Differential neuropeptide Y gene expression in post-mitotic versus dividing neuroblastoma cells driven by an adeno-associated virus vector.
The ability to express exogenous mammalian genes stably in post-mitotic cells such as neurons remains an important goal for those attempting to modulate neurotransmission through gene delivery. We therefore investigated how differentiation to a post-mitotic state affected the expression of an exogenous gene encoding for neuropeptide Y (NPY) following transfection with an adeno-associated virus (AAV) derived vector. This vector (pJDT95npy) was constructed with rat NPY cDNA (551 bp) inserted downstream from the indigenous AAV p5, p19 and p40 promoters to characterize their relative abilities to drive NPY mRNA expression. Transfection of dividing neuroblastoma CHP126 cells with pJDT95npy resulted in the differential expression of chimeric NPY mRNAs derived from each promoter. P40-driven species became dominant after 1 month post-transfection. Vector integration into chromosomal DNA was demonstrated by Southern blot analyses, indicating at least some region-selective integration. In dividing cell extracts, only a low level of pro-NPY immunoreactivity and no mature NPY immunoreactivity was recovered. However, after differentiation of the pJDT95npy-transfected CHP 126 cells to a post-mitotic state, significant levels of pro-NPY and mature NPY were recovered in the cells and media. Differentiation also had a time-dependent effect on mRNA expression: a spike of p5 driven expression on day 3 was followed predominantly by p40-driven expression on day 5. This study indicates that AAV-derived vectors using the p40 promoter may be used to express genes in post-mitotic cells such as neurons.
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville 32610-0267.
P
P
Wu
D
D
Ziska
M A
MA
Bonell
E
E
Grouzmann
W J
WJ
Millard
E M
EM
Meyer
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008938:Mitosis; D009447:Neuroblastoma / Q000378:metabolism*; D009478:Neuropeptide Y / Q000096:biosynthesis*; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D009478:Neuropeptide Y; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003994:Bucladesine