Development of lentiviral vectors for efficient glutamatergic-selective gene expression in cultured hippocampal neurons

Sci Rep. 2018 Oct 11;8(1):15156. doi: 10.1038/s41598-018-33509-5.

Abstract

Targeting gene expression to a particular subset of neurons helps study the cellular function of the nervous system. Although neuron-specific promoters, such as the synapsin I promoter and the α-CaMKII promoter, are known to exhibit selectivity for excitatory glutamatergic neurons in vivo, the cell type-specificity of these promoters has not been thoroughly tested in culture preparations. Here, by using hippocampal culture preparation from the VGAT-Venus transgenic mice, we examined the ability of five putative promoter sequences of glutamatergic-selective markers including synapsin I, α-CaMKII, the vesicular glutamate transporter 1 (VGLUT1), Dock10 and Prox1. Among these, a genomic fragment containing a 2.1 kb segment upstream of the translation start site (TSS) of the VGLUT1 implemented in a lentiviral vector with the Tet-Off inducible system achieved the highest preferential gene expression in glutamatergic neurons. Analysis of various lengths of the VGLUT1 promoter regions identified a segment between -2.1 kb and -1.4 kb from the TSS as a responsible element for the glutamatergic selectivity. Consistently, expression of channelrhodopsin under this promoter sequence allowed for selective light-evoked activation of excitatory neurons. Thus, the lentiviral system carrying the VGLUT1 promoter fragment can be used to effectively target exogenous gene expression to excitatory glutamatergic neurons in cultures.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials
  • Animals
  • Cells, Cultured
  • Fluorescent Antibody Technique
  • GABAergic Neurons / metabolism*
  • Gene Expression*
  • Gene Order
  • Gene Transfer Techniques*
  • Genetic Vectors / genetics*
  • Lentivirus / genetics*
  • Mice
  • Mice, Transgenic
  • Neuroglia / metabolism
  • Organ Specificity / genetics
  • Promoter Regions, Genetic
  • Pyramidal Cells / metabolism*
  • Transgenes
  • Vesicular Glutamate Transport Protein 1 / genetics*

Substances

  • Vesicular Glutamate Transport Protein 1