Establishment Of A Hemophilia B Transgenic Mouse Model On The Basis Of Coagulation Factor Ⅸ Gene Knock-out Mouse

Hemophilia B is a good model for studying gene therapy.Currently,the dog model carrying spontaneous mutation of the factor IX gene and a series of mouse models are available for studying hemophilia B.These models were established with deletions of the factor IX gene.However,the molecular basis of most of hemophilia B patients are point mutations.lt is necessary to establish an apparently faithful model of hemophilia B with point mutation for studying strcture-funtion relationships and gene therapy of human factor IX containing point mutation in vivo.Barrel W.Stafford et al. (1997) generated a murine model of factor IX deficiency in which the endogeous mouse gene encoding factor IX was disrupted from the promoter to the exon 3 by homologous recombination. According to the origin reference,the mouse strain were tested on diffirent levels.The mice offsprings were screened with PCR,RT-PCR and confirmed with Southern blotting on genomic DNA.Clotting activities of the factor IX-knockout mice were measured using plasma from blood withdrawn from the retro-orbital area of anesthetized mice.Based on the defect in the factor IX gene,the lack of factor factor IX transcript and the physical characteristics of the mice,we conclude that the gene structures of mouse strain are stable.The mice strain are a model for hemophlia B.On the basis of this factor IX gene knock-out mouse model,a series of mutations were introduced into the human coagulation factor IX minigene expressing vectors(pMe4bAIXml plasmid),then two of them were selected and transmitted into mouse by pronuclear microinjection. In patients with R338A mutation,the plasma factor IX coagulant activities were there times normal. The pMe4bAIXml-l vector was linearized and injected into 623 fertilized eggsof factor IX gene knock-out mice. The manipulated embryos were transferred into the oviducts of 26 pseudopregnant females respectively,from which 28 offsprings were obtained.None of their genomes stably integrated with copies of the intact pMe4bAIXml-l plasmid. In patients with C361R point mutation,the plasma factor IX coagulant activities and antigen levels were less than 1% of normal. The pMe4bAIXml-2 vector was linearized and injected into 817 fertilized eggs of factor IX gene knock-out mice. The manipulated embryos were transferred into the oviducts of 45 pseudopregnant females respectively,from which 69 offsprings were obtained. The genomic DNAs of these offsprings were analyzed with PCR and genomic Southern blotting. 6 mice(including 1 dead mouse) whose genomes stably integrated with copies of the intact pMe4bAIXml-2 plasmid were obtained and 5 mice were chosen as founders to establish transgenic mouse strain. With analysis of total RNA in factor IX-knockout and transgenic mice by RT-PCR,4 transgenic mice were found who had transcription products.By breeding progress,the mouse model containing the specific point mutation on the basis of factor IX gene knock-out mouse was established.lt could be a more efficient animal model for hemophilia B and for investigating structure-funtion relationships of coagulation factor IX gene.