Transgenic mice can express mutant human coagulation factor IX with higher level of clotting activity.
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 beta-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 mug/mL and 1287% in one founder (F(0)-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.
Yan
Jing-Bin
JB
Institute of Medical Genetics, Shanghai Jiao Tong University, 24/1400 West Beijing Road, Shanghai, PR China.
Wang
Shu
S
Huang
Wen-Ying
WY
Xiao
Yan-Ping
YP
Ren
Zhao-Rui
ZR
Huang
Shu-Zheng
SZ
Zeng
Yi-Tao
YT
D000818:Animals; D001483:Base Sequence; D002364:Caseins / Q000235:genetics; D002460:Cell Line; D005164:Factor IX / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods; D006041:Goats; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008321:Mammary Glands, Animal; D051379:Mice; D008822:Mice, Transgenic; D008892:Milk / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D014914:Whole Blood Coagulation Time
D002364:Caseins; D005164:Factor IX