Abstract
To determine the production of recombinant human protein C (rec-hPC) in milk, we created two homozygous mice lines for the goat β-casein/hPC transgene. Females and males of both lines (#10 and #11) displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg) mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands.
Highlights
Protein C, known as autoprothrombin IIα and blood coagulation factor XIV, is a zymogenic protein, which in its active form plays an important role in regulating blood clotting, inflammation, cell death, and maintaining blood vessel permeability in humans and other animals [1].Its structure is that of a two-chain polypeptide consisting of a light chain and a heavy chain connected by a disulfide bond
Sixty-one pups were born from 22 recipients that had received embryos microinjected with the pBC1-hPC construct
Four transgenic mice were identified by transgene-specific primers for genomic DNA, which was expected to produce 470 bp products (Figure 1)
Summary
Protein C, known as autoprothrombin IIα and blood coagulation factor XIV, is a zymogenic (inactive) protein, which in its active form plays an important role in regulating blood clotting, inflammation, cell death, and maintaining blood vessel permeability in humans and other animals [1].Its structure is that of a two-chain polypeptide consisting of a light chain and a heavy chain connected by a disulfide bond. Protein C, known as autoprothrombin IIα and blood coagulation factor XIV, is a zymogenic (inactive) protein, which in its active form plays an important role in regulating blood clotting, inflammation, cell death, and maintaining blood vessel permeability in humans and other animals [1]. C plays as an anticoagulant, those with deficiencies in protein C, or a resistance to APC, suffer from a significantly increased risk of forming dangerous blood clots (thrombosis). Transgenic animals that produce recombinant proteins in their milk can provide an economical and safe system for the production of medicinally valuable proteins [4,5]. Far, hPC Tg mice failed to lactate normally and were unable to raise litters [12]
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