A factor IX-deficient mouse model for hemophilia B gene therapy

Abstract

We have generated a mouse where the clotting factor IX (FIX) gene has been disrupted by homologous recombination. The FIX nullizygous (-/-) mouse was devoid of factor IX antigen in plasma. Consistent with the bleeding disorder, the factor IX coagulant activities for wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mice were 92%, 53%, and <5%, respectively, in activated partial thromboplastin time assays. Plasma factor IX activity in the deficient mice (-/-) was restored by introducing wild-type murine FIX gene via adenoviral vectors. Thus, these factor IX-deficient mice provide a useful animal model for gene therapy studies of hemophilia B.

Figure 1

Targeting of the FIX gene by homologous recombination. (A) Schematic of the 3′ portion of the murine factor IX gene showing the last exon, the targeting vector, and the expected recombined locus. The targeting construct contains a PGK-neo cassette, flanked by DNA fragments upstream and downstream the putative exon 8. The predicted product of successful homologous recombination is shown at the bottom. (B) Southern blot analysis of XbaI-digested genomic DNA prepared from tail biopsies of FIX +/+, +/−, and −/− mice and hybridized with the probe shown in A. The probe used was the same 800 bp used to identify the homologous recombined ES clones, as shown in A. New genomic fragment of the expected size 9.1 kb is seen in FIX +/− and −/− mice. The 7.8-kb fragment is absent from FIX −/− mice. The 6.7-kb XbaI fragment is upstream of the recombination region and is present in both the normal and mutated allele. (C) Northern blot analysis of FIX RNA. Total RNA was isolated from mouse liver. The FIX probe used was a 2.7-kb BamHI fragment of a murine FIX cDNA that covers the entire coding region of FIX gene. FIX-specific RNA was undetectable in liver RNA from FIX −/− mice. (D) Western blot analysis of plasma from FIX +/+, +/−, and −/− mice. H, human plasma used as a control. The factor IX protein is indicated by an arrow. The star indicates the protein band in mouse plasma that cross-reacts with the rabbit anti-human factor IX antibody A0300 (Dako).

Figure 2

Bleeding disorder in a factor IX-deficient mouse. Photographs of a wild-type (Left) and a factor IX-deficient (−/−) mouse (Right) are shown after trauma generated by pulling through a mouse restrainer. Notice the swollen extremities and hematoma in the knock-out mouse (see arrow), compared with the normal mouse. (Inset) Enlargement of the swollen hemorrhagic foot of the mutant (−/−) mouse.

Figure 3

Factor IX coagulant activity in FIX +/+, +/−, and −/− mice. Factor IX activity was quantitated by using APTT assays. The factor IX activity for wild-type (+/+) mice was 92.1 ± 19.1% (n = 21), for heterozygous (+/−) mice was 52.9 ± 10.4% (n = 12), and for homozygous (−/−) mice was 5.4 ± 2.9% (n = 26).