Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA.

Abstract

The clinical manifestations, epidemiology, and pathology of hepatitis B virus (HBV) infection are well established (1). However, due to the lack of an in vitro culture system in which the virus can be efficiently propagated, little is known about the replicative cycle of the virus or about its direct effect on the metabolism of infected cells. Although these difficulties have not yet been overcome, it is possible to study the expression of viral genes in mammalian cells transfected with cloned HBV DNA sequences (2-4).As we have reported (2), it is possible to obtain expression of viral proteins in HeLa cells after simple transfection with recircularized cloned HBV genomes. Further studies of the expression of HBV genes in transfected HeLa cells were severely limited by the absence of an efficient selection system for isolating the small number of cells producing viral proteins. Here, we describe the selection of 3T3 cells containing HBV DNA by use of a method developed by Wigler et aL (5) for introducing and amplifying nonselectable genetic elements in methotrexate-sensitive mouse cells. Although this method could not be applied to HeLa cells because ofthe high frequency with which they developed methotrexate resistance, it allowed the selection ofHBV-transfected 3T3 clones that efficiently synthesized and secreted HBV surface antigen (HBsAg). We further report that conditions that lead to amplification of methotrexate resistance result not only in amplification of HBV DNA sequences but also in increased HBsAg production.MATERUILS AND METHODS Cell Lines and Culture Conditions. NIH 3T3 cells (mouse fibroblasts) were maintained in Dulbecco's modified Eagle's medium (DME medium) supplemented with 10% fetal bovine serum, penicillin at 250 units/ml, and streptomycin at 0.2 tkg/ ml. A derivative of the hamster line A29 (6), containing at least 40 copies of a mutated gene for dihydrofolate reductase (generously provided by R. Axel) was grown in DME medium with 3x nonessential amino acids supplemented with methotrexate at 40 tkg/ml, 10% calf serum, and antibiotics as above. All cultures were maintained at 370C in a moist atmosphere containing 5% CO2.Construction and Cloning of a Plasmid Containing Tandem Copies of the HBV Genome in a Head-to-Tail Arrangement. HBV DNA sequences were excised from pHBV-1, a recombinant plasmid constructed by inserting EcoRI-cleaved DNA from Dane particles into the EcoRI site of plasmid pBR322 (2). These sequences were purified by electrophoresis on 1% agarose, collected by electroelution, and ligated (7) to pHBV-1 that had been partially digested with EcoRI. The ligated DNA was used to transfect Escherichia coli (strain 294). DNA was extracted from tetracycline-and ampicillin-resistant colonies after chloramphenicol amplification (8).Colonies containing plasmids with tandem head-to-tail HBV insertions were identified by restriction analysis of the isolated plasmid DNA. Fig. 1 shows the restriction analysis of one such plasmid, pTHBV-1, which was used for the transfection experiments descri...

Tripartite motif-containing 22 inhibits the activity of hepatitis B virus core promoter, which is dependent on nuclear-located RING domain

“…After transfection into HepG2 cells, pTHBV-1 can express HBV proteins and initiate HBV replication. 22 Plasmids expressing myc-tagged wild-type TRIM22, RING domain deletion, and point mutants of (⌬R, C15A) were described in our previous study. 23 The SPRY domain deleted TRIM22 or TRIM22 SPRY domain with C-terminal myc epitope was cloned into pcDNA3.1, referred to as pTRIM22-⌬SPRY or pTRIM22-SPRY, respectively.…”

Construction and use of a dominant, selectable marker: a Harvey sarcoma virus-dihydrofolate reductase chimera.

“…The DHFR markers used in previous cotransfection procedures were uncloned cellular genes (10,48). Because of the large size of the cellular DHFR gene, -42 kilobases (kb) (30), biologically active molecular clones of the genomic sequence have been difficult to obtain.…”

“…This limitation can be overcome by use of a selectable gene which acts dominantly with respect to the wild-type genotype of normal cells. An example of such a dominant selectable marker is the dihydrofolate reductase gene (DHFR), whose presence in high copy number confers resistance to high levels of the folate antagonist methotrexate (MTX) (10,38,48 …”

Cloning Vectors Derived from Animal Viruses

“…In this way they succeeded in generating a cell line in which greater than 10~o of the total soluble protein is a polypeptide related to SV40 small t-antigen. Christman et al (1982) and Ringold et al (1982) have performed similar experiments and generated cell lines over-expressing the human hepatitis B virus surface antigen and E. coli XGPRT, respectively. This approach has a great deal of appeal because it does not involve complex manipulations to insert genes into viral vectors, the exogenous gene is carried in the chromosome and the result is a cell line which expresses extremely high levels of the desired protein.…”