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Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017
[Construction of a human factor VIII gene-containing plasmid and its expression in Cos-7 cells]
1Department of Biochemistry, Laboratory of Molecular Biology, and Human Gene Therapy Center, Shanghai Second Medical University, Shanghai 200025.
Objective:
To construct an eukaryotic expressing plasmid--pAd CMV Link F VIII DB and express it in Cos-7 cells.
Methods:
An eukaryotic expressing plasmid--pAd CMV Link F VIII DB was constructed by inserting human factor VIII cDNA (F VIII DB, 4.6 kb), in which most part of B domain was deleted, into an adenovirus sequence-containing plasmid, and then Cos-7 cells were transfected with the constructed plasmid by liposome-mediated gene transfer method. F VIII DB mRNA, F VIII: Ag and F VIII: C in the transfected Cos-7 cells were assayed by RT-PCR, ELISA and one-stage method, respectively, at 24, 48 and 72 hours (hrs) after transfection.
Results:
F VIII DB mRNA was positive and, F VIII: Ag and F VIII: C were 18 ng/10(6) cells/24 hrs and 0.6 U/10(6) cells/24 hrs, respectively at 72 hrs after transfection, which was comparable to 60% of the activity produced by 100 micrograms/L F VIII in normal human plasma.
Conclusion:
The constructed plasmid is proved to be expressed in Cos-7 cells.
