| Co-Investigator(Kenkyū-buntansha) |
IWAKIRI Dai Hokkaido Univ., Institute for Genetic Medicine, Lec., 遺伝子病制御研究所, 助手 (10307853)
KANNNO Hiroyuki Hokkaido Univ., Institute for Genetic Medicine, Asso. Prof., 遺伝子病制御研究所, 助教授 (40252663)
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| Research Abstract |
Transgenesis of EBV genomes are usually accomplished via homologous recombination after transfection of targeting constructs into EBV-infected cells. However, the marker genes introduced into recombinant EBV genomes may affect their virological features. Low efficiency of homologous recombination is another problem. We utilized Cre/loxP-mediated site-specific recombination for removing marker genes from recombinant viruses, as well as for introducing transgenes into EBV genomes. We aimed to make recombinant EBV lacking viral genes encoding small, non-transcribed RNAs, EBERs (EBER-KO EBV), which appear to be responsible for Burkitt lymphoma tumorigenesis. We took advantage of a mutated loxP site, loxP2272, which has two base-pair mutations in its spacer region (Gene 216: 55, 1998). Since loxP2272 cannot recombine with wild-type loxP site, the region flanked with wild-type loxP and loxP2272 can be replaced with DNA sequences flanked with the same combination of loxP sites by means of Cre
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-mediated gene replacement. Recombinant Akata EBV carrying a neomycin-resistant gene inserted into the BamHI X region (XneoEBV) was subject to EBER targeting. First, EBER genes of XneoEBV were replaced with hygromycin-resistant (hygR) gene flanked with wild-type loxP and loxP2272 by means of homologous recombination in Akata cell [EBER(-)hygREBV]. Second, the hygromycin cassette of EBER(-)hygREBV was replaced with a short non-coding DNA sequence by means of Cre-mediated gene replacement in the host cells. Subsequently, virus production was induced, and the produced viruses were used for infecting EBV-negative Akata cells. Infected cells were selected by G418, and drug-resistant cell clones were examined for the presence of recombinant EBV lacking the hygR gene. Cre-mediated gene replacement occurred in 2 of out 76 clones, and those two cell clones produced a large quantity of EBER-KO virus lacking the hygR gene. B lymphocytes purified from cord blood were infected with equivalent amounts of XneoEBV and EBER-KO EBV, respectively. Although these viruses exhibited comparable infectivity, transforming titer of EBER-KO EBV was approximately 100 fold less than XneoEBV, demonstrating the contribution of EBERs for efficient transformation. Less
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