| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 99 === Hemophilia A is an X-linked, recessive bleeding disorder, which is caused by the defective human coagulation factor VIII (hFVIII). At the present time, Hemophilia A patients are treated with recombinant hFVIII produced by chinese hamster ovary (CHO) cells or baby hamster kidney (BHK) cells. However, it is very costly. In this study, we attempted to use zebrafish (Danio rerio) as a bioreactor to produce recombinant hFVIII because of short generation time, high fecundity, simple and cheap culture system, and easy manipulation of gene transfer. We constructed an expression plasmid, in which B domain-deleted hFVIII (hBDD-FVIII) was driven by either CMV promoter. After HEK293T cells were transfected with pCMV-BDFVIII, the total extracted proteins were subjected to western blot analysis using antiserum against hFVIII heavy chain. Results showed that there was a positive band located at 170 kDa, which was corresponding to the recombinant hBDD-FVIII protein. Other expression plasmids pHBDFVIII, pHBDFVIII-EGFP and pZaBDFVIII-EGFP were also constructed, in which either hBDD-FVIII or hBDD-FVIII-EGFP was driven by zebrafish hsp 70/4 promoter or zebrafish a-tubulin promoter, and microinjected into one-celled zebrafish embryos individually. In total, nine G0 lines harboring pHBDFVIII were generated. We extracted genomic DNA from F1 embryos and detected by PCR. A 547 bp PCR-product was amplified and corresponded with the amplification of partial hFVIII A3 domain from transgene. We also extracted total RNA from F1 embryos treated by heat-shock treatment and detected by RT-PCR. A 547 bp PCR-product was also amplified and corresponded with the amplification of transgene, suggesting that these lines can transcribe the recombinant B domain-deleted human factor VIII transgene after induction. Furthermore, the total proteins that were extracted from F1 embryos treated by heat-shock treatment were subjected to western blot analysis using antiserum against hFVIII heavy chain. However, results showed that there was no signal corresponding to the recombinant hBDD-FVIII protein. It suggested that these lines can not translate the recombinant B domain-deleted human factor VIII protein after induction.
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