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Generation and validation of GLUL -/- HeLaS3 cell lines.( A ) Schematic representation of the GLUL gene structure, showing three transcript variants with exons represented as boxes and coding sequences highlighted in yellow. Exon 5, where the protein catalytic site is located, is also highlighted in orange. ( B ) <t>T7</t> <t>endonuclease</t> I assay of PCR-amplified genomic DNA from nucleofected cells. Original gel is presented in Supplementary Figure . ( C ) Phase-contrast microscopy images of GLUL -/- clones GKO1, GKO2, and GKO3 at Day 0, Day 3, and Day 10 post single-cell seeding. White arrows indicate clonal outgrowth. ( D ) Western blot analysis of GLUL protein expression in parental HeLaS3 (GLUL +/+ ) and GLUL -/- clones. β-actin was used as a loading control. Original blots are presented in Supplementary Figure .
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Generation and validation of GLUL -/- HeLaS3 cell lines.( A ) Schematic representation of the GLUL gene structure, showing three transcript variants with exons represented as boxes and coding sequences highlighted in yellow. Exon 5, where the protein catalytic site is located, is also highlighted in orange. ( B ) T7 endonuclease I assay of PCR-amplified genomic DNA from nucleofected cells. Original gel is presented in Supplementary Figure . ( C ) Phase-contrast microscopy images of GLUL -/- clones GKO1, GKO2, and GKO3 at Day 0, Day 3, and Day 10 post single-cell seeding. White arrows indicate clonal outgrowth. ( D ) Western blot analysis of GLUL protein expression in parental HeLaS3 (GLUL +/+ ) and GLUL -/- clones. β-actin was used as a loading control. Original blots are presented in Supplementary Figure .

Journal: Scientific Reports

Article Title: Streamlined rAAV HeLaS3 producer cell line generation via GS selection

doi: 10.1038/s41598-025-34826-2

Figure Lengend Snippet: Generation and validation of GLUL -/- HeLaS3 cell lines.( A ) Schematic representation of the GLUL gene structure, showing three transcript variants with exons represented as boxes and coding sequences highlighted in yellow. Exon 5, where the protein catalytic site is located, is also highlighted in orange. ( B ) T7 endonuclease I assay of PCR-amplified genomic DNA from nucleofected cells. Original gel is presented in Supplementary Figure . ( C ) Phase-contrast microscopy images of GLUL -/- clones GKO1, GKO2, and GKO3 at Day 0, Day 3, and Day 10 post single-cell seeding. White arrows indicate clonal outgrowth. ( D ) Western blot analysis of GLUL protein expression in parental HeLaS3 (GLUL +/+ ) and GLUL -/- clones. β-actin was used as a loading control. Original blots are presented in Supplementary Figure .

Article Snippet: CRISPR-Cas9 editing efficiency assay was performed based on the T7 Endonuclease I-based mutation detection method with the EnGen® Mutation Detection Kit (NEB #E3321) following manufacturer ́s protocol.

Techniques: Biomarker Discovery, T7EI Assay, Amplification, Microscopy, Clone Assay, Western Blot, Expressing, Control