skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

Abstract

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product.more » The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Lab. of Cell Biology and Genetics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Disease, Bethesda, MD 20205
OSTI Identifier:
5996838
Resource Type:
Journal Article
Journal Name:
Mol. Cell. Biol.; (United States)
Additional Journal Information:
Journal Volume: 4:10
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADENOVIRUS; STRUCTURE-ACTIVITY RELATIONSHIPS; TRANSFERASES; ENZYME ACTIVITY; GENES; RECOMBINANT DNA; BIOASSAY; CHLORAMPHENICOL; DNA REPLICATION; GENE OPERONS; GENE REGULATION; GENETIC ENGINEERING; HELA CELLS; MAN; PLASMIDS; SENSITIVITY; ANIMALS; ANTI-INFECTIVE AGENTS; ANTIBIOTICS; CELL CONSTITUENTS; DNA; DRUGS; ENZYMES; MAMMALS; MICROORGANISMS; NUCLEIC ACID REPLICATION; NUCLEIC ACIDS; ONCOGENIC VIRUSES; ORGANIC COMPOUNDS; PARASITES; PRIMATES; VERTEBRATES; VIRUSES; 550400* - Genetics; 550200 - Biochemistry

Citation Formats

Tratschin, J D, West, M H.P., Sandbank, T, and Carter, B J. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase. United States: N. p., 1984. Web. doi:10.1128/MCB.4.10.2072.
Tratschin, J D, West, M H.P., Sandbank, T, & Carter, B J. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase. United States. doi:10.1128/MCB.4.10.2072.
Tratschin, J D, West, M H.P., Sandbank, T, and Carter, B J. Mon . "A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase". United States. doi:10.1128/MCB.4.10.2072.
@article{osti_5996838,
title = {A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase},
author = {Tratschin, J D and West, M H.P. and Sandbank, T and Carter, B J},
abstractNote = {The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.},
doi = {10.1128/MCB.4.10.2072},
journal = {Mol. Cell. Biol.; (United States)},
number = ,
volume = 4:10,
place = {United States},
year = {1984},
month = {10}
}