RNAi can block retroviral infection in vertebrates. The tissue electroporation method described here should allow RNAi to be used widely to study gene function and control of infection in vertebrate animals.

Early during retroviral infection, a fraction of the linear reverse-transcribed viral DNA genomes become circularized by cellular enzymes, thereby inactivating the genomes for further replication. Prominent circular DNA forms include 2-long-terminal repeat (LTR) circles, made by DNA end joining, and 1-LTR circles, produced in part by homologous recombination. These reactions provide a convenient paradigm for analyzing the cellular machinery involved in DNA end joining in vertebrate cells. In previous studies, we found that inactivating components of the nonhomologous DNA end-joining (NHEJ) pathway--specifically Ku, ligase 4, or XRCC4--blocked formation of 2-LTR circles. Here we report that inactivating another NHEJ component, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), had at most modest effects on 2-LTR circle formation, providing informative parallels with other end-joining reactions. We also analyzed cells mutant in components of the RAD50/MRE11/NBS1 nuclease and found a decrease in the relative amount of 1-LTR circles, opposite to the effects of NHEJ mutants. In MRE11-mutant cells, a MRE11 gene mutant in the nuclease catalytic site failed to restore 1-LTR circle formation, supporting a model for the role of MRE11 in 1-LTR circle formation. None of the cellular mutations showed a strong effect on normal integration, consistent with the idea that the cellular pathways leading to circularization are not involved in productive integration.

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.

Introduction Detection of oncogenic fusions has been of great importance for understanding tumorigenesis and for precision oncology in enhancing diagnosis and selection of targeted therapies. Herein, we describe an extended Oncomine targeted RNA sequencing assay for detection of fusion transcripts and intragenic rearrangements (exon deletion/skipping). For multiple key driver genes we also supplemented the panel with a complementary transcript-based expression imbalance assay designed to identify gene fusions in a partner agnostic manner. Methods Based on evidence from Oncomine™ Knowledgebase and collaboration with leading oncology researchers, we designed an Ion AmpliSeq™ panel to target > 1,200 fusion breakpoints in > 50 driver genes, > 40 intragenic rearrangements (e.g., MET exon 14 skipping, ARv7, EGFRvIII) in 7 genes, and 5 RNA expression controls. In addition, the panel supports detection and reporting of non-targeted fusions (i.e., novel combinations of drivers and partners). We supplemented the panel with exon tiling expression imbalance assays, using amplicons tiling the exon junctions of ALK, RET, NTRK1, NTRK2 and NTRK3 to measure 3'/5' expression imbalance signatures. We developed a bioinformatic tool to call fusions from a normalized and corrected expression imbalance profile per gene (using a baseline from normal formaldehyde fixed paraffin embedded [FFPE samples]). We optimized the gene fusion algorithms and integrated them as workflows into the Ion ReporterTM Software to facilitate the summary of the results with relevant annotations, rich data visualizations and easily interpretable reports. Results We sequenced hundreds of positive and negative fusion samples including commercial reference standards, cell lines and FFPE clinical research samples on the Ion GeneStudioTM S5 sequencer. To assess the feasibility of the combined panel, we sequenced the Seraseq® FFPE tumor fusion RNA reference, 7 fusion positive cell lines with ALK, RET, ROS1, NTRK1, FGFR1, FGFR2 and FGFR3 rearrangements, and cohorts of FFPE samples using 20ng RNA as input and successfully detected the expected fusion isoforms or other RNA rearrangements in each sample. We applied the exon tiling fusion detection method for ALK, RET and NTRK1 and observed perfect concordance between the true isoform in the positive samples and the predicted breakpoint position and magnitude of 3'/5' expression imbalance indicated by the exon tiling method. Conclusions We developed an extended, multiplexed RNA panel for fusions and intragenic rearrangements that retains the simple workflow and fast turn-around time of previous Oncomine fusion panels and significantly expands the scope of fusion isoform detection including methods to detect gene fusions in a partner agnostic manner. For research use only. Not for use in diagnostic procedures. Citation Format: Amir Marcovitz, Rajesh K. Gottimukkala, Gary G. Bee, Jennifer M. Kilzer, Vinay K. Mital, Elain Wong-Ho, Chenchen Yang, Yu-Ting Tseng, Scott P. Myrand, Paul D. Williams, Seth Sadis, Fiona C. Hyland. RNA sequencing based gene fusion detection with oncomine comprehensive assay plus [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 177.

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