A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9
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 amenabi...
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Wiley
2020
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oai:doaj.org-article:dfb6426d17994ee9ae5dbf1dce097efe2021-11-15T06:10:44ZA rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas92475-037910.1002/rth2.12280https://doaj.org/article/dfb6426d17994ee9ae5dbf1dce097efe2020-01-01T00:00:00Zhttps://doi.org/10.1002/rth2.12280https://doaj.org/toc/2475-0379Abstract 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.Jessica GarciaVeronica H. FloodSandra L. HaberichterScot A. FahsJeremy G. MattsonAron M. GeurtsMark ZoggHartmut WeilerQizhen ShiRobert R. MontgomeryWileyarticleCRISPRfactor VIIIrat modelsevere von Willebrand diseasevon Willebrand factorDiseases of the blood and blood-forming organsRC633-647.5ENResearch and Practice in Thrombosis and Haemostasis, Vol 4, Iss 1, Pp 64-71 (2020) |
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CRISPR factor VIII rat model severe von Willebrand disease von Willebrand factor Diseases of the blood and blood-forming organs RC633-647.5 |
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CRISPR factor VIII rat model severe von Willebrand disease von Willebrand factor Diseases of the blood and blood-forming organs RC633-647.5 Jessica Garcia Veronica H. Flood Sandra L. Haberichter Scot A. Fahs Jeremy G. Mattson Aron M. Geurts Mark Zogg Hartmut Weiler Qizhen Shi Robert R. Montgomery A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9 |
| description |
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. |
| format |
article |
| author |
Jessica Garcia Veronica H. Flood Sandra L. Haberichter Scot A. Fahs Jeremy G. Mattson Aron M. Geurts Mark Zogg Hartmut Weiler Qizhen Shi Robert R. Montgomery |
| author_facet |
Jessica Garcia Veronica H. Flood Sandra L. Haberichter Scot A. Fahs Jeremy G. Mattson Aron M. Geurts Mark Zogg Hartmut Weiler Qizhen Shi Robert R. Montgomery |
| author_sort |
Jessica Garcia |
| title |
A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9 |
| title_short |
A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9 |
| title_full |
A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9 |
| title_fullStr |
A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9 |
| title_full_unstemmed |
A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9 |
| title_sort |
rat model of severe vwd by elimination of the vwf gene using crispr/cas9 |
| publisher |
Wiley |
| publishDate |
2020 |
| url |
https://doaj.org/article/dfb6426d17994ee9ae5dbf1dce097efe |
| work_keys_str_mv |
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