Transgenic Mice Can Express Mutant Human Coagulation Factor IX with Higher Level of Clotting Activity |
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Authors: | Jing-Bin Yan Shu Wang Wen-Ying Huang Yan-Ping Xiao Zhao-Rui Ren Shu-Zheng Huang Yi-Tao Zeng |
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Affiliation: | (1) Institute of Medical Genetics, Shanghai Jiao Tong University, 24/1400 West Beijing Road, Shanghai, 20040, P.R. China |
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Abstract: | To improve the available values of transgenic animals, we produced a mutant human coagulation factor IX minigene (including
cDNA and intron I) with arginine at 338 changed to alanine (R338A-hFIX) by using a direct mutation technique. The R338A-hFIX
minigene was then cloned into a plasmid carrying the goat β-casein promoter to get a mammary gland-specific expression vector.
The clotting activity in the supernatant of the transfected HC-11 cells increased to approximately three times more than that
of wild-type hFIX. Nine transgenic mice (three females and six males) were produced, and the copy number of the foreign gene
was very different, ranging from 1 to 43 in different lines. ELISA, Western blot, and clotting assay experiments showed that
the transgenic mice could express R338A-hFIX, showing higher average levels of clotting activity than wild-type hFIX in the
milk (103.76% vs. 49.95%). The highest concentration and clotting activity of hFIX reached 26 μg/mL and 1287% in one founder
(F0-7), which was over 10 times higher than that in human plasma. Furthermore, RT-PCR, APTT assay, and histological analysis
indicated that hFIX was expressed specifically in the mammary gland without affecting the intrinsic coagulation pathway and
physiologic performance of the local tissue. |
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Keywords: | mutant human coagulation factor IX transgenic mouse expression |
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