Development of various double transgenic liver cancer models using hydrodynamic transfection

Abstract

Liver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. This study aims to develop transgenic liver cancer models using a hydrodynamics-based transfection method with the Sleeping Beauty transposon system. Using this time-saving and cost-effective methodology, transgenic models have been developed that express various types of oncogenes, individually or in combination to elucidate the genetic mechanisms underlying liver cancer. Transposon vectors each encoding an oncogene or down-regulating a tumor suppressor gene (HrasG12V, β-cateninS33Y, cMyc, SmoM2, and shP53) were constructed. Tumors were observed when livers were hydrodynamically transfected with HrasG12V plus shP53, cMyc plus shP53 and cMyc plus HrasG12V. In tumor of HrasG12V plus shP53 model, expression levels of genes related to cell cycle and epithelial-mesenchymal transition were significantly elevated when determined by a quantitative RT-PCR method. Along with the simple transgenic approach, Cre-mediated conditional transgenic mice were developed using the traditional transgenic methodology that express two oncogenes (KrasG12D and P53R172H) and two reporter genes (green fluorescent protein and luciferase) upon DNA recombination by Cre. Liver tumors were induced using adenovirus encoding Cre and tumor development was subsequently monitored in vivo using bioluminescence imaging. Also, transposons expressing various isoforms of oncogenic Ras (KrasG12V, NrasG12V, KrasG12D, Hras61L, and Nras61K) were generated and used for hydrodynamic transfection. Although different tumorigenic potentials among Ras isoforms have been suggested in liver, we found no difference in intrinsic hepato-carcinogenic

potentials among the isoforms.Lastly, using the hydrodynamic transfection model which successfully expressed HrasG12V and shP53, we have observed repression of liver cancer growth in low-carbohydrate diet groups.The methodology described here is expected to accelerate and facilitate in vivo studies of tumorigenic potentials of cancer-related genes by means of oncogenic cooperation. Due to the versatility of our transposon-based liver cancer models in testing the efficacy of anti-cancer therapeutics, they can be applied to various anti-cancer research leading to the development of new drugs and novel therapeutic strategies.