recombinant spcas9 protein  (New England Biolabs)

Journal: Bioactive Materials

Article Title: Novel strategy for tumor immunotherapy using FITC-folate bispecific adapter bridged CAR immune cell cocktails

doi: 10.1016/j.bioactmat.2025.06.025

Figure Lengend Snippet: (A) Schematic of anti-FITC CAR construct and targeted knock-in strategy at the AAVS1 safe harbor locus. (B) Schematic illustration of optimized neutrophil and NK cells differentiation from CAR-modified human pluripotent stem cells, and tumor treatment process.

Article Snippet: Cells were then singularized by Accutase for 8–10 min, and 1–2.5 × 10 6 hPSCs were nucleofected with 6 μg SpCas9 AAVS1 gRNA T2 (Addgene; #79888) and 6 μg CAR donor plasmids (the anti-FITC CAR plasmid with CD8a signal peptide, anti-fluorescein scFv, CD8a extracellular and intracellular domains, 4-1BB co-stimulatory domain and CD3ζ signaling domain was previously constructed in our lab [ ] in 100 μL human stem cell nucleofection solution (Lonza; #VAPH‐5012) or 200 μL room temperature PBS−/− using program B-016 in a Nucleofector 2b.

Techniques: Construct, Knock-In, Modification

Journal: Bioactive Materials

Article Title: Novel strategy for tumor immunotherapy using FITC-folate bispecific adapter bridged CAR immune cell cocktails

doi: 10.1016/j.bioactmat.2025.06.025

Figure Lengend Snippet: Construction of anti-FITC chimeric antigen receptor (CAR) expressing H9 human pluripotent stem cells. (A) Schematic of anti-FITC CAR construct and targeted knock-in strategy at the AAVS1 safe harbor locus. Anti-FITC CAR is composed of a CD8a signaling peptide (CD8a-SP), an extracellular anti-FITC scFv, extracellular hinge and transmembrane domains of CD8a (CD8a-hg-tm), a co-stimulatory domain 4-1BB, and an intracellular signal transduction domain CD3ζ. (B) PCR genotyping of hPSC clones after puromycin selection is shown, and the expected PCR product for correctly targeted AAVS1 site is 991 bp (red arrow) with and efficiency of 13 clones from a total of 13. A homozygosity assay was performed on the knock-in clones, and those without ∼204 bp PCR products were homozygous (blue arrow). (C) Representative flow cytometry analysis of anti-FITC CAR expression on wildtype and CAR knock-in hPSCs. (D) Immunostaining analysis of OCT-4 and SSEA-4 expression by fluorescence images. Scale bar, 100 μm.

Article Snippet: Cells were then singularized by Accutase for 8–10 min, and 1–2.5 × 10 6 hPSCs were nucleofected with 6 μg SpCas9 AAVS1 gRNA T2 (Addgene; #79888) and 6 μg CAR donor plasmids (the anti-FITC CAR plasmid with CD8a signal peptide, anti-fluorescein scFv, CD8a extracellular and intracellular domains, 4-1BB co-stimulatory domain and CD3ζ signaling domain was previously constructed in our lab [ ] in 100 μL human stem cell nucleofection solution (Lonza; #VAPH‐5012) or 200 μL room temperature PBS−/− using program B-016 in a Nucleofector 2b.

Techniques: Expressing, Construct, Knock-In, Transduction, Clone Assay, Selection, Flow Cytometry, Immunostaining, Fluorescence