A novel carcinogenic mouse model by site-directed insertion of tandem human HRAS large DNA fragment into 15E1 site
Susu Liu1,2, Yanwei Yang3, Guitao Huo3
1Division of Animal Model Research, Institute for Laboratory Animal Resources, National Institutes for Food and Drug Control (NIFDC), Beijing, China.
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).
Insights
A new genetically modified mouse model, NF-hHRAS, was developed for precise insertion of large DNA fragments. These mice show high tumor susceptibility and genetic stability, making them valuable for drug safety evaluations.
Area of Science:
- Genetics
- Genomics
- Animal Models
Background:
- Precise insertion of large DNA fragments (>3-5 kb) is a significant challenge in creating genetically modified mouse models.
- Development of advanced genetic engineering techniques is crucial for improving the generation of these models.
Purpose of the Study:
- To develop a novel genetically modified mouse model (NF-hHRAS) by inserting a large DNA fragment containing the human HRAS gene.
- To evaluate the genetic stability, phenotype consistency, and carcinogen susceptibility of the developed NF-hHRAS mouse model.
Main Methods:
- A 21 kb DNA fragment with three HRAS gene copies was inserted into C57BL/6J mice.
- Whole-genome and Sanger sequencing confirmed precise insertion and copy number.
- Transgene expression stability was assessed using digital PCR, western blot, and DNA sequencing.
- Tumor susceptibility was evaluated after N-Nitroso-N-methylurea (MNU) administration, followed by histopathological examination (H&E staining).
Main Results:
- The HRAS fragment was accurately inserted into mouse chromosome 15E1.
- NF-hHRAS mice demonstrated stable inheritance and consistent phenotypes across generations.
- NF-hHRAS mice exhibited high susceptibility to MNU, with earlier mortality and 100% tumor development compared to wild-type littermates.
- Tumor types in NF-hHRAS mice, such as squamous cell carcinoma, mirrored those in the rasH2 model, with low spontaneous tumor incidence.
Conclusions:
- The NF-hHRAS mouse model exhibits excellent genetic stability, a reproducible phenotype, and high carcinogen susceptibility.
- This model is suitable for non-clinical safety evaluations of pharmaceuticals, aligning with ICH S1B guidelines.
- The NF-hHRAS model offers a valuable tool for preclinical research and drug development.
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