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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

Authors

  • A.G. Menzorov
  • K.E. Orishchenko
  • V.S. Fishman
  • A.A. Shevtsova
  • R.V. Mungalov
  • I.E. Pristyazhnyuk
  • E.A. Kizilova
  • N.M. Matveeva
  • N. Alenina
  • M. Bader
  • N.B. Rubtsov
  • O.L. Serov

Journal

  • Journal of Cellular Biochemistry

Citation

  • J Cell Biochem

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.


DOI

doi:10.1002/jcb.28981