Abstract
The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100--1800 μg ml−1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100--400 μg ml−1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein
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References
Anon. (1995) Points to consider in the manufacture and testing of therapeutic products for human use derived from transgenic animals, Docket No. 95D-0131. Center for Biologics Evaluation and Research, US Food and Drug Administration.
Brettler, D.B., Forsberg, A.D., Levine, P.H., Aledort, L.M., Hilgartner, M.W., Kasper, C.K., Lusher, J.M., McMillan, C., Roberts, H., Watson-Williams, E.J., Thomas, P., Stafford, B., Czapek, E., Goldsmith, J., Harkins, G., Friend, S., Kitchens, C., Garcia, A., Waddell, C., Goodnight, S., Pasquale, D., Luper, W., Hathaway, W., Jones, R.L. (1989) The use of porcine factor VIII concentrate (Hyate:C) in the treatment of patients with inhibitor antibodies to factor VIII. Arch. Intern. Med., 149, 1381-85.
Burdon, T., Sankaran, L., Wall, R., Spencer, M. and Hennighausen, L. (1991) Expression of a whey acidic protein transgene during mammary development. J. Biol. Chem., 266, 6909- 914.
Carver, A.S., Dalrymple, M.A., Wright, G., Cottom, D.S., Reeves, D.B., Gibson, Y.H., Keenan, J.L., Barrass, J.B., Scott, A.R., Colman, A., and Garner, I. (1993) Transgenic livestock as bioreactors: stable expression of human alpha-1antitrypsin by a flock of sheep. Bio/Technology 11, 1263-70.
Colman, A. (1995) Production of vitamin K-dependent protein in the milk of various transgenic animals. IBC Third International Symposium on Exploiting Transgenic Technology for Commercial Development, San Diego, CA.
Dawson, M., Wells, G.A.H., Parker, B.N.J. and Scott, A.C. (1990) Primary parenteral transmission of bovine spongiform encephalopathy to the pig. Vet. Rec., 126, 338.
Degener, A., Belew, M. and Velander, W.H. (1996) Expanded bed purification of a recombinant protein from the milk of transgenic livestock. American Chemical Society, 211th National Meeting, 24-28 March 1996, New Orleans, LA (abstract).
Drews, R., Paleyanda, R.K., Lee, T.K., Chang, R.R., Rehemtulla, A., Kaufman, R.J., Drohan, W.N. and Lubon, H. (1995) Proteolytic maturation of protein C upon engineering the mouse mammary gland to express furin. Proc. Natl Acad. Sci. USA 92, 10462-6.
Drohan, W.N., Zhang, D.-W., Paleyanda, R.K., Chang, R., Wroble, M., Velander, W. and Lubon, H. (1994) Inefficient processing of human protein C in the mouse mammary gland. Transgenic Res. 3, 355-64.
Fodor, W.L., Williams, B.L., Matis, L.A., Madri, J.A., Rollins, S.A., Knight, J.W., Velander, W. and Squinto, S.P. (1994) Expression of a functional human complement inhibitor in a transgenic pig as a model for the prevention of xenogeneic hyperacute organ rejection. Proc. Natl Acad. Sci. USA 91, 11153-57.
Gatti, L. and Mannucci, P.M. (1984) Use of porcine factor VIII in the management of seventeen patients with factor VIII antibodies. Thromb. Haemostas (Stuttgart). 51, 379-84.
Heeb, M.J., Schwarz, H.P., White, T., Lammle, B., Berrettini, M. and Griffin, J.H. (1988) Immunoblotting studies of the molecular forms of protein C in plasma. Thrombosis Res. 52, 33-43.
Joklik, W.K., Willett, H.P., Amos, D.B. and Wilfert, C.M. (1988) Zinsser Microbiology. Appleton and Lange, Norwalk, pp. 648-658.
Lantz, G.C., Badylak, S.F., Hiles, M.C., Coffey, A.C., Geddes, L.A., Kokini, K., Sandusky, G.E. and Morff, R.J. (1993) Small intestinal submucosa as a vascular graft: a review. J. Invest. Surgery. 6, 297-310.
Lee, T. K., Drohan, W.N. and Lubon, H. (1995) Proteolytic processing of human protein C in swine mammary gland. J. Biochem. 118, 81-87.
Lollar, P., Parker, C.G. and Tracy, R.P. (1988) Molecular characterization of commercial porcine factor VIII concentrate. Blood 71, 137-43.
Lubon, H. and Hennighausen, L. (1987) Nuclear proteins from lactating mammary glands bind to the promoter of a milk protein gene. Nucl. Acids Res. 15, 2103-21.
Lubon, H., Paleyanda, R.K., Velander, W.H. and Drohan, W.N. (1996) Blood proteins from transgenic animal bioreactors. Transfusion Medicine Revs 10, 131-43.
McKnight, R., Wall, R. and Hennighausen, L. (1995) Expression of genomic and cDNA transgenes after co-integration in transgenic mice. Transgenic Res., 4, 39-43.
Pinkert, C.A., Galbreath, E.J., Yang, C.W. and Striker, L.J. (1994) Liver, renal, and subcutaneous histopathology in PEPCK-bGH transgenic pigs. Transgenic Res. 3, 401-5.
Pittius, C.W., Hennighausen, L., Lee, E., Westphal, H., Nicols, E., Vitale, J. and Gordon, K. (1988) A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice. Proc. Natl Acad. Sci. USA 85, 5874-8.
Pursel, V.G., Pinkert, C.A., Miller, K.F., Bolt, D.J., Campbell, R.G., Palmiter, R.D., Brinster, R.L. and Hammer, R.E. (1989) Genetic engineering of livestock. Science 244, 1281-88.
Pursel, V.G. and Rexroad, C.B. (1993) Recent progress in the transgenic modification of swine and sheep. Molecular Reprod. Development 36, 251-4.
Sandusky, G.E., Badylak, S.F., Morff, R.J., Johnson, W.D. and Lantz, G. (1992) Histologic findings after in vivoplacement of small intestine submucosal vascular grafts and saphenous vein grafts in the carotid artery in dogs. Am. J. Pathology 140, 317-24.
Shamay, A., Solinas, S., Pursel, V.G., McKnight, R.A., Alexander, L., Beattie, C., Hennighausen, L. and Wall, R.J. (1991) Production of the mouse whey acidic protein in transgenic pigs during lactation. J. Anim. Sci. 69, 4552-62.
Shamay, A., Pursel, B.G., Wall, R.J. and Hennighausen, L. (1992) Induction of lactogenesis in transgenic virgin pigs: evidence for gene and integration site-specific hormonal regulation. Mol. Endocrinology 6, 191-7.
Sharma, A., Martin, M.J., Okabe, J.F. et al.(1994) An isologous promoter permits high level expression of human hemoglobin in transgenic swine. Bio/Technology 12, 55-9.
Van Cott, K.E., Williams, B.L., Gwazdauskas, F., Lee, T.K., Lubon, H., Drohan, W.N. and Velander, W.H. (1997) Affinity purification of biologically active and inactive forms of recombinant human protein C produced in the porcine mammary gland. J. Molecular Recognition, 9, 407-14.
Velander, W.H., Johnson, J.L., Page, R.L., Russell, C.G., Subramanian, A., Wilkins, T.D., Gwazdauskas, F.C., Pittius, C. and Drohan, W.N. (1992a) High-level expression of a heterologous protein in the milk of transgenic swine using the cDNA encoding human protein C. Proc. Natl Acad. Sci. USA 89, 12003-7.
Velander, W.H., Page, R.L., Morcol, T.M., Russell, C.G., Canseco, R., Young, J., Drohan, W.N., Gwazdauskas, F.C., Wilkins, T.D. and Johnson, J.L. (1992b) Production of biologically active human protein C in the milk of transgenic mice. Annals NY Acad. Sci. 665, 391-403.
Wall, R.J., Pursel, V.G., Shamay, A., McKnight, R.A., Pittius, C.W. and Hennighausen, L. (1991) High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine. Proc. Natl Acad. Sci. USA 88, 1696-1700.
Wall, R.J., Hawk, H.W. and Nel, N. (1992) Making transgenic livestock: genetic engineering on a large scale. J. Cellular Biochem. 49, 113-20.
Wattanavijarn, W., Tesprateep, T. and Burke, D.S. (1985) Absence of porcine parvovirus transmission to man. Transactions Royal Soc. Tropical Med. Hygiene 79, Correspondence.
Weiss, R., Teich, N., Varmus, H. and Coffin, J. (1984) RNA Tumor Viruses. Cold Spring Harbor Laboratory. New York. pp. 120- 121.
Whittemore, C. (1993) The Science and Practice of Pig Production. Longman Scientific and Technical, UK.
Wilmut, I., Archibald, A.L., Harris, S., McClenaghan, M., Simons, J.P., Whitelaw, C.B.A. and Clark, A.J. (1990) Modification of milk composition. J. Reprod. Fert., Suppl. 41, 135-46.
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Van Cott, K.E., Lubon, H., Russell, C.G. et al. Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C. Transgenic Res 6, 203–212 (1997). https://doi.org/10.1023/A:1018442124584
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DOI: https://doi.org/10.1023/A:1018442124584