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Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies
1Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Abstract:
Previously we created two strains of factor VIII-deficient mice by insertion of a neo gene into (1) the 3' end of exon 16 and (2) exon 17 of the factor VIII gene. Affected mice of both strains have no plasma factor VIII activity, yet are healthy with no spontaneous bleeding. Factor VIII-deficient females bred with affected males survive pregnancy and delivery. We used reverse transcriptase-polymerase chain reaction of liver RNA to characterize factor VIII mRNA processing. Factor VIII mRNA of the exon 16 knockout strain contains neo sequences plus 17 bp of intron 16 due to use of a cryptic donor site in intron 16. All factor VIII mRNA of the exon 17 knockout strain lacks exon 17 and neo sequences. In skipping exon 17, the intron 16 donor site or a cryptic donor site 46 bp 3' to the intron 16 donor site are used. Thus, factor VIII deficiency in exon 16 knockout mice is due to truncated protein, while in exon 17 knockout mice it is due to either truncated or partially deleted protein. After immunizing exon 16 knockout mice with human recombinant factor VIII, two monoclonal antibodies were obtained that recognize < 100 pg of mouse factor VIII light chain. Assay of cryoprecipitate from the plasma of affected mice failed to show factor VIII light chain.
Insights
Two mouse models of hemophilia A were developed. These models exhibit no factor VIII activity but remain healthy, aiding research into factor VIII gene function and deficiency.
Area of Science:
- * Genetics
- * Molecular Biology
- * Hematology
Background:
- * Two strains of factor VIII-deficient mice were generated by targeted gene disruption.
- * Both strains exhibit complete absence of plasma factor VIII activity.
- * Despite factor VIII deficiency, mice are phenotypically healthy with no spontaneous bleeding.
Purpose of the Study:
- * To investigate the molecular mechanisms of factor VIII deficiency in the generated mouse models.
- * To characterize factor VIII mRNA processing in exon 16 and exon 17 knockout strains.
- * To explore the potential for developing diagnostic tools for factor VIII deficiency.
Main Methods:
- * Creation of two factor VIII gene knockout mouse strains via neo gene insertion.
- * Reverse transcriptase-polymerase chain reaction (RT-PCR) of liver RNA to analyze mRNA.
- * Production and characterization of monoclonal antibodies against mouse factor VIII light chain.
Main Results:
- * Exon 16 knockout strain: Factor VIII mRNA includes neo sequences and intron 16 due to cryptic splice site usage.
- * Exon 17 knockout strain: Factor VIII mRNA lacks exon 17 and neo sequences, utilizing alternative splice sites.
- * Monoclonal antibodies identified, but no factor VIII light chain detected in affected mouse plasma.
Conclusions:
- * Factor VIII deficiency in exon 16 knockout mice results from truncated protein production.
- * Factor VIII deficiency in exon 17 knockout mice results from truncated or partially deleted protein.
- * The generated mouse models provide valuable tools for studying hemophilia A and factor VIII function.
