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Figure 1 | Genome Biology

Figure 1

From: Polygenic in vivovalidation of cancer mutations using transposons

Figure 1

Transposon mediated in vivo validation of cancer mutations. (a) Schematic for design of transposon construct, not to scale. Arrows show relative primer positions for subsequent genotyping and PCR assays. ATG, translation start codon of incorporated cDNA; IRES, encephalomyocarditis virus internal ribosome entry site; pA, bovine growth hormone poly-adenylation signal; PB, piggyBac; SA, Engrailed splice acceptor; SB, Sleeping Beauty; TR, terminal repeat. (b) Distribution of unique transposon species in pooled electroporation. A pool of 24 unique sequence-tagged transposons containing the neomycin resistance marker is electroporated into murine ES cells and colonies selected for in media supplemented with geneticin. Distribution shown is for 73 picked clones that are geneticin-resistant. (c) List of kinase mutations and the tumour type where they were observed in human patients. For each kinase both the wild-type and mutant versions of the cDNA were constructed and incorporated into individually tagged transposon constructs. (d) Schematic of the experimental strategy for in vivo validation of candidate cancer gene alleles using transposon. (e) Genotyping PCR with forward primer in cDNA and reverse primer in sequence tag (see panel a) to detect presence of individual transposons. Each row shows an individual animal with the 25 PCR reactions to the different transposons, 3’F1 progeny from a litter are shown. (f) In F1 pups with SB transposase, both intact transposon (detected by the junction primers) and SB mobilisation (detected by the flanking primers) are detectable (primers shown in Figure 1a). Loss of the nested SB transposon results in the PCR reaction amplifying a product of similar size to the terminal repeat junctions.

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