Hemophilia is a group of genetic bleeding disorders that affect the blood’s ability to clot. The common feature of this disease is the generation of abnormal clotting factors, which leads to prolonged clotting time and increased risk of bleeding after minor injuries. In severe cases, spontaneous bleeding can occur even without obvious trauma. As an X-linked recessive disorder, Hemophilia B is more common in males, with approximately 1 in every 30,000 newborn males worldwide being affected ^[1]. Hemophilia B is caused by mutations in the F9 (FIX) gene, which leads to a deficiency of clotting factor IX. The severity of the disease is usually correlated with the activity level of factor IX in the blood plasma. Mild patients (IX factor activity >5%, >0.05 IU/mL) do not experience spontaneous bleeding, but the amount of bleeding after injury or surgery may increase. Moderate patients (IX factor activity 1%-5%, 0.01-0.05 IU/mL) rarely experience spontaneous bleeding, but even minor injuries can cause prolonged bleeding. Severe patients (IX factor activity <1%, <0.01 IU/mL) experience spontaneous bleeding, soft tissue or joint bleeding, and severe subcutaneous hematomas ^[2]. According to the Centers for Disease Control and Prevention (CDC) in the United States, severe Hemophilia B patients account for 30%-40% of all diagnosed patients ^[3]. The F9 gene encodes coagulation factor IX, a vitamin K-dependent serine protease that plays a key role in the intrinsic coagulation pathway. Factor IX circulates in the blood as an inactive zymogen and is converted to its active form, factor IXa, by the cleavage of its activation peptide by factor XIa. Factor IXa then interacts with Ca2+ ions, membrane phospholipids, and factor Ⅷ to activate factor X in the coagulation cascade. The body can normally stop bleeding when the levels of factors Ⅷ and IX are ≥50% of normal values ^[4]. The deficiency of the F9 gene can lead to a clotting disorder with insufficient factor IX, causing X-linked recessive hemophilia B. F9 KO mice are Hemophilia B disease models constructed by knocking out the mouse F9 gene. F9 KO mice lack F9 mRNA expression and exhibit coagulation dysfunction and other Hemophilia B-related phenotypes. They can be used to study the genetic mechanisms and clinical phenotypes of Hemophilia B in humans and to assist in developing, screening, and evaluating therapeutic drugs. The homozygotes are viable and fertile. Tail docking may lead to significant bleeding. Immediate hemostasis, such as cauterizing the tail incision, is advised to prevent health complications in homozygous mice. Usually, ear tags are applied to mice at 2 to 3 weeks of age (a small notch is made with scissors for identification). After tail clipping for genotyping, the tail wound should be promptly cauterized (using metal forceps heated with an alcohol lamp) to prevent fatal bleeding. Following cauterization, place the mouse in a clean cage to prevent wound infection and add environmental enrichment.

Hemophilia is a group of inherited bleeding disorders primarily caused by deficiency or dysfunction of coagulation factor VIII or IX, leading to impaired coagulation. Patients typically present with prolonged clotting time, easy bleeding even after minor trauma, and in severe cases, spontaneous bleeding, commonly occurring in joints and deep tissues. Hemophilia is mainly classified into three types: type A (factor VIII deficiency), type B (factor IX deficiency), and type C (factor XI deficiency). Among these, types A and B are the most prevalent. Hemophilia A is caused by mutations in the F8 gene, resulting in factor VIII deficiency, while hemophilia B is caused by mutations in the F9 gene, leading to factor IX deficiency ^[1]. Coagulation factor IX, encoded by the F9 gene, is activated to FIXa during coagulation and works in concert with FVIIIa, Ca2+, and membrane phospholipids to activate factor X. Hemophilia A and B are both X-linked recessive genetic disorders with a higher incidence in males. The incidence of hemophilia B is approximately 1/25,000 to 1/30,000, accounting for about 15%-20% of all hemophilia cases ^[2]. Currently, coagulation factor replacement therapy is the primary treatment for hemophilia ^[2]. For hemophilia A, the treatment is intravenous injection of factor VIII concentrates; for hemophilia B, factor IX concentrates are injected to maintain normal levels of coagulation factors in patients. However, this therapy is a supplementary approach, requiring lifelong regular injections, which may not only cause side effects but also impose a substantial economic burden on patients. Therefore, gene therapy, particularly for hemophilia B, is considered a highly promising research direction. Etranacogene dezaparvovec (brand name Hemgenix) is the first gene therapy for hemophilia B approved by the U.S. FDA ^[3-4]. This therapy utilizes adeno-associated virus vector AAV5 to deliver the coagulation factor IX gene to patient hepatocytes, thereby increasing FIX activity in vivo and reducing bleeding events ^[3]. Gene therapy is considered a potential curative approach for hemophilia B. Considering the genetic differences between animals and humans, and that most gene therapies target human genes, humanizing mouse genes will help accelerate the drug pipeline of gene therapies into the clinical stage. This strain is a humanized mouse F9 gene model, which can be used for preclinical evaluation of hemophilia B pathogenesis and therapeutic drugs. Homozygotes of this model are viable and fertile. In addition, based on the independently developed TurboKnockout fusion BAC recombination technology innovation, Cyagen can also provide disease models of popular point mutations based on this model, and can also provide customized services for different point mutations to meet the experimental needs of researchers in the pharmacodynamics of hemophilia B.

Gabra4 is located on chromosome 5 of mice. Nuclease Technology will be used to design sgRNA; Gabra4 knockout mice will be obtained by applying high-throughput electroporation of fertilized eggs. After sexual maturity, sperm were collected for cryopreservation.

Efnb3 is located on chromosome 11 of mice. SgRNA and ssDNA will be designed using Nuclease Technology; Efnb3 conditional knockout mice will be obtained by high-throughput electroporation of fertilized eggs. After sexual maturity, sperm will be collected for cryopreservation.