Study On The Specific Expression Of Human Recombinant Coagulation Factor ? In Mouse Mammary Glands

It is well known that mammary gland bioreactor has many advantages,such as remarkable activity,high yields,easy collection and less pollution.Generating transgenic mammary gland bioreactor to produce medical protein drugs is one of the hottest scientific studies all over the world now.Human coagulation factor ?(F ?)is an effective drug for the prevention and treatment of hemophilia A.Nowadays,the F ?drug products made in China are mainly extracted from plasma.While recombinant coagulation F? can only be bought from the international markets.The aim of this study is trying to generate transgenic mice,which could express recombinant human coagulation F? by microinjecting two different mammary gland specific expression vectors(?-casein promoter and aSl-casein promoter)into embryos.Then the expression feature of two different vectors will be examined.Mammary gland specific expression vectors were amplified in E.coli cells.Expression fragments were digested from vectors and then purified and filtrated by kits.The pBCl-rhFV? vector was digested by Sal ?and Not ?,and the 807-1 plasmid was digested by Not I.Then,these two fragments were injected into embryo pronuclear by microinjection manipulation separately.Injected embryos were transplanted into pseudopregnant surrogate mothers.Offspring were delivered at term development.Transgenic mice were selected by PCR amplication.The next two generations of transgenic mice were produced from that founder mice mated with normal mice.The milk of positive transgenic mice(F0)and their offspring was analyzed by Enzyme-linked immunosorbent assay(ELISA)and Western blot.454 embryos were injected with pBCl-rhF? fragment and of which 409 were transferred into recipients.95 mice were born and 12 were detected existing transgenic vector by PCR amplication.705 embryos were injected with 807-1 vector and of which 535 were transferred into recipients.124 mice were born and 9 were detected existing transgenic vector by PCR amplication.The integration rates of pBC1-rhF? and 807-1 were 12.6%and 7.3%.Milk from F01 mice was analyzed by ELISA,and the two types of the transgenic mice both expressed rFV?.Average expression level was 0.22 IU/mL in four pBCl-rhF?I F0 mice,and 0.19 IU/mL in three 807-1 F0 mice.The expression of rF? was confirmed by Western Blot.In conclusion,transgenic mice can be generated with these two expression vectors.By the direction of mammary gland-specific expression regulatory elements in vectors,rF? can express specifically in mammary gland.