Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies
Introduction
Certain types of neurons in the brain are difficult to study because they cannot be identified by location or morphological criteria alone. One approach to identify such neurons is to tag them with a reporter protein. Recent work on the gonadotropin-releasing hormone (GnRH) neuronal network has demonstrated that such an approach can be quite powerful, yielding important new information about the anatomical distribution and electrophysiological activity of a neuronal population, as well as the promoter elements required for its specific expression of genes. This review discusses several commonly used reporter proteins and their past, present, and future application, with particular emphasis on the GnRH neuronal network.
Section snippets
Reporter proteins
A reporter protein is a protein whose expression is linked to the expression of a gene of interest and that can be visualized as a result of its bioluminescence, fluorescence, or, if it is an enzyme, due to the bioluminescence, fluorescence, or color of the product of the reaction it catalyzes. Compared to non-enzymatic reporter proteins, enzymatic reporter proteins are more sensitive detectors of gene expression because each molecule of an enzymatic reporter protein can catalyze the reaction
GnRH neurons and GnRH-reporter mice
GnRH–LacZ and GnRH–GFP mice, in which the GnRH gene promoter drives the expression of the lacZ gene or a humanized, red-shifted variant of the GFP gene, are striking examples of tagging a neuronal population with a reporter gene for gene promoter, anatomical, and physiological studies. GnRH–LacZ and GnRH–GFP mice, along with GnRH–luciferase and GnRH–Bla mice, are discussed below following a brief description of the GnRH neuronal network.
Simultaneous use of two or more reporter genes
Tagging a selected neuronal population with two or more reporter genes can result in both high sensitivity of detection in fixed tissue and identification of reporter-tagged neurons in live tissue. Such tagging can be obtained by breeding mice with transgenes containing different reporters, or by using a transgene with a bi-directional cytomegalovirus promoter that can be transactivated by a cell type- or brain region-specific transactivator to drive the expression of two different reporters.
Future developments
Likely future developments include the generation of even brighter GFP's and the enhancement of red fluorescent proteins from species other than jellyfish (Matz et al., 1999, Tsien, 1999). In combination with GFP, the red fluorescent proteins should provide a convenient and powerful approach to double-label neurons that contain two peptides or proteins of interest for physiological studies.
Conclusion
The results from the GnRH–LacZ and GnRH–GFP mice described in this review clearly demonstrate that using reporter genes to tag selected neuronal populations is an important approach that enables one to perform gene promoter, anatomical, and physiological investigations that were previously difficult or impossible. Such an approach, in its various present and future forms, some of which have been described in this review, will undoubtedly assist greatly in deepening our understanding of neuronal
Acknowledgements
D.J.S. was supported by an Alexander von Humboldt Foundation Research Fellowship, by SFB Grants 317 and 488 of the Deutsche Forschungsgemeinschaft, and by an NIH Individual National Research Service Award (F32-NS10085). Funded in part by grants from the Volkswagen Foundation and the German Chemical Society to P.H.S.
References (44)
- et al.
Visualizing unstained neurons in living brain slices by infrared-DIC videomicroscopy
Brain Res.
(1990) - et al.
Codon usage limitation in the expression of HIV-1 envelope glycoprotein
Curr. Biol.
(1996) - et al.
Engineering green fluorescent protein for improved brightness, longer wavelengths, and fluorescence energy transfer
Curr. Biol.
(1996) - et al.
Sequence homologies in the mouse protamine 1 and 2 genes
Biochem. Biophys. Acta
(1988) - et al.
The dopamine beta-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice
Neuron
(1991) - et al.
5′ upstream DNA sequence of the rat tyrosine hydroxylase gene directs high-level and tissue-specific expression to catecholaminergic neurons in the central nervous system of transgenic mice
Mol. Brain Res.
(1994) - et al.
Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy
Biophys. J.
(1997) - et al.
Double labelling of subcellular structures with organelle-targeted GFP mutants in vivo
Curr. Biol.
(1996) - et al.
Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells
Curr. Biol.
(1995) - et al.
Cell-specific expression of the human gonadotropin-releasing hormone gene in transgenic animals
J. Biol. Chem.
(1996)
Directing gene expression to cerebellar granule cells using gamma-aminobutyric acid type A receptor alpha-6 subunit transgenes
Proc. Natl. Acad. Sci. USA
Circular permutation and receptor insertion within green fluorescent proteins
Proc. Natl. Acad. Sci. USA
Green fluorescent protein as a marker for gene expression
Science
In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene
Science
Fast application of agonists to isolated membrane patches
Two subgroups of gonadotropin-releasing hormone neurons control gonadotropin secretion in rats
News Physiol. Sci.
Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice
Proc. Natl. Acad. Sci. USA
Local regulation of gonadotroph function by pituitary gonadotropin-releasing hormone
Endocrinology
Modulating mechanisms of neuroendocrine cell activity — the LHRH pulse generator
Cell. Mol. Neurobiol.
Fluorescent proteins from nonbioluminescent Anthozoa species
Nature Biotechnol.
Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins
Nature
Dynamic and quantitative Ca2+ measurements using improved cameleons
Proc. Natl. Acad. Sci. USA
Cited by (138)
Methods of detection of β-galactosidase enzyme in living cells
2021, Enzyme and Microbial TechnologyNew insights into the detection mechanism of β-galactosidase in living cells with fluorescent probes
2021, Chemical Physics LettersCitation Excerpt :For instance, β-gal is often inextricably linked to the generation of primary ovarian cancer, and it is usually found to be overexpressed [1,2]. At the same time, it is also considered as an essential biomarker for diagnosing cellular senescence and ovarian cancer [3,4]. Therefore, establishing a sensitive and efficient method to detect β-gal in living cells is critical for developing medicine and understanding its physiological role in pathological conditions.
A fluorescent probe for specific detection of β-galactosidase in living cells and tissues based on ESIPT mechanism
2021, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyDevelopment of a red-emissive two-photon fluorescent probe for sensitive detection of beta-galactosidase in vitro and in vivo
2020, Sensors and Actuators, B: ChemicalRecent Advances of Molecular Optical Probes in Imaging of β-Galactosidase
2019, Bioconjugate Chemistry