5'-flanking sequences from the rat preprotachykinin gene direct high-level expression of a reporter gene in adult rat sensory neurons transfected in culture by microinjection.
The rat preprotachykinin-A gene, which encodes substance P, is expressed in response to nerve growth factor in a subpopulation of dorsal root ganglion sensory neurons. To investigate mechanisms regulating preprotachykinin-A transcription, we transfected adult rat sensory neurons in culture by microinjection of plasmids containing genomic DNA sequences linked to a lacZ (beta-galactosidase) reporter gene. Expression of beta-galactosidase was seen in 10-15% of neurons receiving injections of prPPT-betaGAL1, which contained the preprotachykinin transcription start site and 3356 bp of 5'-flanking DNA. Deletion analysis showed that expression was directed by 865 bp lying immediately upstream of the transcription start site. Extension of the prPPT-betaGAL1 sequence to include the first intron of preprotachykinin increased beta-galactosidase two- to threefold. Functional promoter and enhancer sequences from the rat prolactin gene failed to direct expression in sensory neurons, indicating neuronal selectivity for preprotachykinin sequences. Expression of prPPT-betaGAL1, measured relative to a construct containing the Rous sarcoma virus promoter, was approximately fivefold higher in neurons than in nonneuronal cells. This suggests selectivity by preprotachykinin 5'-flanking sequences for neuronal expression. However, prPPT-betaGAL1 expression was not restricted to the neuronal subpopulation containing immunoreactive substance P nor was it dependent upon nerve growth factor. Therefore, it does not share all the characteristics of endogenous preprotachykinin expression implying the need for additional regulatory sequences or the involvement of post-transcriptional regulation. Our results show that transfection of differentiated neurons in culture by microinjection has considerable potential in studies of neuron-specific gene expression.
Mulderry
P K
PK
MRC Brain Metabolism Unit, Royal Edinburgh Hospital, Morningside Park, Edinburgh EH10 5HF, Scotland, U.K.
Chapman
K E
KE
Lyons
V
V
Harmar
A J
AJ