title: Highly efficient site-specific transgenesis in cancer cell lines.
abstract: BACKGROUND
  Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI) for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells.
  
  
  METHODS
  According to this system, a "docking-site" was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an "incoming" vector containing the gene of interest was specifically inserted in the docking-site using PhiC31.
  
  
  RESULTS
  Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate format.
  
  
  CONCLUSIONS
  The novel PhiC31-IMSI system described in this study represents a powerful tool that can facilitate the characterization of cancer-related genes.
authors.affiliation: Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada.
authors.forename: Iacovos P
authors.initials: IP
authors.lastname: Michael
authors.forename: Claudio
authors.initials: C
authors.lastname: Monetti
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Chiu
authors.forename: Puzheng
authors.initials: P
authors.lastname: Zhang
authors.forename: Takeshi
authors.initials: T
authors.lastname: Baba
authors.forename: Koichiro
authors.initials: K
authors.lastname: Nishino
authors.forename: Siamak
authors.initials: S
authors.lastname: Agha-Mohammadi
authors.forename: Knut
authors.initials: K
authors.lastname: Woltjen
authors.forename: Hoon-Ki
authors.initials: HK
authors.lastname: Sung
authors.forename: Andras
authors.initials: A
authors.lastname: Nagy
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D023061:Gene Order; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D057166:High-Throughput Screening Assays; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000235:genetics*; D015203:Reproducibility of Results; D019076:Transgenes; D014183:Transplantation, Heterologous
chemical_list: D004318:Doxycycline