Immortalization of primary marmoset skin fibroblasts by CRISPR-Cas9-mediated gene targeting

Immortalized cell lines can be used for diverse in vitro experiments, providing invaluable data before conducting in vivo studies Callithrix jacchus, the common marmoset, is a non-human primate model utilized for studying various human diseases. However, only a few immortalized marmoset cell lines are currently available. In the present study, we reveal that CRISPR-Cas9-mediated targeting of the p53 gene or CDKN2A locus is an effective means for immortalizing primary marmoset skin fibroblasts. In addition to frameshift mutations that result in premature stop codons, in-frame mutations potentially destroying the DNA-binding motif of p53 are frequently detected in immortalized cells. Like Cdkn2a-deficient mouse cells, CDKN2A-deficient marmoset cells express wild-type p53 proteins normally respond to genotoxic stresses, including adriamycin and etoposide. Taken together, these findings indicate that Cas9- mediated gene targeting of the p53 gene or CDKN2A locus is an effective tool for establishing immortalized marmoset cell lines with defined genetic alterations.</p

Functional pathway analysis.

<p>(A) Predicted FGFR signaling pathway map showing genes obtained from our comparative analyses in red. Whole mount <i>in situ</i> hybridization using RNA probes corresponding to the genes in black was performed. (B) Table summarizing gene expression in neural crest cells. pNCC, premigratory neural crest cells: mNCC, migratory neural crest cells: -, not detected: +, detected: ++, expressed at high level. (C) Transverse sections of chick embryos showing gene expression in premigratory and/or emigrating neural crest cells at stage HH 8–9 (a–f) and in migrating neural crest cells at the midbrain level at HH stage 9–10 (a′–f′); (a, a′) frs2, (b, b′) gab1, (c, c′) pik3r2, (d, d′) shc1, (e, e′) sos1, (f, f′) sos2. Arrows indicate emigrating neural crest cells (a–d, f) or premigratory neural crest cells (e). Arrowheads indicate head mesenchymal cells (a,a′).</p

Vertebrate orthologs of genes essential for cell migration in <i>C. elegans</i> are expressed in premigratory neural crest cells in chicken embryos.

<p>Whole mount <i>in situ</i> hybridization was performed using RNA probes corresponding to orthologs of each nematode gene (upper panel). Of the twenty-five genes examined in this study, fourteen chicken orthologs were expressed in the premigratory neural crest domain in the neural folds, as clearly shown in sections (lower panel); Crk (ced-2), Dock180 (ced-5), p21-Rac1 (<i>ced-10</i>), Chx10 (<i>ceh-10</i>), NR1I3 (<i>daf-12</i>), Fgfr1 (<i>egl-15</i>), Fgf18 (<i>egl-17</i>), Fbln2 (<i>fbl-1</i>), Frizzled10 (<i>lin-17</i>), HoxA4 (<i>lin-39</i>), HoxB6 (<i>mab-5</i>), Grb2 (<i>sem-5</i>), DCC (<i>unc-40</i>) and Kinesin (<i>vab-8</i>). arrow, plane of section; arrowhead, gene expression in neural fold.</p

Putative chicken orthologs of nematode cell migration genes are conserved in migrating cranial neural crest cells.

<p>Chicken embryos were subjected to whole mount <i>in situ</i> hybridization using RNA probes corresponding to the vertebrate orthologs of each nematode gene. (A) Whole mount chicken embryos (upper panel) and tissue sections at the midbrain level of each embryo (lower panel) show that ten vertebrate orthologs are expressed in neural crest cells migrating from the neural tube in chicken embryos at HH stage 9–10; Crk (<i>ced-2</i>), Dock180 (<i>ced-5</i>), p21-Rac1 (<i>ced-10</i>), NR1I3 (<i>daf-12</i>), Fgfr1 (<i>egl-15</i>), Fgf18 (<i>egl-17</i>), Fbln2 (<i>fbl-1</i>), HoxB6 (<i>mab-5</i>), Grb2 (<i>sem-5</i>) and Ulk2 (<i>unc-51</i>). Arrowhead indicates migrating neural crest cells. (B) Five vertebrate orthologs are expressed in migrating neural crest cells at the hindbrain level in HH stage 11–13 embryos; Dock180 (<i>ced-5</i>), p21-Rac1 (<i>ced-10</i>), NR1I3 (<i>daf-12</i>), Fgfr1 (<i>egl-15</i>) and Kinesin (<i>vab-8</i>). Arrows indicate gene expression in migrating cranial neural crest cells in rhombomere 4 and/or rhombomere 6.</p