Characteristics Of Retrovirus Research Articles

Discovery and analysis of an active long terminal repeat-retrotransposable element in Aspergillus oryzae

Wild-type Aspergillus oryzae RIB40 contains two copies of the AO090005001597 gene. We previously constructed A. oryzae RIB40 strain, RKuAF8B, with multiple chromosomal deletions, in which the AO090005001597 copy number was found to be increased significantly. Sequence analysis indicated that AO090005001597 is part of a putative 6,000-bp retrotransposable element, flanked by two long terminal repeats (LTRs) of 669 bp, with characteristics of retroviruses and retrotransposons, and thus designated AoLTR (A. oryzae LTR-retrotransposable element). AoLTR comprised putative reverse transcriptase, RNase H, and integrase domains. The deduced amino acid sequence alignment of AoLTR showed 94% overall identity with AFLAV, an A. flavus Tf1/sushi retrotransposon. Quantitative real-time RT-PCR showed that AoLTR gene expression was significantly increased in the RKuAF8B, in accordance with the increased copy number. Inverse PCR indicated that the full-length retrotransposable element was randomly integrated into multiple genomic locations. However, no obvious phenotypic changes were associated with the increased AoLTR gene copy number.

Enhanced pancreatic tumor regression by a combination of adenovirus and retrovirus-mediated delivery of the herpes simplex virus thymidine kinase gene.

We have evaluated the effectiveness of combining the different characteristics of retrovirus and adenovirus to apply the herpes simplex virus thymidine kinase gene (HSVtk) and ganciclovir (GCV) treatment for gene therapy of pancreatic cancer. Transduction of NP-18 human pancreatic cells in culture by either the adenoviral vector (ADV/tk) or the retroviral vector (Rv/tk) followed by GCV treatment resulted in a GCV dose-dependent cytotoxic effect. A bystander effect was determined, both in NP-18 cultures and in xenogeneic cell mixtures of NP-18 and PA317 cells. Studies in vivo indicated that the effectiveness of tumor regression after HSVtk gene transfer and GCV treatment was dependent first on the tumor size at the time of viral injection and secondly, in large tumors, on the type of virus administered. The administration of the viral combination (ADV/tk + vector producer cells VPC-Rv/tk) was the best approach tested and resulted in a dramatic reduction in tumor mass after 4 days of GCV treatment which was maintained for the treatment period. Remarkably, two animals presented a complete eradication of the tumor. Thus, the HSVtk/GCV system when administered using a viral combination (ADV/tk + VPC-Rv/tk), may be a promising suicide gene therapy for pancreatic carcinomas.

Efficient targeting of retrovirally FRT-tagged chromosomal loci

▼The isolation of recombinant mammalian cells with defined expression levels requires extensive screening. Due to random integration, transfection or injection of DNA leads to cell clones in which the integration site and multicopy number affects foreign gene expression. To overcome these problems efforts have been made to create cell lines with predictable gene expression. One approach is to target the expression cassette to predefined chromosomal loci in the cell line of interest. FRT or loxP-tagged reporter genes, introduced into favorable chromosomal loci, permit analysis of cells with relevant expression. Subsequently, FLPor Crerecombinase enable sequence-specific targeting of these loci. However, the FRT/Flpand loxP/Crerecombinase systems still demand screening for specific integration events (Ref. 1, 2, 3). By contrast with embryonic stem cells, homologous and sequence specific recombination in fibroblastoid cells is considered to be very inefficient. While for embryonic stem cells a protocol has recently been published that allows efficient targeting using the FRT/Flp system (Ref. 4), in fibroblastoid cells the FRT/Flp system was not considered favorable (Ref. 5). Here, we present an improved targeting method that allows the isolation of targeted recombinants in one step in fibroblastoid cells. Cell lines containing a FRTtagged retroviral reporter construct can be targeted with the gene of interest with 100% efficiency. The strength of this system lies in the unique combination of the characteristics of retroviruses, the improved double FRT/Flp system and a stringent selection for the targeting event. In our system a retroviral vector pCMVGALEO is used to screen for appropriate integration sites. The retroviral vector pCMVGALEO contains β-galactosidase (LacZ), followed by the fusion gene hygromycin B phosphotransferase/thymidine kinase as a positive/negative selection marker. The 3′ LTR of pCMVGALEO contains two FRT-sites in tandem, a wild type (F1) and a mutant FRT form (F2),