Profiling neurotransmitter receptor expression in mouse gonadotropin-releasing hormone neurons using green fluorescent protein-promoter transgenics and microarrays
Section snippets
Animals
All experimentation was approved by both the Babraham Institute and University of Otago Animal Welfare and Ethics Committees. Experiments were designed to minimize the number of mice used and any suffering. Mice were maintained under 12-h lighting conditions (lights on 07:00 h) with food and water available ad libitum. Transgenic GnRH-GFP (C57BL6/J) mice (Spergel et al., 1999) were kindly provided by Dr. Dan Spergel (Department of Paediatrics, University of Chicago, IL, USA) and bred at the
Single cell RT-PCR for GnRH
All 10 of the fluorescent cells harvested from a GnRH–GFP mouse were shown to express GnRH transcripts following nested single cell RT-PCR whereas the no-RT and water controls did not generate an amplicon (Fig. 1). This demonstrates the very high likelihood that 100% of the fluorescent cells harvested for the microarray study were GnRH neurons.
Microarray profile
The hybridizations from the four pools of GnRH neurons resulted in the positive hybridization of 1007–1975 spots on each of the microarrays. Although the
Discussion
We report here a comparatively straightforward strategy for generating a qualitative profile of the neurotransmitter receptor mRNAs expressed by a defined neuronal cell population. The present microelectrode strategy provides an alternative to laser capture microdissection and has the advantage of being able to access mRNA from living cells without the need for any prior fixation or histochemical staining that may have deleterious effects on RNA quality (Hinkle et al., 2004). A further major
Acknowledgments
We thank Tom Freeman and Debbie Williams (Medical Research Council, Hinxton, UK) for the microarrays and Dan Spergel (Department of Pediatrics, University of Chicago, USA) for generous provision of the GnRH-GFP mice. Drs. Christine Jasoni and Rebecca Campbell are thanked for critical review of the manuscript. Research supported by the Biotechnology and Biological Sciences Research Council (UK) and The Wellcome Trust.
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