Somatic integration of an oncogene-harboring Sleeping Beauty transposon models liver tumor development in the mouse

Abstract

The Sleeping Beauty (SB) transposon system can integrate foreign sequences of DNA in the genome of mouse somatic cells eliciting long-term expression in vivo. This technology holds great promise for human gene therapy as a nonviral technology to deliver therapeutic genes. SB also provides a means to study the effects of defined genetic elements, such as oncogenes, on somatic cells in mice. Here, we test the ability of the SB transposon system to facilitate somatic integration of a transposon containing an activated NRAS oncogene in mouse hepatocytes to elicit tumor formation. NRAS oncogene-driven tumors developed when such vectors were delivered to the livers of p19Arf-null or heterozygous mice. Delivery of the NRAS transposon cooperates with Arf loss to cause carcinomas of hepatocellular or biliary origin. These tumors allowed characterization of transposon integration and expression at the single-cell level, revealing robust NRAS expression and both transposase-mediated and random insertion of delivered vectors. Random integration and expression of the SB transposase plasmid was also observed in one instance. In addition, studies using effector loop mutants of activated NRAS provide evidence that mitogen-activated protein kinase activation alone cannot efficiently induce liver carcinomas. This system can be used to rapidly model tumors caused by defined genetic changes.cancer ͉ gene therapy ͉ Nras ͉ p19Arf

Cyclin-dependent kinase 1 (Cdk1) is essential for cell division and suppression of DNA re-replication but not for liver regeneration

“…Hydrodynamic tail-vein injections and Sleeping Beauty transposon-induced liver tumorigenesis were performed as described previously (25,35). Six-to 10-wk-old mice were injected with a plasmid mixture diluted in lactated Ringer's solution.…”

“…To investigate tumor formation in vivo, we used tail-vein injections of activated Ras alone (in a p53 +/− background) or in combination with an shRNA to silence p53 expression (25). Control mice (Cdk1 FLOX/FLOX ) developed many liver tumors within 3-4 mo (Fig.…”

Sleeping Beautytransposon system for genetic etiological research and gene therapy of cancers

“…For example, through hydrodynamic tail vein injection, SB transposon system containing an activated N-RAS oncogene can elicit multifocal liver cancer in p19Arf-null or heterozygous mice. 62 Subcutaneous injection of SB transposon system harboring oncogenes, including c-Myc, H-RAS, and short hairpin RNA against the transformation related protein 53 (Trp53) gene (shp53), into female C57BL/6 mice can induce sarcomatoid carcinomas in skin ( Table 1). 63 The SB system can also be applied in other mammalians, such as rats.…”

piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice

“…CRISPR/Cas9 | piggyBac transposon | liver cancer | tumorigenesis | screening F or the last decade, transposon mutagenesis and RNA interference-mediated screens have been the main methods for in vivo screening and validation of cancer genes in mice (1)(2)(3)(4)(5)(6). However, because of their low efficiency, these two methods have not been widely used.…”