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

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Item Type:	Article
Title:	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
Creators Name:	Menzorov, A.G., Orishchenko, K.E., Fishman, V.S., Shevtsova, A.A., Mungalov, R.V., Pristyazhnyuk, I.E., Kizilova, E.A., Matveeva, N.M., Alenina, N., Bader, M., Rubtsov, N.B. and Serov, O.L.
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, Animals, Mice
Source:	Journal of Cellular Biochemistry
ISSN:	0730-2312
Publisher:	Wiley
Volume:	120
Number:	10
Page Range:	17208-17218
Date:	October 2019
Official Publication:	https://doi.org/10.1002/jcb.28981
PubMed:	View item in PubMed

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