Generation of a landing-pad T cell line useful for T cell receptor customization

Presented by

Stacey Ward, PhD

About this talk

T cell biology is integral to the study of normal immune regulation as well as cancer biology, Car-T cells, epitope specificity and antigen presentation. However, primary T cells can be difficult to propagate in culture for the length of time necessary for functional assays. In addition, primary T cells express variant T cell receptor (TCR) heterodimers that can be challenging to identify and may not be optimal for downstream studies. We sought to simplify this system using transformed T cells which can be grown in culture for extended periods of time. We engineered a floxed landing pad sequence into the safe harbor AAVS1 genetic locus using CompoZr zinc finger nucleases. Both the promoter and landing pad expression cassette are flanked by unique lox sites, allowing swapping of either the promoter and/or expression cassette as needed. We ensured that only one copy of this sequence was found within the genome to avoid any complications associated with random insertion events. We also generated a landing pad cell line null for the endogenous TCR using Cas9/CRISPR ribonucleotide complexes. Both the TCR alpha and beta loci were rendered null due to non-homologous end joining and the presence of insertions and deletions culminating in premature stop codons were genotyped using next generation sequencing. The absence of a functional TCR was validated using flow cytometry staining for surface TCR and CD3. This cell line was then used to generate a knock-in of the desired exogenous TCR heterodimer to the landing pad locus, verified using flow cytometry staining. These lines will be very useful for a multitude of studies where a researcher needs to express a gene of interest in a discrete genetic locus or wants to generate a panel of TCR expressing cell lines.

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