TY - CHAP
T1 - Cultivation of testicular germ cell cancer cell lines and establishment of gene-edited subclones using crispr/cas9
AU - Jostes, Sina
AU - Nettersheim, Daniel
AU - Schneider, Simon
AU - Schorle, Hubert
N1 - Publisher Copyright:
© Springer Science+Business Media, LLC, part of Springer Nature 2021.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - CRISPR/Cas9
KW - Cell culture
KW - Gene editing
KW - Germ cell tumors
KW - Transfection
UR - http://www.scopus.com/inward/record.url?scp=85090079014&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-0860-9_7
DO - 10.1007/978-1-0716-0860-9_7
M3 - Chapter
C2 - 32852759
AN - SCOPUS:85090079014
T3 - Methods in Molecular Biology
SP - 85
EP - 97
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -