Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells*24?hrs-1, and the FⅧ antigen was 500?ng/106 cells*24?hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12?U/106 cells*24?hrs-1 and 62.4?ng/106 cells*24?hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells.Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.
Objective To establish an effective laboratory examination system for carrier detection and prenatal diagnosis of haemophilia A (HA) with a variety of molecular biological methods which are simple, rapid and easy to use. Methods Detection of inversion involving intron 22 in the FVIII gene was completed by long distance polymerase chain reaction ( PCR) and linkage analysis was performed by using several genetic polymorphisms including an intragenic Bcl I RFLP, 2 STRs and an extragenic St14 VNTR. Results Intron 22 inversion was observed in 10 out of the 21 (47.6%) pedigrees examined. Prenatal diagnosis was completed in 3 pedigrees. A further combination of the four intragenic and extragenic polymophic loci gave an informative rate of 94.7%. Conclusions Female relatives in HA families with inversion can be detected with direct diagnostic procedure. The application of long distance PCR makes the detection much more simple and rapid. For families without inversions, it is easier and more cost-effective to undertake linkage analysis of genetic polymorphism based on PCR.
