Send to

Choose Destination
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

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


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.


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


Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center