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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:Preprint
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. and Orishchenko, K. and Fishman, V. and Shevtsova, A. and Mungalov, R. and Pristyazhnyuk, I. and Kizilova, E. and Matveeva, N. and Alenina, N. and Bader, M. and Rubtsov, N. and Serov, O.
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: 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 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 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.
Keywords:Mouse Embryonic Stem Cells, Tubulin beta 3 Class III, Tryptophan Hydroxylase 2, mEos2, Neuronal Differentiation, Targeted Genomic Integration
Source:bioRxiv
Publisher:Cold Spring Harbor Laboratory Press (U.S.A.)
Article Number:490243
Date:7 December 2018
Official Publication:https://doi.org/10.1101/490243
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https://edoc.mdc-berlin.de/18246/Final version

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