Quantification of transcriptional activities of reporter gene constructs in primary cultures of sympathetic neurons.
Primary cultures of sympathetic neurons provide an attractive cellular model for investigating the mechanisms of neurotransmitter phenotypic plasticity. However, it has not been possible to transfect these neurons by conventional techniques, and this has been a major impediment to molecular investigations of neuronal gene expression in this system. Here, reporter plasmids were transferred into the nuclei of cultured sympathetic neurons by microinjection. We developed and improved this procedure and were able to measure the transcriptional activities of two coinjected promoters in small groups of neurons, and even from a single neuron. Promoter activities can thus be quantified and normalized relative to that of a constitutively expressed promoter, allowing correction for variability in the injection and assay procedures. High and low promoter activities can be reliably quantified. Importantly, this method can be used not only for reporter plasmids but also for DNA fragments containing only a promoter and reporter gene without any vector sequence that might interfere with promoter. Using this approach, we measured neuronal promoter activities and found that one promoter region of the gene encoding choline acetyltransferase was up-regulated by more than sevenfold by leukemia inhibitory factor. This method thus provides the means to investigate the function of neuronal genes and the mechanisms that regulate their transcription in cultured sympathetic neurons.
Pajak
Fabrice
F
Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs, Centre National de la Recherche Scientifique UMR 7091, Hôpital de la Pitié-Salpétrière, Paris, France.
De Gois
Stéphanie
S
Houhou
Leïla
L
Védrine
Christophe
C
Mallet
Jacques
J
Berrard
Sylvie
S
D000320:Adrenergic Fibers / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D001517:Coleoptera; D017930:Genes, Reporter / Q000187:drug effects / Q000502:physiology*; D008156:Luciferases / Q000096:biosynthesis; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010957:Plasmids / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*
D008156:Luciferases