• Factor IX, also known as Christmas Factor, is a 52 kDa zymogen of plasma serine proteases that initiates coagulation through activation by Tissue Factor (TF) and Factor VIIa. The activated form of Factor IX, known as Factor IXa, further activates an intrinsic activation complex that consists of factor X and factor VIIIa in the presence of Ca2+, and phospholipids. Mutations that cause a deficiency in Factor IX lead to x-linked hemophilia B, whereas overproduction of Factor IX has been shown to be a risk factor in venous thromboembolism (VTE). B-hFIX/F8 KO mice serve as a suitable preclinical model for hemophilia research.

Targeting strategy:

• The exons 3~8 of mouse F9 gene were replaced by full length human F9 exons 1~8 in  B-hFIX mice. The 3’UTR region of the mouse gene are replaced by 3’UTR region of human gene. The promoter, 5'UTR also use human sequences. The exons 16~20 of mouse F8 gene were knocked out, there by F8 protein was not expressed in B-hFIX/F8 KO mice anymore.

Protein expression analysis：

• Human FIX was exclusively detectable in homozygous B-hFIX/F8 KO mice by ELISA.

mRNA expression analysis:

• Mouse F9 and F8 mRNA was only detectable in wild-type C57BL/6JNifdc mice. Human F9 mRNA was only detectable in homozygous B-hFIX/F8 KO mice.

Functional Analysis:

• No significant differences were observed in APTT (A), TT (B), or PT (C) values between homozygous B-hFIX mice and wild-type mice. However, homozygous B-hFIX/F8 KO mice exhibited a significantly prolonged APTT (A) compared to the homozygous B-hFIX mice, while their TT (B) and PT (C) values showed no significant changes.

Gene targeting strategy for B-hFIX/F8 KO mice.

• The exons 3~8 of mouse F9 gene were replaced by full length human F9 exons 1~8 in  B-hFIX mice.
• The 3’UTR region of the mouse gene are replaced by 3’UTR region of human gene. The promoter, 5'UTR also use human sequences.
• The exons 16~20 of mouse F8 gene were knocked out, there by F8 protein was not expressed in B-hFIX/F8 KO mice anymore.

Strain specific FIX expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hFIX/F8 KO mice by ELISA. Plasma was collected from wild-type C57BL/6JNifdc mice (+/Y) and homozygous B-hFIX/F8 KO mice (H/Y, -/Y) (n=6, male, 8-week-old). Protein expression level of FIX was analyzed by ELISA (Abcam, ab108831). Human FIX was exclusively detectable in homozygous B-hFIX/F8 KO mice. Values are expressed as mean ± SEM.

Strain specific analysis of F8 and F9 mRNA expression in wild-type C57BL/6JNifdc mice and B-hFIX/F8 KO mice by RT-PCR. Liver mRNA were isolated from wild-type C57BL/6JNifdc mice (+/Y) and homozygous B-hFIX/F8 KO mice (H/Y, -/Y), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human F9 primers and mouse F8 primer. Mouse F8 and F9 was detectable only in wild-type mice. Human F9 mRNA was detectable only in homozygous B-hFIX/F8 KO mice but not in wild-type mice.

Blood coagulation analysis in wild-type C57BL/6JNifdc mice, homozygous B-hFIX mice and homozygous B-hFIX/F8 KO mice. Plasma was collected from wild-type C57BL/6JNifdc mice (+/Y), homozygous B-hFIX mice (H/Y) and homozygous B-hFIX/F8 KO mice (H/Y, -/Y) (male, 8-week-old, n=6). No significant differences were observed in APTT (A), TT (B), or PT (C) values between homozygous B-hFIX mice and wild-type mice. However, homozygous B-hFIX/F8 KO mice exhibited a significantly prolonged APTT (A) compared to the homozygous B-hFIX mice, while their TT (B) and PT (C) values showed no significant changes. APTT: activated partial thromboplastin time, TT: Thrombin time, PT: prothrombin time. Values are expressed as mean ± SEM. Significance was determined by Ordinary one-way ANOVA, ns: non-significant, *p < 0.05, **p< 0.01, ***p < 0.001.