A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase
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.
- Research Organization:
- Lab. of Cell Biology and Genetics, National Institute of Arthritis, Diabetes, and Digestive and Kidney Disease, Bethesda, MD 20205
- OSTI ID:
- 5996838
- Journal Information:
- Mol. Cell. Biol.; (United States), Vol. 4:10
- Country of Publication:
- United States
- Language:
- English
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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