Generation Of Recombinant HFâ…§ Transgenic Rabbit And Preliminary Identification

Human factor Ⅷ gene located in the remote end of X chromosome long arm (Xq28), is186Kb long, which accounts for0.1%of the length of X chromosome, contains26exons (9kb) and25introns (177kb). After splicing, FⅧ mRNA is approximately9kb long, which is an open reading frame coding for a polypeptide of2351amino acids. FⅧ gene is enormous, with various gene mutation and highly heterogeneity.Hemophilia A (HA) is a hereditary hemorrhagic disease, due to hereditary defects or genetic mutations of the FⅧ, which causes serious blood clotting disorders of the patients. Patients often can’t stop bleeding, no matter which is idiopathic or after a trauma. It can happen at any position, but the most frequent location is the joint. Repeated joint bleeding can lead to disability or even the death of patients. This disease is a concatenate recessive inherited disease of X sex chromosome. The incidence rate in men is from1/5000to1/10,000. At present, the treatment of patients mainly relies on the injection of fresh blood or FⅧ concentrated preparation. However, due to the rare resource and high price, it cannot meet the needs of HA patients. In1984, the success of FⅧ gene extracorporeal expression demonstrates a large quantity of rFⅧ with high purity can be obtained through biological technology, serving as the substitute of FⅧ for clinical uses.A2.0-kb promoter sequence of bovine alpha-lactalbumin (aLA) was generated by polymerase chain reacion (PCR) amplification using a genomic DNA from high milk-producing Holstein cow, which obtained from National Taiwan University Farm, as the template.This PCR product containing entire aLA promoter and15-aa signal peptide sequence. This promoter and signal peptide sequences was then subsequently inserted into the T-protruding pCR3vector.For the full-length hFⅧ construction, the resulting7.0-kb plasmid containing aLA promoter and its intact signal peptide sequence was double digested with Mlul and PstI and treated with calf intestinal phosphatase. The pCMV5/hFVⅧ plasmid containing the intact human FⅧ coding sequence was also double digested with MluI and PstI. The9.7-kb transgene consisting of2.0-kb bovine aLA promoter,7.2-kb FⅧ cDNA, and0.5-kb bovine GH gene polyadenosine signal sequence was separated from plasmid pCR-aLA/FⅧ-5using Clal and XbaI digestion and purified for micro injection with QIAEX gel extration kit.For the B domain-deleted FⅧ construction, the resulting7.0-kb plasmid containing aLA promoter and its signal peptide sequence was double digested with HpaI and Xhol and treated with calf intestinal phosphatase. The pCMV5/FⅧ plasmid containing the intact human FⅧ coding sequence was used as template to generate a2233-bp rhFⅧ A-domain fragment using PCR, and a2085-bp rhFⅧ C-domain fragment respectively. Equal molar ratio of A-domain segment, C-domain segment and pCR3-aLA vector were co-ligated and transformed into host competent cells. The6.8-kb transgene consisting of2.0-kb bovine aLA promoter and aS1-casein leader sequence,4.3-kb hFⅧ cDNA, and0.5-kb bovine GH gene polyadenosine signal sequence was separated from plasmid pCR-aLA/hFⅧ(△B) using ClaI and XbaI digestion and purified for microinjection with QIAEX gel extration kit. PCR, restriction endonuclease analysis and DNA sequencing were used to identify the recombinant plasmids. The results indicated that the fragments were successfully inserted.Recombinant plasmid pCR-aLA/hFⅧ (△B) is supposed to be digested by restriction enzyme ClaI and XbaI through which gene segment with6.8kb degree of purity are recycled. It includes2.0kb aLA promoter4.3kb hFⅧ (△B) gene and0.5kb bovin somatotropin poly A. Applying FSH and HCG on the test of6-9months superovulated female New Zealand White (NZW) rabbits, they are found that after mating with males NZW rabbits,754fertilized eggs are obtained. aLA-hFⅧ (△B)-bGHpA segments are injected into the male pronucleus of fertilized eggs through pronucleus micro injection. After injection, the number of transplantation eggs is328, the number of receptors is13. And there are12pregnant rabbits giving birth to52pups. Through PCR detection, there are four transgenic positive rabbits. Since the transgenic positive rabbits are only three months, they have no offspring.