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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

Highly efficient site-specific transgenesis in cancer cell lines

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Iacovos P Michael, Claudio Monetti, Anthony C Chiu, Puzheng Zhang, Takeshi Baba, Koichiro Nishino, Siamak Agha-Mohammadi, Knut Woltjen, Hoon-Ki Sung, Andras Nagy
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-89) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contribution

IPM design the overall study, conducted the majority of experiments, collected and analyzed the data. CM, KN, TB, KW, HS contributed to experimental design. CM, ACC, PZ, HS conducted experiments. SAM offered reagents. AN guided the study. IPM and AN wrote the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.

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.
Zusatzmaterial
Additional file 1: Highly efficient site-specific transgenesis in cancer cell lines. (PDF 9 MB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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Authors’ original file for figure 5
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Literatur
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