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A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9
- Source :
- Research and Practice in Thrombosis and Haemostasis, Research and Practice in Thrombosis and Haemostasis, Vol 4, Iss 1, Pp 64-71 (2020)
- Publication Year :
- 2019
- Publisher :
- John Wiley and Sons Inc., 2019.
-
Abstract
- Background Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species‐specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF. Objectives To develop a VWF knock out (VWF–/–) rat by excision of all 52 exons of the VWF locus. Methods The entire VWF gene was eliminated in Sprague‐Dawley (Crl:SD) rats via CRISPR/Cas9‐mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF–/– rats were infused with FVIII–/– rat platelet poor plasma (PPP) to determine response of plasma FVIII. Results Breeding of VWF ± rats yielded VWF–/– offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF–/– rats. VWF–/– rats bled longer and more than VWF+/– and VWF+/+ rats when challenged. Transfusion of FVIII‐deficient platelet‐poor plasma induced a rapid rise in endogenous FVIII:C in VWF–/– rats. Conclusion This rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.
- Subjects :
- medicine.medical_specialty
congenital, hereditary, and neonatal diseases and abnormalities
animal diseases
030204 cardiovascular system & hematology
von Willebrand factor
03 medical and health sciences
Exon
0302 clinical medicine
Antigen
Von Willebrand factor
Bleeding time
Internal medicine
hemic and lymphatic diseases
Original Articles: Hemostasis
medicine
Von Willebrand disease
Diseases of the blood and blood-forming organs
Protein precursor
030304 developmental biology
Platelet-poor plasma
0303 health sciences
medicine.diagnostic_test
biology
rat model
Hematology
Phlebotomy
medicine.disease
severe von Willebrand disease
Endocrinology
factor VIII
CRISPR
biology.protein
cardiovascular system
Original Article
RC633-647.5
circulatory and respiratory physiology
Subjects
Details
- Language :
- English
- ISSN :
- 24750379
- Volume :
- 4
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Research and Practice in Thrombosis and Haemostasis
- Accession number :
- edsair.doi.dedup.....7801619efc791b4cc3c0d14c6260f156