cleavage.

Article Snippet: The gRNA entry vector was cloned by infusion cloning of a EF1a promoter into pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W vector (a gift from Kosuke Yusa, Addgene plasmid # 67974) using BamHI and EcoRI sites, and then cloned a EGFP-CAAX to swap the EGFP sequence using XhoI and NotI sites.

Techniques: Expressing, Sequencing, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Over Expression, Transduction, Flow Cytometry, Immunofluorescence, Functional Assay, Western Blot, Molecular Weight

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Schematic of the TP63 locus repair template design and final product. E14, exon 14; 5’ HA, 5’ homology arm; 3’ HA, 3’ homology arm. Arrow indicates the position of the gRNA. ( b ) Representative images showing TP63-T2A-Rox-EF1a-Rox-Venus-NLS heterozygous organoid. Nuclear-localised Venus fluorescence can be observed under epifluorescent microscope. Scale bar indicates 50 μm. ( c ) Schematic of the TP63 genotyping strategy. 5’ and 3’ junction amplicons consist of a primer inside the Venus sequence and another primer upstream, or downstream, of the homology arm, respectively. The expected lengths of each amplicon are labelled below the schematic. ( d ) Representative gel image showing correct colony genotyping results. The WT bands were excised for Sanger sequencing in lane #3. ( e ) Schematic of the final TP63 reporter genotyping strategy to validate EF1a removal. ( f ) Representative gel image showing correct colony genotyping results after Dre recombinase.

Article Snippet: The gRNA entry vector was cloned by infusion cloning of a EF1a promoter into pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W vector (a gift from Kosuke Yusa, Addgene plasmid # 67974) using BamHI and EcoRI sites, and then cloned a EGFP-CAAX to swap the EGFP sequence using XhoI and NotI sites.

Techniques: Fluorescence, Microscopy, Sequencing, Amplification