title: Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene.
abstract: ESSENTIALS: Anti-factor VIII (FVIII) inhibitory antibody formation is a severe complication in hemophilia A therapy. We genetically engineered and characterized a mouse model with complete deletion of the F8 coding region. F8(TKO) mice exhibit severe hemophilia, express no detectable F8 mRNA, and produce FVIII inhibitors. The defined background and lack of FVIII in F8(TKO) mice will aid in studying FVIII inhibitor formation.
  
  
  BACKGROUND
  The most important complication in hemophilia A treatment is the development of inhibitory anti-Factor VIII (FVIII) antibodies in patients after FVIII therapy. Patients with severe hemophilia who express no endogenous FVIII (i.e. cross-reacting material, CRM) have the greatest incidence of inhibitor formation. However, current mouse models of severe hemophilia A produce low levels of truncated FVIII. The lack of a corresponding mouse model hampers the study of inhibitor formation in the complete absence of FVIII protein.
  
  
  OBJECTIVE
  We aimed to generate and characterize a novel mouse model of severe hemophilia A (designated the F8(TKO) strain) lacking the complete coding sequence of F8 and any FVIII CRM.
  
  
  METHODS
  Mice were created on a C57BL/6 background using Cre-Lox recombination and characterized using in vivo bleeding assays, measurement of FVIII activity by coagulation and chromogenic assays, and anti-FVIII antibody production using ELISA.
  
  
  RESULTS
  All F8 exonic coding regions were deleted from the genome and no F8 mRNA was detected in F8(TKO) mice. The bleeding phenotype of F8(TKO) mice was comparable to E16 mice by measurements of factor activity and tail snip assay. Similar levels of anti-FVIII antibody titers after recombinant FVIII injections were observed between F8(TKO) and E16 mice.
  
  
  CONCLUSIONS
  We describe a new C57BL/6 mouse model for severe hemophilia A patients lacking CRM. These mice can be directly bred to the many C57BL/6 strains of genetically engineered mice, which is valuable for studying the impact of a wide variety of genes on FVIII inhibitor formation on a defined genetic background.
authors.affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD, USA.
authors.forename: B N
authors.initials: BN
authors.lastname: Chao
authors.affiliation: Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Emory University, Atlanta, GA, USA.
authors.forename: W H
authors.initials: WH
authors.lastname: Baldwin
authors.affiliation: Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Emory University, Atlanta, GA, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Healey
authors.affiliation: Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Emory University, Atlanta, GA, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Parker
authors.affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD, USA.
authors.forename: K
authors.initials: K
authors.lastname: Shafer-Weaver
authors.affiliation: Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Emory University, Atlanta, GA, USA.
authors.forename: C
authors.initials: C
authors.lastname: Cox
authors.affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD, USA.
authors.forename: P
authors.initials: P
authors.lastname: Jiang
authors.affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD, USA.
authors.forename: C
authors.initials: C
authors.lastname: Kanellopoulou
authors.affiliation: Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Emory University, Atlanta, GA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Lollar
authors.affiliation: Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Emory University, Atlanta, GA, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Meeks
authors.affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Lenardo
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood; D001780:Blood Coagulation Tests; D002863:Chromogenic Compounds; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D017353:Gene Deletion; D020022:Genetic Predisposition to Disease; D006467:Hemophilia A / Q000097:blood / Q000188:drug therapy / Q000235:genetics* / Q000276:immunology; D006487:Hemostasis / Q000187:drug effects / Q000235:genetics; D006490:Hemostatics / Q000276:immunology / Q000494:pharmacology; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010641:Phenotype; D012720:Severity of Illness Index
chemical_list: D000906:Antibodies; D002863:Chromogenic Compounds; D006490:Hemostatics; C078147:F8 protein, human; D005169:Factor VIII
keywords: Animal model; blood coagulation factor inhibitors; factor VIII; hemophilia A; knockout mouse