Differential neuropeptide Y gene expression in post-mitotic versus dividing neuroblastoma cells driven by an adeno-associated virus vector

Ping Wu, David Ziska, Manuel A. Bonell, Eric Grouzmann, William J. Millard, Edwin M. Meyer

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)27-33
Number of pages7
JournalMolecular Brain Research
Volume24
Issue number1-4
DOIs
StatePublished - 1994
Externally publishedYes

Fingerprint

Dependovirus
Neuropeptide Y
Neuroblastoma
Gene Expression
Transfection
Messenger RNA
Genes
Neurons
Southern Blotting
Cell Extracts
Synaptic Transmission
Complementary DNA
adjuvant P40
DNA

Keywords

  • Adeno-associated virus
  • Gene expression
  • Neuroblastoma
  • Neuropeptide Y

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience

Cite this

Differential neuropeptide Y gene expression in post-mitotic versus dividing neuroblastoma cells driven by an adeno-associated virus vector. / Wu, Ping; Ziska, David; Bonell, Manuel A.; Grouzmann, Eric; Millard, William J.; Meyer, Edwin M.

In: Molecular Brain Research, Vol. 24, No. 1-4, 1994, p. 27-33.

Research output: Contribution to journalArticle

Wu, Ping ; Ziska, David ; Bonell, Manuel A. ; Grouzmann, Eric ; Millard, William J. ; Meyer, Edwin M. / Differential neuropeptide Y gene expression in post-mitotic versus dividing neuroblastoma cells driven by an adeno-associated virus vector. In: Molecular Brain Research. 1994 ; Vol. 24, No. 1-4. pp. 27-33.
@article{5ed450c42696498db061a42672ab7b64,
title = "Differential neuropeptide Y gene expression in post-mitotic versus dividing neuroblastoma cells driven by an adeno-associated virus vector",
abstract = "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.",
keywords = "Adeno-associated virus, Gene expression, Neuroblastoma, Neuropeptide Y",
author = "Ping Wu and David Ziska and Bonell, {Manuel A.} and Eric Grouzmann and Millard, {William J.} and Meyer, {Edwin M.}",
year = "1994",
doi = "10.1016/0169-328X(94)90114-7",
language = "English (US)",
volume = "24",
pages = "27--33",
journal = "Brain Research",
issn = "0006-8993",
publisher = "Elsevier",
number = "1-4",

}

TY - JOUR

T1 - Differential neuropeptide Y gene expression in post-mitotic versus dividing neuroblastoma cells driven by an adeno-associated virus vector

AU - Wu, Ping

AU - Ziska, David

AU - Bonell, Manuel A.

AU - Grouzmann, Eric

AU - Millard, William J.

AU - Meyer, Edwin M.

PY - 1994

Y1 - 1994

N2 - 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.

AB - 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.

KW - Adeno-associated virus

KW - Gene expression

KW - Neuroblastoma

KW - Neuropeptide Y

UR - http://www.scopus.com/inward/record.url?scp=0028352489&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028352489&partnerID=8YFLogxK

U2 - 10.1016/0169-328X(94)90114-7

DO - 10.1016/0169-328X(94)90114-7

M3 - Article

VL - 24

SP - 27

EP - 33

JO - Brain Research

JF - Brain Research

SN - 0006-8993

IS - 1-4

ER -