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利用精子载体技术通过人工授精观察人凝血因子Ⅷ基因在转基因小鼠体内的表达。将含有人FⅧBD(B-domain deleted)cDNA的质粒pRC/RSV-hFⅧBD转染至小鼠精子,通过人工授精使母鼠受孕。新生小鼠出生后第4周,应用PCR筛查hFⅧBD cDNA阳性转基因幼鼠,取转有人FⅧBD cDNA幼鼠的血液检测血浆中的人FⅧ抗原和抗体,分别用Northern印迹和Western印迹检测脾、肝、肺和肾组织中人FⅧBD cDNA的转录和表达。结果发现,人工授精后20只受体母鼠7只受孕,共产下11只仔鼠,存活9只。PCR筛检出3只转有人FⅧBD cDNA的阳性鼠。3只阳性鼠血浆中的人FⅧ抗原量分别为8.65 ng/ml,7.84 ng/ml和8.44 ng/ml,人FⅧ的抗体均为阴性。Northern印迹和Western印迹检测显示3只F1代阳性幼鼠的脾、肝、肺和肾组织中均有人FⅧBD cDNA的转录和表达。结论提示,利用精子载体法可以制备转有人凝血因子Ⅷ基因的转基因小鼠,并表达人FⅧ蛋白,这为用精子载体技术生产转基因动物并将之作为生物反应器生产人凝血因子Ⅷ提供了实验依据。
The expression of human factor Ⅷ gene in transgenic mice was observed by artificial insemination using sperm vector technology. The plasmid pRC / RSV-hFⅧBD containing the human FⅧBD cDNA was transfected into mouse spermatozoa, and the female rats were fertilized by artificial insemination. At 4 weeks after birth, hFVIIIBD cDNA positive transgenic mice were screened by PCR. Human FⅧ antigen and antibody in plasma were obtained from the blood of the human FⅧBD cDNA vaccine. Northern blotting and Western blotting were used to detect the spleen and liver , Transcription and expression of human FⅧBD cDNA in lung and kidney tissues. The results showed that, after artificial insemination, only 20 recipient maternal 7 conceive, co-produce 11 offspring, survived 9. Three positive mice that were transfected with human F Ⅷ BD cDNA were screened by PCR. The plasma levels of human FⅧ antigen in the three positive mice were 8.65 ng / ml, 7.84 ng / ml and 8.44 ng / ml, respectively, and the antibody against human FⅧ was negative. Northern blotting and Western blotting showed that the F F1BD cDNA was expressed and expressed in the spleen, liver, lung and kidney of all 3 F1-positive pups. The results suggest that transgenic mice transfected with human factor Ⅷ gene can be prepared by sperm vector method and expressed human F Ⅷ protein. This provides an experiment for producing transgenic animals by sperm vector technology and producing human factor Ⅷ as a bioreactor in accordance with.