Related Experiment Video
Updated: Aug 5, 2025
In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity
Published on: November 2, 2020
Evaluation of Radiation Sensitivity Differences in Mouse Liver Tumor Organoids Using CRISPR/Cas9-Mediated Gene
Wan Jeon1,2, Se Yeon Jung3, Chae Young Lee3
1Department of Radiation Oncology, 222204Dongnam Institute of Radiological and Medical Sciences, Busan, Republic of Korea.
Background:
To assess the radiosensitivity of liver tumors harboring different genetic mutations, mouse liver tumors were generated in vivo through the hydrodynamic injection of clustered regularly interspaced short palindromic repeat/caspase 9 (CRISPR/Cas9) constructs encoding single-guide RNAs (sgRNAs) targeting Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, or Rb1.
Methods:
The plasmid vectors were delivered to the liver of adult C57BL/6 mice via hydrodynamic tail vein injection. The vectors were injected into 10 mice in each group. Organoids were generated from mouse liver tumors. The radiation response of the organoids was assessed using an ATP cell viability assay.
Results:
The mean survival period of mice injected with vectors targeting Nf2 (4.8 months) was lower than that of other mice. Hematoxylin and eosin staining, immunohistochemical (IHC) staining, and target sequencing analyses revealed that mouse liver tumors harbored the expected mutations. Tumor organoids were established from mouse liver tumors. Histological evaluation revealed marked morphological similarities between the mouse liver tumors and the generated tumor organoids. Moreover, IHC staining indicated that the parental tumor protein expression pattern was maintained in the organoids. The results of the ATP cell viability assay revealed that the tumor organoids with mutated Nf2 were more resistant to high-dose radiation than those with other gene mutations.
Conclusions:
This study developed a radiation response assessment system for mouse tumors with mutant target genes using CRISPR/Cas9 and organoids. The Tp53 and Pten double mutation in combination with the Nf2 mutation increased the radiation resistance of tumors. The system used in this study can aid in elucidating the mechanism underlying differential intrinsic radiation sensitivity of individual tumors.
Insights
This study used CRISPR/Cas9 and organoids to assess liver tumor radiosensitivity in mice. Nf2 mutations, especially with Tp53 and Pten, increased tumor radiation resistance.
Area of Science:
- Oncology
- Genetics
- Radiation Biology
Background:
- Liver tumors were induced in mice using CRISPR/Cas9 technology targeting specific genes.
- Genetic mutations investigated include Tp53, Pten, Nf1, Nf2, Tsc2, Cdkn2a, and Rb1.
Purpose of the Study:
- To evaluate the radiosensitivity of liver tumors with distinct genetic mutations.
- To establish a novel system for assessing tumor response to radiation.
Main Methods:
- Hydrodynamic tail vein injection was used to deliver CRISPR/Cas9 constructs into mouse livers.
- Tumor organoids were derived from induced mouse liver tumors for radiosensitivity assays.
- Adenosine triphosphate (ATP) cell viability assays were employed to assess radiation response.
Main Results:
- Mice with Nf2-targeted mutations exhibited a reduced mean survival period.
- Established tumor organoids accurately reflected the histology and protein expression of parental tumors.
- Organoids with Nf2 mutations demonstrated increased resistance to high-dose radiation compared to other genetic alterations.
Conclusions:
- A novel system combining CRISPR/Cas9 and organoids was developed for assessing tumor radiosensitivity.
- Combined Tp53, Pten, and Nf2 mutations significantly enhanced tumor radioresistance.
- This system can help elucidate mechanisms of differential tumor radiation sensitivity.
