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J Cell Biochem. 2019 Oct;120(10):17208-17218. doi: 10.1002/jcb.28981. Epub 2019 May 20.

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.

Author information

1
Department of Molecular Mechanisms of Development, Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
2
Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russia.
3
Laboratory of Molecular Genetic Technologies of the Institute for Living Systems, Immanuel Kant Baltic Federal University, Kaliningrad, Russia.
4
Cell Biology Department, Institute of Cytology and Genetics of the Russian Academy of Sciences, Novosibirsk, Russia.
5
Laboratory of Molecular Biology of Peptide Hormones, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Abstract

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.

KEYWORDS:

mEos2; mouse embryonic stem cells; neuronal differentiation; targeted genomic integration; tryptophan hydroxylase 2; tubulin beta 3 class III

PMID:
31106442
DOI:
10.1002/jcb.28981

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