A novel carcinogenic mouse model by site-directed insertion of tandem human HRAS large DNA fragment into 15E1 site.

BACKGROUND: The precise insertion of large DNA fragments (>3-5 kb) remains one of the key obstacles in establishment of genetically modified murine models. METHODS: A 21 kb large DNA fragment containing three tandemly linked copies of the human HRAS gene was inserted into the genome of C57BL/6J mouse, generating a mouse model designated as KI.C57-ras (or named NF-hHRAS). Whole-genome sequencing and Sanger sequencing were utilized to it confirm precise insertion and copy number. The stability of transgene expression among different generations was verified from multiple aspects using by digital PCR, western blot and DNA sequencing. To assess tumor susceptibility in the mouse model, N-Nitroso-N-methylurea (MNU) was administered at a dosage of 75 mg/kg. Histopathological examinations were conducted using hematoxylin and eosin (H&E) staining. RESULTS: The HRAS DNA fragment was inserted into mouse chromosome 15E1 site, locating between 80 623 202 bp and 80 625 020 bp. NF-hHRAS mice exhibited stable inheritance and displayed consistent phenotypes across individuals. Moreover, this mouse model exhibited a high susceptibility to carcinogens. Upon administration of MNU the earliest mortality onset was earlier than that of wild-type littermates (day 65 vs. day 78 for male and day 56 vs. day 84 for female). Notably, 100% of the NF-hHRAS transgenic mice developed tumors, with approximately 84% of male NF-hHRAS mice exhibiting specific tumor types, such as squamous cell carcinoma or squamous cell papilloma, which was consistent with the previously reported carcinogenic rasH2 mouse model. The types of tumors and the target organs exhibited diversity in NF-hHRAS mice, while the spontaneous tumor incidence remained low (1/50). CONCLUSIONS: The NF-hHRAS mice demonstrated excellent genetic stability, a reproducible phenotype, and high susceptibility to carcinogens, indicating their potential utility in non-clinical safety evaluations of drugs as per the S1B guidelines issued by the ICH (The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use).