Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts

Matveeva, N. M.; Fishman, Veniamin; Захарова, И. С.; Shevchenko, Alexander I.; Pristyazhnyuk, Inna E.; Мензоров, А. Г.; Serov, O. L.

doi:10.1038/s41598-017-18352-4

Cited by 6 publications

(9 citation statements)

References 53 publications

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“…In our previous study, we evaluated the expression of Tubb3 in undifferentiated E14Tg2aSc4TP6.3 ES cells. 10 Actually, in our experiment, Tubb3 was detectable in undifferentiated ES cells, and its expression level was only 10 times lower than that of such pluripotency markers as Sox2 and Nanog. It is still unclear, whether this Tubb3 expression is the result of a partial differentiation or normal for undifferentiated ES cells.…”

Section: Discussionmentioning

confidence: 49%

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Мензоров

Orishchenko

Fishman

et al. 2019

J of Cellular Biochemistry

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Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type–specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock‐in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan‐neural marker class III β‐tubulin (Tubb3) promoter and mEos2 under serotonergic neuron‐specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3‐EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3‐positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2‐TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction‐based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.

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Section: Discussionmentioning

confidence: 49%

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Мензоров

Orishchenko

Fishman

et al. 2019

J of Cellular Biochemistry

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“…The second explanation is corroborated by the known fact that partially differentiated cells are always present among ES cells. In our previous study we evaluated expression of Tubb3 in undifferentiated E14Tg2aSc4TP6.3 ES cells (Matveeva et al 2017). Actually, in our experiment, Tubb3 was detectable in undifferentiated ES cells, and its expression level was only ten times lower than that of such pluripotency markers as Sox2 and Nanog.…”

Section: Egfp Expression In Undifferentiated Dges1-tubbegfp Es Cells mentioning

confidence: 70%

“…Cytogenetic analysis for ES cell lines was carried out during passages 6-10. Preparation of metaphase chromosomes from the cells was performed as previously described (Matveeva, Fishman, Zakharova, Shevchenko, Pristyazhnyuk, Menzorov and Serov, 2017). Metaphase plates were analyzed using a Carl Zeiss Axioplan 2 imaging microscope (Jena, Germany) with CoolCube1 CCD-camera and processed using the ISIS (In Situ Imaging System, MetaSystems GmbH, Altlussheim, Germany) software in the Public Center for Microscopy SB RAS, Novosibirsk.…”

Section: Es Cell Cytogenetic Analysismentioning

confidence: 99%

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Мензоров

Orishchenko

Fishman

et al. 2018

Preprint

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Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type-specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: EGFP under panneural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP ES cells into neurons revealed that thoughTubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on mRNA level using PCR-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared to random genomic integration. KeywordsMouse embryonic stem cells, tubulin beta 3 class III, Tryptophan hydroxylase 2, mEos2, neuronal differentiation, targeted genomic integration

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“…At the same time, it was shown that primary mouse carcinoma cells and malignant melanoma cell line remain amenable to reprogramming to iPSCs [ 10 , 29 , 30 ]. Also it was shown that immortalized mouse fibroblast cells m5S can be efficiently reprogrammed via cell fusion with ESCs [ 44 ]. The two situations, however, cannot be compared one-to-one, because mechanism of cell fusion-based reprogramming might be significantly different from reprogramming driven by OKSM [ 21 , 44 , 45 ].…”

Section: Resultsmentioning

confidence: 99%

“…Also it was shown that immortalized mouse fibroblast cells m5S can be efficiently reprogrammed via cell fusion with ESCs [ 44 ]. The two situations, however, cannot be compared one-to-one, because mechanism of cell fusion-based reprogramming might be significantly different from reprogramming driven by OKSM [ 21 , 44 , 45 ].…”

Section: Resultsmentioning

confidence: 99%

Immortalized murine fibroblast cell lines are refractory to reprogramming to pluripotent state

2018

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To date different cell types of various mammalian species have been reprogrammed to induced pluripotent stem cells (iPSCs) using Yamanaka's cocktail of transcription factors (Oct4, Klf4, Sox2, and cMyc). It has been shown that several primary human cancer cell lines could be reprogrammed to iPSCs. We sought if immortalized mouse fibroblast cell lines could also be reprogrammed to iPSCs. The approach of generating iPSCs from such cells should be valuable in different experimental settings as it allows clonally derive cell lines carrying mutations whose impact on reprogramming could be next evaluated. Therefore, we investigated reprogramming of widely used immortalized cell lines (NIH3T and STO), as well as of de novo immortalized fibroblast line (tKM) with the use of highly effective lentiviral polycistronic OKSM expression system. Our reprogramming experiments have shown that in contrast to mouse embryonic fibroblasts (MEFs), none of the immortalized cell lines can be reprogrammed to pluripotent state. Contrary to colonies derived from MEFs, those derived from the immortalized cells lines (1) developed much later, (2) contained large round cells, not typical for iPSCs, and (3) were negative for trusted markers of matured iPSCs, Nanog and SSEA1. Immortalized cell lines NIH3T and STO are known to be mostly aneuploid, whereas tKM population includes cells with normal karyotype, however, neither cell type can be reprogrammed. Thus our data argue that aneuploidy per se is not a reason for the observed refractoriness of mouse immortalized cells to reprogramming to pluripotent state.

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Alternative dominance of the parental genomes in hybrid cells generated through the fusion of mouse embryonic stem cells with fibroblasts

Cited by 6 publications

References 53 publications

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Immortalized murine fibroblast cell lines are refractory to reprogramming to pluripotent state

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