Immortalization of human primary prostate epithelial cells via CRISPR inactivation of the CDKN2A locus and expression of telomerase

Abstract

Background Immortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16 INK4A knockdown. Materials and methods Here, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene. Results Using this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin. Conclusions Use of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.

Expanding the Prostate Cancer Cell Line Repertoire with ACRJ-PC28, an AR-negative Neuroendocrine Cell Line Derived From an African-Caribbean Patient

“…Establishment of ACRJ-PC28 cells was accomplished through transduction with pLV-hTERT-IRES-hygro (Addgene, Watertown, Massachusetts, USA; #85140) and lentiCRISPRv2-sgCDKN2A, as described previously [20]. Briefly, primary PCa cells at passage 4, were cotransduced with lentivirus conditioned medium of HEK-293T cells (RRID:CVCL_ZK70) transfected with pLV-hTERT-IRES-hygro (RRID:Addgene_85140), lentiCRISPRv2-sgCDKN2A, and packaging constructs using calcium phosphate.…”

“…The lentivirus containing media was replaced with fresh lentiviral medium 24 h after the initial transduction and two infections were done 24 h apart. Cells were then washed with PBS and incubated with fresh media for 72 hours before addition of hygromycin (Millipore Sigma #H3274, 25 µg/mL) and puromycin (Millipore Sigma #P8833, 0.25 µg/mL; [20].…”

Alternatives for obtaining a continuous cell line from Apis mellifera

“…The deletion of the p53 gene in a canine cell culture made it possible to obtain several populations with infinite useful life, resistance to genotoxicity and absence of carcinogenic characteristics (EUN et al, 2019). The inactivation of two loci of CDKN2A, responsible for the expression of p16INK4A and p14ARF, precursors of the activation of tumor suppressors p53 and pRB, associated with ectopic expression of hTERT, was able to immortalize human prostate epithelial cells without any phenotypic changes (ZHAO et al, 2020). By not directly inactivating the tumor suppressors p53 and pRB, it is expected that other properties of the cells remain normal, such as the characteristic cell phenotype, protein production, and genomic stability (MCKINLEy & CHEESEMAN, 2017;ZHAO et al, 2020).…”

Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells

“…At present, hTERT is a typical factor for the immortalization of bovine cells [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. However, the ectopic expression of hTERT has unknown effects due to the introduction of genes from heterologous animals.…”

“…The CRISPR/Cas9 system, a genome precise editing technology, can achieve precise knockout and insertion, which has been widely used in livestock breeding and selection [14][15][16]. Recently, CRISPR/Cas9 also has applications in immortalizing human and mouse cells [17,18]. TERT, the catalytic subunit of the telomerase complex, is a positive regulator of telomerase.…”