Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat embryonic germ cells

Northrup, Emily; Eisenblätter, R.; Glage, Silke; Rudolph, Cornelia; Dorsch, Martina; Schlegelberger, Brigitte; Hedrich, Hans-Jürgen; Zschemisch, Nils-Holger

doi:10.1016/j.yexcr.2011.04.013

Cited by 8 publications

(12 citation statements)

References 39 publications

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“…This correlates with the previously found results that primordial germ cells from embryos carrying the ter mutation are more easily transformed into pluripotent cells in culture than their wild type counterparts [36].…”

Section: Discussionsupporting

confidence: 92%

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The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

et al. 2012

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A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LOD = 3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1ter/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders.

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Section: Discussionsupporting

confidence: 92%

“…We have shown that the homozygous ter mutation facilitates the transformation of germ cells from embryonic day 14.5 post coitum (pc) into pluripotent cells in culture, whereas the cultivation of germ cells from embryonic day 10.5 pc is not influenced [36].…”

Section: Introductionmentioning

confidence: 99%

The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

et al. 2012

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“…We have also been able to derive embryonic germ (EG) cell lines from rat primordial germ cells that satisfied all but the last of these criteria (Leitch et al, 2010). Subsequently, other studies have employed similar systems to derive germline competent rat ES and EG cells (Kawamata and Ochiya, 2010; Hirabayashi et al, 2010; Northrup et al, 2011), and to propagate rat induced pluripotent stem (iPS) cells (Hamanaka et al, 2011)…”

Section: Introductionmentioning

confidence: 99%

Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells

et al. 2011

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SummaryThe ability of cultured pluripotent cells to contribute to the germline of chimaeric animals is essential to their utility for genetic manipulation. In the three years since rat embryonic stem (ES) cells were first reported the anticipated proliferation of genetically modified rat models from this new resource has not been realised. Culture instability, karyotypic anomalies, and strain variation are postulated to contribute to poor germline colonisation capacity. The resolution of these issues is essential to bring pluripotent cell-based genetic manipulation technology in the rat to the level of efficiency achieved in the mouse. Recent reports have described various alternative methods to maintain rat ES cells that include provision of additional small molecules and selective passaging methods. In contrast, we report that euploid, germline competent rat ES and embryonic germ (EG) cell lines can be maintained by simple adherent culture methods in defined medium supplemented with the original two inhibitors (2i) of the mitogen-activated protein kinase (ERK1/2) cascade and of glycogen synthase kinase 3, in combination with the cytokine leukaemia inhibitory factor (LIF). We demonstrate genetic modification, clonal expansion and transmission through the germline of rat ES and EG cell lines. We also describe a marked preference for full-term chimaera contribution when SD strain blastocysts are used as recipients for either DA or SD pluripotent stem cells.

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“…Blastocysts were washed three times in modified M16 medium followed by 30 minutes incubation in a 1:8 dilution of guinea pig complement (Sigma-Aldrich) and additional three washing steps in modified M16 medium. Then the ICMs were dissociated from the trophoblast cells by pipetting, and cultured in 2i with 1000 units rat LIF/ml (Northrup et al 2011 ), YPAC medium (Kawamata and Ochiya 2010 ) or 4i (YPAC without serum supplemented with 1000 units/ml rat LIF) on mitotic inactivated TRF-O3 as feeder cells (Northrup et al 2011 ).…”

Section: Methodsmentioning

confidence: 99%

“…Besides the serum-free 2i medium supplemented with MEK/ERK pathway and GSK3 inhibitors and 3i medium additionally containing a FGF receptor inhibitor (Buehr et al 2008 ; Li et al 2008 ) Kawamata and Ochiya used the serum-containing YPAC medium furthermore comprising chemical inhibitors for the TGF-β receptor Alk5 and rho kinase inhibitor (ROCKi) for rat ESC culture (Kawamata and Ochiya 2010 ). In contrast to feeder-free conditions developed for pluripotent mouse ESC in vitro culture a feeder layer seemed to be essential for rat ESCs, embryonic germ cells (EGC) and induced pluripotent stem cells (iPS) (Furue et al 2005 ; Nichols and Ying 2006 ; Blair et al 2011a ; Northrup et al 2011 ). In this study we showed that the immortalized tumor rat fibroblast cell line TRF-O3 as innovative feeder cells supported the culture of rat pluripotent and germ-line transmissible ESCs.…”

Section: Introductionmentioning

confidence: 99%

Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm

et al. 2014

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Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-β and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-588) contains supplementary material, which is available to authorized users.

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Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat embryonic germ cells

Cited by 8 publications

References 39 publications

The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells

Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm

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