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Validation for the function of protein C in mouse models
Ya Liu1, Maoping Cai1, Yan Chen1
1Zhanjiang Institute of Clinical Medicine, Central People's Hospital of Zhanjiang, Guangdong Medical University, Zhanjiang, Guangdong, China.
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.
Insights
Mice with a Protein C (PC) deficiency mutation showed altered blood clotting and organ damage, validating PC’s crucial role in anticoagulation and hemostasis.
Area of Science:
- Biochemistry
- Genetics
- Hematology
Background:
- Protein C (PC) is a vital anticoagulant protein encoded by the PROC gene.
- Understanding PC's function is crucial for managing thrombotic and bleeding disorders.
- Mouse models are essential for studying human genetic diseases.
Purpose of the Study:
- To create and characterize a mouse model of autosomal recessive Protein C deficiency (PCD).
- To investigate the in vivo function of Protein C using gene editing technology.
- To validate the PROC c.1198G>A (p.Gly400Ser) mutation in a mammalian model.
Main Methods:
- CRISPR/Cas9 gene editing was employed to introduce the specific PROC mutation in mice.
- Genotyping, phenotypic analysis, and blood coagulation tests were performed on heterozygous and wild-type mice.
- Histopathological examination (H&E staining) and immunohistochemistry were used to assess organ damage and fibrin deposition.
Main Results:
- Heterozygous mice for the PC mutation exhibited prolonged clotting times (PT, APTT, TT) and decreased fibrinogen levels.
- Reduced Protein C activity was observed in heterozygous mice, indicating impaired anticoagulant function.
- Histopathology revealed liver steatosis, renal tubule damage, splenic hemorrhage, and significant fibrin deposition in multiple organs.
Conclusions:
- The developed mouse model successfully recapitulates key features of Protein C deficiency.
- The study validates the critical role of Protein C in maintaining hemostasis and preventing thrombosis.
- The findings highlight the potential for organ damage secondary to PC deficiency.
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