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

Abstract

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 106 to 107 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 106 total clones or one clone of EBO-pcD-Leu-2 per 2 x 104 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.There are presently only a few general strategies for cloning a eucaryotic gene. In a very limited number of cases. the mRNA of the gene is so highly expressed in a tissue or cell that it may be physically purified and converted into a relatively pure cDNA probe or cDNA clone (2. 8, 15, 23. 29).However, the vast majority of mammalian mRNAs are of such low abundance that this direct approach cannot usually be applied. For a few low-abundance mRNAs, conditions for differential expression allow the generation of a cDNA probe enriched for the sought-after gene. This type of approach has been used to clone several developmentally regulated genes (7, 43) as well as the genes for human fibroblast interferon (39), the T-cell receptor (14,44), and the 5-opioid receptor (Law et al., Proc. Natl. Acad. Sci. USA, in press). In situations where neither RNA nor enriched cDNA is available but the protein product of the gene is abundant, the following approach has often been successful (4. 12, 28-30, 38-40): the protein is purified, the partial amino acid sequence is determined, and oligonucleotide probe(s) based upon the peptide sequence are synthesized and then used to screen an appropriate library for a clone that hybridizes with the probe(s). However, this approach is of limited utility when the protein product is not known, ver...

Modulatory Proteins Can Rescue a Trafficking Defective Epileptogenic Nav1.1 Na+Channel Mutant

“…The plasmid EBO-pcD-CD8 expressing human lymphocyte T-killer CD8 receptor has been described previously (Margolskee et al, 1988). We cotransfected pCDM8-YFPhNa v 1.1-M1841 with EBO-pcD-CD8 and tested the cells after 48 h identifying cotransfected cells by YFP fluorescence and labeling with Dynal anti-CD8 antibody-coated beads (Dynabeads; Invitrogen).…”

Mutations in CD8 that affect interactions with HLA class I and monoclonal anti-CD8 antibodies.

“…Human CD8a cDNA (23) was subcloned into the HindIII-BamHI site of Bluescript SK`. Mouse CD8a was cut out of the PM7-CD8L vector (24) with XbaI and Pstl and subcloned into the BamHI-Pstl sites of Bluescript SK' .…”

The EBNA- 3 gene family proteins disrupt the G2/M checkpoint

“…EBO-EBNA-2: The 10.5 kbp shuttle vector EBO-pLPP (Margolskee et al, 1988), which contains the oriP/EBNA-1 replicon for episomal maintenance of the vector, the hygromycin resistance gene for eukaryotic selection and an SV40 transcription-cassette was used for constructing a plasmid stably expressing the EBNA-2 ORF of EBV. The B95.8 EBNA-2A gene was restricted with SacI and SalI and subcloned into EBO-pLPP restricted with the same enzyme sites.…”