Fig. 1: Generation of hES cell lines with conditional LMNA::NTRK1 fusion expression. | Oncogenesis

Fig. 1: Generation of hES cell lines with conditional LMNA::NTRK1 fusion expression.

From: Generating in vitro models of NTRK-fusion mesenchymal neoplasia as tools for investigating kinase oncogenic activation and response to targeted therapy

Fig. 1

A Strategy for conditional LMNA::NTRK1 fusion gene expression. A donor template with homology arms (shaded boxes) and a promoter-less selectable marker (hygromycin or puromycin) is introduced at the DSB sites (scissors) by HDR. A splice acceptor (SA) sequence allows the expression of the marker from the LMNA promoter. Induction of the fusion occurs after removal of the selectable marker by expression of the CRE recombinase. B Dual color FISH analysis showing the NTRK1 locus and the rearranged allele in clones 1 (hygromycin positive; red, centromeric; green, telomeric) and 3 (puromycin positive; green, centromeric; red, telomeric) after loss of the centromeric probe either by t(1;1) or ID. C RT-PCR analysis in hES cells showing the LMNA::NTRK1 fusion level over a period of 30 days after expression of the CRE recombinase in clones 1 and 3. D Dual color FISH analysis on clone 13, (from NHEJ strategy, see S1D) showing loss of 5′ NTRK1 probe (red, centromeric) and RT-PCR confirming the presence of the LMNA::NTRK1 fusion in two distinct clones. E Western blot analysis showing the LMNA::TRKA fusion protein in clones isolated after removal of the marker (clones 1.14 and 3.3) and clone 13. Phosphorylation of TRKA is not detected.

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