TY - JOUR

T1 - 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

AU - Menzorov, Aleksei G.

AU - Orishchenko, Konstantin E.

AU - Fishman, Veniamin S.

AU - Shevtsova, Anastasia A.

AU - Mungalov, Roman V.

AU - Pristyazhnyuk, Inna E.

AU - Kizilova, Elena A.

AU - Matveeva, Natalia M.

AU - Alenina, Natalia

AU - Bader, Michael

AU - Rubtsov, Nikolai B.

AU - Serov, Oleg L.

N1 - © 2019 Wiley Periodicals, Inc.

PY - 2019/10

Y1 - 2019/10

N2 - 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.

AB - 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.

KW - mEos2

KW - mouse embryonic stem cells

KW - neuronal differentiation

KW - targeted genomic integration

KW - tryptophan hydroxylase 2

KW - tubulin beta 3 class III

KW - EMBRYONIC STEM-CELLS

UR - http://www.scopus.com/inward/record.url?scp=85071162546&partnerID=8YFLogxK

U2 - 10.1002/jcb.28981

DO - 10.1002/jcb.28981

M3 - Article

C2 - 31106442

AN - SCOPUS:85071162546

VL - 120

SP - 17208

EP - 17218

JO - Journal of Cellular Biochemistry

JF - Journal of Cellular Biochemistry

SN - 0730-2312

IS - 10

ER -