Cultivation of Testicular Germ Cell Cancer Cell Lines and Establishment of Gene-Edited Subclones Using CRISPR/Cas9.

Abstract

Type II testicular germ cell tumors (GCTs) can be classified as seminoma or embryonal carcinoma. Both subtypes present distinct cellular morphologies and characteristics. Seminomas closely resemble primordial germ cells (PGCs) with respect to their transcriptome and epigenetic signature (DNA hypomethylation). They express the pluripotency markers LIN28, NANOG, and OCT3/4 and the PGC markers SOX17, PRDM1, TFAP2C, DMRT1, and cKIT. Embryonal carcinomas show increased levels of DNA methylation (hypermethylation). They also express the pluripotency markers LIN28, NANOG, and OCT3/4, but additionally DNMT3B and SOX2. In contrast to seminomas, these tumors are pluripotent to totipotent and thus able to differentiate into cells of all three germ layers (teratoma) and extraembryonic tissues (yolk-sac tumor, choriocarcinoma). This protocol summarizes the essential techniques for standard cultivation of seminoma (TCam-2), embryonal carcinoma (NCCIT, NT2/D1, 2102EP), and choriocarcinoma (JEG-3, JAR) cell lines, as well as the methods to establish gene-edited subclones using the CRISPR/Cas9 system.