Production of High-Titer Epstein-Barr Virus Recombinants Derived from Akata Cells by Using a Bacterial Artificial Chromosome System

Abstract

An Epstein-Barr virus (EBV) genome in Burkitt's lymphoma-derived cell line Akata was cloned into a bacterial artificial chromosome (BAC) vector. The BAC clone, designated AK-BAC, was rapidly and precisely modified by means of efficient homologous recombination in Escherichia coli. This system was used to produce recombinant EBVs with transgenes. An expression cassette of green fluorescent protein (GFP) was inserted into AK-BAC, and the resultant BAC clone, AK-BAC-GFP, was transfected into Akata cells. We found that transfected BAC plasmids efficiently formed episomes in EBV-positive Akata cells. Mixtures of wild-type and AK-BAC-GFP viruses were then produced and used to infect EBV-negative Akata cells. We obtained cell clones that harbored only AK-BAC-GFP but no wild-type episome. These cell clones produced infectious viruses after stimulating virus production, and the recombinant viruses of AK-BAC-GFP efficiently immortalized primary B lymphocytes. We further revised the method so that any kind of cDNA could be rapidly inserted into the unique I-PpoI site that had been artificially introduced into AK-BAC. The AK-BAC system will have a broad range of applications, such as genetic analyses of various viral gene products and development of viral vectors for human gene therapy.

HLA-restricted presentation of WT1 tumor antigen in B-lymphoblastoid cell lines established using a maxi-EBV system

“…22 A kanamycin-resistance gene derived from PUC4K was inserted into the EcoRI site of double-I-PpoI to make double-I-PpoI-km. An mCherry gene or a complementary DNA clone of WT1 (variant D) 25 were cloned into the HincII site of double-I-PpoI to make double-I-PpoI-mCherry or double-IPpoI-WT1, respectively.…”

“…22 Recombinant virus production HEK293 cells (4 Â 10 5 cells) were plated in six-well tissue culture dishes and transfected with BAC clone DNA (1 mg, prepared with the Nucleobond BAC100 kit) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Two days after transfection, the transfected cells were re-plated at a density of 4000 cells per well in 96-well tissue culture plates in medium containing 150 mg of hygromycin per ml.…”

“…Thus, the artificially created I-PpoI site is a unique site within the modified BAC clone, and enables the insertion of complementary DNA cassettes by simple in vitro ligation. 22 As a proof of concept, we used the strategy to generate a recombinant EBV genome with a red fluorescent protein (mCherry) transgene. 31 An mCherry gene was inserted into the created I-PpoI site to obtain EBV-BAC-mCherry (Figure 1).…”

“…22 We recently established a new BAC clone of B95-8 strain of EBV and improved the method to produce pure recombinant viruses with higher transforming titers. 23 The newly obtained BAC clone (174-kb BAC) is similar to a precedent called 'maxi-EBV', 16 but it is unique in that it stably maintains a viral repetitive sequence (designated as Family of Repeats) and enables production of recombinant EBVs with excellent transgene expression.…”

Cd40 doesn't Complement The Function Of Epstein-barr Virus (Ebv) Latent Membrane Protein 1 In B Cells Transformation

“…Previously established AK-BAC-GFP (recombinant EBV containing Bacterial artificial chromosome (BAC) and Green fluorescent protein (GFP) gene in their genome) [15] and d.LMP1-EBV (LMP1 knockout AK-BAC-GFP) [16] were maintained in RPMI 1640 medium (Sigma-Aldrich Fine Chemicals, St. Louis, Mo.) containing 10% fetal bovine serum at 37°C in a 5% humidified CO 2 atmosphere.…”