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Figure 1 | Molecular Cancer

Figure 1

From: A novel approach to identify driver genes involved in androgen-independent prostate cancer

Figure 1

LV-mediated mutagenesis screen. A) Schematic of LV-SFFVEGFP vector. The strong spleen focus-forming viral (SFFV) promoter drives the expression of enhanced green fluorescent protein (EGFP). The vector includes a R6Kγ origin of replication (R6Kori) and kanamycin resistance gene (KanR) for rescue in E.coli. Both long terminal repeats are self-inactivating (SIN.LTR). B) Experimental outline for in vitro LV-mediated insertional mutagenesis screen. Mutagenized sample (SVM-A) became androgen-independent before control, non-mutagenized cells. At this time genomic DNA was extracted and analyzed by our shuttle vector rescue approach. C) Shuttle vector rescue. Genomic DNA is sheared into smaller fragments. The ends are polished and the fragments are ligated into plasmids. Plasmids are transformed into electrocompetent E. coli. Using an LTR-specific primer, the junction between the provirus LTR and the chromosome DNA is sequenced. D) To identify the proviral integration site the chromosomal DNA at the junction is used as a query and aligned to the human genome to identify the integration site.

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