Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.
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 hygromycin 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 lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-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 hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(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.
Margolskee
R F
RF
Department of Biochemistry, Stanford University School of Medicine, California 94305.
Kavathas
P
P
Berg
P
P
D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics; D016827:CD8 Antigens; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004261:DNA Replication; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D010957:Plasmids; D014162:Transfection
D000945:Antigens, Differentiation, T-Lymphocyte; D016827:CD8 Antigens; D007041:Hypoxanthine Phosphoribosyltransferase