cloning prepared oligonucleotides  (Addgene inc)

Journal: Molecular biology reports

Article Title: Protein kinase C iota (PKCι) and pVHL are both needed for lysosomal degradation of α5 integrin in renal carcinoma cells.

doi: 10.1007/s11033-025-10272-1

Figure Lengend Snippet: Fig. 2 Knockout of PKCι in pVHL-expressing 786-O cells disrupts tight junction forma- tion. 786-O cells without pVHL (1st row) and with pVHL rein- troduced (rows 2–4) were grown on coverslips to confluence and assayed by indirect immuno- fluorescence staining for ZO-1 as a tight junction marker. Left panels show ZO-1 localization, center panels show stain for DAPI (nuclei), and the right panel depicts a merged image of the ZO-1 and DAPI stains. Cell lines used and their sgRNA targets are indicated on the left of each row. The images were taken at 1000× magnification

Article Snippet: LentiCRISPRv2 plasmid was digested with BsmBI restriction enzyme and ligated with annealed sgRNA oligonucleotides as described in the Target Guide Sequence Cloning Protocol found at the Addgene website provided. sgRNA sequences targeting PKCι were TGT CTC GAA CCT CAT TGC AA (exon 2, antisense strand) and TCC AAG CCA AGC GTT TCA AC (exon 5, sense strand), hereafter named PKCι sgRNA 1 and 2, respectively. sgRNA sequence targeting PKCζ was CCA TCC ATC CCA TCG ATA AC (exon 9, antisense strand).

Techniques: Knock-Out, Expressing, Fluorescence, Staining, Marker

Journal: Molecular biology reports

Article Title: Protein kinase C iota (PKCι) and pVHL are both needed for lysosomal degradation of α5 integrin in renal carcinoma cells.

doi: 10.1007/s11033-025-10272-1

Figure Lengend Snippet: Fig. 4 Cycloheximide and bafilomycin assays reveal that pVHL-mediated down- regulation of α5 integrin levels requires lysosomal degrada- tion. (A) Parental 786-O cells (VHL-) and those with reintro- duced VHLp19 (VHL+) were incubated with cycloheximide (100 µg/ml for various time points and then a western blot for α5 integrin was performed, with an α-tubulin western blot as a loading control. Band intensities were divided by their corresponding α-tubulin band intensities (from the same lane) and presented as a percent, with the first lane set to 100%. (B) Similar analysis was performed as in (A) with 786-O cell lines with pVHL reintroduced (VHL+) containing sgRNA targeting LacZ (as a control) or PKCι (PKCι 1 and PKCι 2). (C) 786-O cell lines used in (A) and (B) were incubated with vehicle or bafilomycin (10 nM) overnight and a western blot for α5 integrin was performed, with an α-tubulin western blot performed as a loading control. Note that in (A), (B), and (C), the α-tubulin western blots were performed by reblotting the membranes in the top panel. (D) Cells with reintroduced VHLp19 (VHL+) were incu- bated with DMSO (vehicle) or bafilomycin (10 nM) overnight before ZO-1 immunostaining, as previously described. Images were taken at 1000× magnifica- tion

Article Snippet: LentiCRISPRv2 plasmid was digested with BsmBI restriction enzyme and ligated with annealed sgRNA oligonucleotides as described in the Target Guide Sequence Cloning Protocol found at the Addgene website provided. sgRNA sequences targeting PKCι were TGT CTC GAA CCT CAT TGC AA (exon 2, antisense strand) and TCC AAG CCA AGC GTT TCA AC (exon 5, sense strand), hereafter named PKCι sgRNA 1 and 2, respectively. sgRNA sequence targeting PKCζ was CCA TCC ATC CCA TCG ATA AC (exon 9, antisense strand).

Techniques: Incubation, Western Blot, Control, Immunostaining

Journal: BIO-PROTOCOL

Article Title: Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1

doi: 10.21769/bioprotoc.5108

Figure Lengend Snippet: Figure 4. AsCpf1-mediated mutation formation in hPSCs. A. Representative fluorescent micrographs of hPSCs electroporated with the corresponding constructs on the left. B. Disruption of tdTomato expression in H1-tdT cells by CAGGS promoter driven AsCpf1 with a tdT crRNA. A 4-nucleotide deletion was identified in cells that lost tdTomato expression but not in control cells. Scale bar: 100 μm. G. Knockin of hPSC and multiplex genome editing

Article Snippet: Anneal the tdT-targeting crRNA oligos and ligate to CAGGS-AsCpf1-2A-GFP-U6-crRNA-cloning vector (Addgene, #159281) based on the procedures described in section B, giving rise to the CAGGS-AsCpf12A-GFP-U6-tdT-crRNA plasmid (Addgene, #194724).

Techniques: Mutagenesis, Construct, Disruption, Expressing, Control, Knock-In, Multiplex Assay

Journal: BIO-PROTOCOL

Article Title: Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1

doi: 10.21769/bioprotoc.5108

Figure Lengend Snippet: Figure 7. AsCpf1-mediated targeting in the INS locus in hPSCs enables monitoring of INS expression. A. The design of an INS targeting crRNA. The PAM sequence is highlighted in magenta, and the guide sequence is highlighted in yellow. B. The targeting strategy for generating INS luciferase and tdTomato reporter INS- luciferase-tdT allele by knocking the 2A-luciferase-2A-tdt cassette to the 3′ end of the INS coding sequence. C. Southern blots with internal probe (left panel) and external probe (right panel) to identify clones with a single copy of the 2A-luciferase-2A-tdt cassette inserted into the INS locus. Correct clones were labeled in green. The double bands in the control samples for the external probe (right panels) correspond to two wild-type INS alleles of the H1-OCT4-GFP cells. D. Expression of tdTomato in cells during the differentiation of the INS-luciferase- tdT reporter hPSCs toward pancreatic β-like cells. E. Representative confocal micrographs of INS-luciferase- tdT hPSC differentiated β-like cells with anti-tdTomato staining (red) and anti-C-peptide staining (green). F.

Article Snippet: Anneal the tdT-targeting crRNA oligos and ligate to CAGGS-AsCpf1-2A-GFP-U6-crRNA-cloning vector (Addgene, #159281) based on the procedures described in section B, giving rise to the CAGGS-AsCpf12A-GFP-U6-tdT-crRNA plasmid (Addgene, #194724).

Techniques: Expressing, Sequencing, Luciferase, Clone Assay, Labeling, Control, Staining