Version 1
: Received: 28 August 2024 / Approved: 29 August 2024 / Online: 29 August 2024 (09:20:29 CEST)
How to cite:
Zhiyong, H.; Cole, K.; He, H.-J. Immortalization of Human Primary CD8+ T cells by Inserting a Single Copy of Human Telomerase Reverse Transcriptase via CRISPR/Cas9. Preprints2024, 2024082129. https://doi.org/10.20944/preprints202408.2129.v1
Zhiyong, H.; Cole, K.; He, H.-J. Immortalization of Human Primary CD8+ T cells by Inserting a Single Copy of Human Telomerase Reverse Transcriptase via CRISPR/Cas9. Preprints 2024, 2024082129. https://doi.org/10.20944/preprints202408.2129.v1
Zhiyong, H.; Cole, K.; He, H.-J. Immortalization of Human Primary CD8+ T cells by Inserting a Single Copy of Human Telomerase Reverse Transcriptase via CRISPR/Cas9. Preprints2024, 2024082129. https://doi.org/10.20944/preprints202408.2129.v1
APA Style
Zhiyong, H., Cole, K., & He, H. J. (2024). Immortalization of Human Primary CD8<sup>+</sup> T cells by Inserting a Single Copy of Human Telomerase Reverse Transcriptase via CRISPR/Cas9. Preprints. https://doi.org/10.20944/preprints202408.2129.v1
Chicago/Turabian Style
Zhiyong, H., Kenneth Cole and Hua-Jun He. 2024 "Immortalization of Human Primary CD8<sup>+</sup> T cells by Inserting a Single Copy of Human Telomerase Reverse Transcriptase via CRISPR/Cas9" Preprints. https://doi.org/10.20944/preprints202408.2129.v1
Abstract
Background: Existing human cell immortalization methods made the cells obtain oncogenesis phenotype and/or caused the cells gain and/or lose chromosomes. Immortalized normal human T cells lines provide critical in vitro models for basic research and therapeutic products development.Methods: We utilized a CRISPR/Cas9 system to replace a single copy of the exon 2 of the cell cycle inhibitor gene CDKN2A (encoding p16 and p14 proteins) with a single copy of human telomerase reverse transcriptase (hTERT) to immortalize human primary CD8+ T cells (hCD8+T-TERT). By using Cas9 protein and low donor DNA copies/cell, we successfully immortalized hCD8+T cells with a single copy of hTERT transgene, which also avoided uncontrolled insertion of Cas9 gene and guide RNA vector.Results: Human primary CD8+ cells were immortalized and expanded more than 2.6 × 107 times. Characterization of the cells revealed that the immortalized CD8+ T-TERT cells retained most of the cell surface markers and normal karyotype. The CD8+ T-TERT cells also retained the dependence of IL-2 and CD3/CD28 activator for survival and expansion.Conclusion: we established a stable immortalized CD8+ T cell line that had a phenotype consistent with T cells and will be a useful research model.
Keywords
Human CD8+ T cells; Immortalization; Telomerase reverse transcriptase (TERT); CRISPR/Cas9; CDKN2A; p16; Large DNA fragment insertion; Homologous recombination
Subject
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
