Research Paper | Published:

Transgenic Production of a Variant of Human Tissue-Type Plasminogen Activator in Goat Milk: Generation of Transgenic Goats and Analysis of Expression

Abstract

We report the first successful production of transgenic goats that express a heterologous protein in their milk. The production of a glycosylation variant of human tPA (LAtPA - longer acting tissue plasminogen activator) from an expression vector containing the murine whey acid promoter (WAP) operatively linked to the cDNA of a modified version of human tPA was examined in transgenic dairy goats. Two transgenic goats were identified from 29 animals born. The first animal, a female, was mated and allowed to carry the pregnancy to term. Milk was obtained upon parturition and was shown to contain enzymatically active LAtPA at a concentration of 3 μg/ml.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Hennighausen, L. 1990. The mammary gland as a bioreactor: production of foreign proteins in milk. Protein Expression and Purification 1: 3–8.

  2. 2

    Ebert, K.M. and Selgrath, J.P. 1991. Changes in domestic livestock through genetic engineering. In: Applications of Basic Research in Mammalian Development. Pedersen, R. A., McLaren, A. and First, N. (Eds.). Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

  3. 3

    Gordon, K., Lee, E., Vitale, J.A., Smith, A.E., Westphal, H. and Henninghausen, L. 1987. Production of human tissue plasminogen activator in transgenic mice. Bio/Technology 5: 1183–1187.

  4. 4

    Pittius, C.W., Henninghausen, 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: 5874–5878.

  5. 5

    Clark, A.J., Bessos, H., Bishop, J.O., Brown, P., Harris, S., Lathe, R., McClenaghan, M., Prowse, C., Simons, J.P., Whitelaw, C.B.A. and Wilmut, I. 1989. Expression of human anti-hemophilic factor IX in the milk of transgenic sheep. Bio/Technology 7: 487–492.

  6. 6

    Yu, S.H., Deen, K.C., Lee, E., Hennighausen, L., Sweet, R.W., Rosenberg, M. and Westphal, H. 1989. Functional human CD4 protein produced in milk of transgenic mice. Mol. Biol. Med. 6: 255–261.

  7. 7

    Buhler, Th.A., Bruyere, Th., Went, D.F., Stranzinger, G. and Burki, K. 1990. Rabbit β-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits. Bio/Technology 8: 140–143.

  8. 8

    Archibald, A.L., McClenaghan, M., Hornsey, V., Simons, J.P. and Clark, A.J. 1990. High-level expression of biologically active human α1-antitrypsin in the milk of transgenic mice. Proc. Natl. Acad. Sci. USA 87: 5178–5182.

  9. 9

    Meade, H., Gates, L., Lacy, E. and Lonberg, N. 1990. Bovine alpha S1-casein gene sequences direct high level expression of active human urokinase in mouse milk. Bio/Technology 8: 443–446.

  10. 10

    Wall, R.J., Pursel, V.G., Shamay, A., McKnight, R.A., Pittius, C.W. and Henninghausen, 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.

  11. 11

    Simos, E., Voutsinas, L.P. and Pappas, C.P. 1991. Composition of milk of native Greek goats in the region of Metsovo. Small Ruminant Research 4: 47–60.

  12. 12

    Lau, D., Kuzma, G., Wei, C.M., Livingstron, D.J. and Hsiung, N. 1987. A modified human tissue plasminogen activator with extended half-life in vivo. Bio/Technology 5: 953–958.

  13. 13

    Selgrath, J.P., Memon, M.A., Smith, T.E. and Ebert, K.M. 1990. Collection and transfer of microinjectable embryos from dairy goats. Theriogenology 34: 1195–1205.

  14. 14

    Cuthbertson, R.A. and Klintworth, G.K. 1988. Biology of Disease: Transgenic mice - a gold mine for furthering knowledge in pathobiology. Laboratory Investigation 58: 484–502.

  15. 15

    Ebert, K.M., Smith, T.E., Buonomo, F.C., Overstrom, E.W. and Low, M.J. 1990. Porcine growth hormone gene expression from viral promoters in transgenic swine. Animal Biotechnology 1: 145–159.

  16. 16

    Hammer, R.E., Pursel, V.G., Rexroad, C.E., Wall, R.J., Bolt, D.J., Ebert, K.M., Palmiter, R.D. and Brinster, R.L. 1985. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315: 680–683.

  17. 17

    Feinberg, A. and Vogelstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 72: 248–254.

  18. 18

    Thomas, P. 1980. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. 77: 5201–5205.

  19. 19

    Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Further reading