title: Novel lentiviral-inducible transgene expression systems and versatile single-plasmid reporters for in vitro and in vivo cancer biology studies.
abstract: Many of the cancer cell lines derived from solid tumors are difficult to transfect using commonly established transfection approaches. This hurdle for some DNA transfection systems has hindered cancer biology studies. Moreover, there are limited tools for studying pathway activities. Therefore, highly efficient improved gene transfer and versatile genetic tools are required. In this study, we established and developed a comprehensive set of new lentiviral tools to study gene functions and pathway activities. Using the optimized conditions, cancer cell lines achieved >90% transduction efficiency. Novel lentiviral doxycycline-regulated pTet-IRES-EGFP (pTIE) systems for transgene expression and TRE reporters used for pathway activity determination were developed and tested. The pTIE Tet-Off system showed in vitro doxycycline-sensitive responses with low or undetectable leakage of protein expression and in vivo tumor suppression as illustrated using candidate tumor suppressors, Fibulin-2 and THY1. In contrast, the Tet-On system showed dose-dependent responses. The pTRE-EGFP (pTE) and pTRE-FLuc-EF1α-RLuc (pT-FER) reporters with the NFκB p65 subunit consensus sequence showed GFP and firefly luciferase responses, which were directly correlated with TNFα stimulation, respectively. Taken together, these newly developed lentiviral systems provide versatile in vitro and in vivo platforms to strengthen our capabilities for cancer biology studies.
authors.affiliation: Department of Clinical Oncology, University of Hong Kong, Hong Kong (SAR), China.
authors.forename: W H
authors.initials: WH
authors.lastname: Shuen
authors.affiliation: Department of Clinical Oncology, University of Hong Kong, Hong Kong (SAR), China.
authors.forename: R
authors.initials: R
authors.lastname: Kan
authors.affiliation: Department of Clinical Oncology, University of Hong Kong, Hong Kong (SAR), China.
authors.forename: Z
authors.initials: Z
authors.lastname: Yu
authors.affiliation: 1] Department of Clinical Oncology, University of Hong Kong, Hong Kong (SAR), China [2] Center for Cancer Research, University of Hong Kong, Hong Kong (SAR), China [3] Center for Nasopharyngeal Carcinoma Research, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong (SAR), China.
authors.forename: H L
authors.initials: HL
authors.lastname: Lung
authors.affiliation: 1] Department of Clinical Oncology, University of Hong Kong, Hong Kong (SAR), China [2] Center for Cancer Research, University of Hong Kong, Hong Kong (SAR), China [3] Center for Nasopharyngeal Carcinoma Research, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong (SAR), China.
authors.forename: M L
authors.initials: ML
authors.lastname: Lung
mesh_terms: D000818:Animals; D019556:COS Cells; D063646:Carcinogenesis / Q000473:pathology; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D049410:Luciferases, Renilla / Q000096:biosynthesis / Q000235:genetics; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000473:pathology / Q000628:therapy*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D049410:Luciferases, Renilla