Validation for the function of protein C in mouse models

Ya Liu; Maoping Cai; Yan Chen; Guocai Wu; Songyu Li; Zhanghui Chen

doi:10.7717/peerj.17261

Validation for the function of protein C in mouse models

PeerJ. 2024 Apr 24:12:e17261. doi: 10.7717/peerj.17261. eCollection 2024.

Authors

Ya Liu^#¹, Maoping Cai^#¹, Yan Chen¹, Guocai Wu², Songyu Li¹, Zhanghui Chen¹

Affiliations

¹ Zhanjiang Institute of Clinical Medicine, Central People's Hospital of Zhanjiang, Guangdong Medical University, Zhanjiang, Guangdong, China.
² Department of Hematology, Central People's Hospital of Zhanjiang, Guangdong Medical University, Zhanjiang, Guangdong, China.

^# Contributed equally.

Abstract

Objectives: Protein C (PC) is an anticoagulant that is encoded by the PROC gene. Validation for the function of PC was carried out in mouse models.

Methods: In this study, autosomal recessive PC deficiency (PCD) was selected as the target, and the specific mutation site was chromosome 2 2q13-q14, PROC c.1198G>A (p.Gly400Ser) which targets G399S (GGT to AGC) in mouse models. To investigate the role of hereditary PC in mice models, we used CRISPR/Cas9 gene editing technology to create a mouse model with a genetic PCD mutation.

Results: The two F0 generation positive mice produced using the CRISPR/Cas9 gene editing technique were chimeras, and the mice in F1 and F2 generations were heterozygous. There was no phenotype of spontaneous bleeding or thrombosis in the heterozygous mice, but some of them were blind. Blood routine results showed no significant difference between the heterozygous mice and wild-type mice (P > 0.05). Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were prolonged in the heterozygous mice, while the level of fibrinogen content (FIB) decreased, suggesting secondary consumptive coagulation disease. The protein C activity of heterozygous mice was significantly lower than that of wild-type mice (P < 0.001), but there was no significant difference in protein C antigen levels (P > 0.05). H&E staining showed steatosis and hydrodegeneration in the liver of heterozygous mice. Necrosis and exfoliated epithelial cells could be observed in renal tubule lumen, forming cell or granular tubules. Hemosiderin deposition was found in the spleen along with splenic hemorrhage. Immunohistochemistry demonstrated significant fibrin deposition in the liver, spleen, and kidney of heterozygous mice.

Conclusion: In this study, heterozygotes of the mouse model with a PC mutation were obtained. The function of PC was then validated in a mouse model through genotype, phenotype, and PC function analysis.

Keywords: CRISPR/Cas9; Mouse model; Protein C deficiency; Venous thromboembolism.

Publication types

Research Support, Non-U.S. Gov't
Validation Study

MeSH terms

Animals
Blood Coagulation / genetics
CRISPR-Cas Systems / genetics
Disease Models, Animal*
Female
Gene Editing / methods
Heterozygote
Male
Mice
Mutation
Partial Thromboplastin Time
Protein C Deficiency / genetics
Protein C* / genetics
Protein C* / metabolism

Substances

Protein C

Grants and funding

This work was supported by the National Natural Science Foundation of China (No. 81971886, No. 82170053 and No. 82370099), the Zhujiang Talent Program (No. 2019QN01Y279), the Zhanjiang Science and Technology Project (No. 2021A05137, No. 2022A01107 and No. 2022A01075) and the Discipline Construction Fund of Zhanjiang Central Hospital, Guangdong Medical University (No. 2022A03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.