Epstein-barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells

Journal Article · Fri Jul 01 00:00:00 EDT 1988 · Mol. Cell. Biol.; (United States)

DOI:https://doi.org/10.1128/MCB.8.7.2837· OSTI ID:6272750

Margolskee, R F; Kavathas, P; Berg, P

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromcying B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastodi cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10/sup 6/ to 10/sup 7/ independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBP-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthing-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2. Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10/sup 6/ total clones or one clone of EBO-pcD-Leu-2 per 2 x 10/sup 4/ total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.

• Research Organization: Dept. of Neurosciences, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ (US)
• OSTI ID: 6272750
• Journal Information: Mol. Cell. Biol.; (United States), Vol. 8:7
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA-CLONING
LYMPHOCYTES
CELL TRANSFORMATIONS
PLASMIDS
DNA REPLICATION
ANTIGENS
BIOLOGICAL MARKERS
CLONE CELLS
EXPERIMENTAL DATA
HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE
MAN
ONCOGENIC VIRUSES
PHENOTYPE
REPLICONS
SURFACE PROPERTIES
ANIMAL CELLS
ANIMALS
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL CONSTITUENTS
CELL CULTURES
CLONING
CONNECTIVE TISSUE CELLS
DATA
DNA HYBRIDIZATION
ENZYMES
GENES
GLYCOSYL TRANSFERASES
HYBRIDIZATION
INFORMATION
LEUKOCYTES
MAMMALS
MATERIALS
MICROORGANISMS
NUCLEIC ACID REPLICATION
NUMERICAL DATA
PARASITES
PENTOSYL TRANSFERASES
PRIMATES
SOMATIC CELLS
TRANSFERASES
VERTEBRATES
VIRUSES
550200* - Biochemistry