Pharming B.v.

Other

Universiteit Leiden

Genpharm International, Inc.

Academic Hospital

Eramus Universiteit

Frank R Pieper

Rein Strijker

Paul J Krimpenfort

Herbert L Heyneker

Sang He Lee

Gerard Platenburg

Herman A DeBoer

Robert M Kay

Anton J Berns

Richard A Berg

Gerard Johannes Platenburg

Costas N Karatzas

Paul David Toman

Herman A Deboer

Ineke De Wit

Martin Ph Verbeet

Ans T Van Der Ploeg

Arnold J Reuser

The invention provides transgenic nonhuman mammals capable secreting exogenous procollagen or collagen into their milk. The mammals are healthy and capable of producing procollagen or collagen at high levels, usually in trimeric form. Suitable transgenes for incorporation into the mammals are also provided.

Production of collagen in the milk of transgenic mammals

Transgenes for producing recombinant polypeptides transgenic bovine species. A transgene for producing recombinant polypeptides in the milk of transgenic bovine species comprises at least one expression regulation sequence, a secretory DNA sequence encoding a secretory signal sequence which is functional in mammary secretory cells of the bovine species and a recombinant DNA sequence encoding the recombinant polypeptide. Also included are methods for producing transgenic bovine species. The method includes introducing the above transgene into an embryonal target cell of a bovine species, transplanting the transgenic embryonic target cell formed thereby into a recipient bovine parent and identifying at least one female offspring which is capable of producing the recombinant polypeptide in its milk. The invention also includes transgenic bovine species capable of producing recombinant polypeptides in transgenic milk as well as the milk from such transgenic bovine species and food formulations containing one or more recombinant polypeptide. Methods are also provided for producing transgenic non-human mammals having a desirable phenotype. The method comprises first methylating a transgene followed by introduction into fertilized oocytes. The oocytes are then cultured to form pre-implantation embryos. Thereafter, at least one cell is removed from each of the pre-implantation embryos and the DNA digested with a restriction endonuclease capable of cleaving the methylated transgene but incapable of cleaving the unmethylated form of the transgene. Those pre-implantation embryos which have integrated the transgene contain DNA which is resistant to cleavage by the restriction endonuclease in the region containing the transgene.

Production of recombinant polypeptides by bovine species and transgenic

The invention provides transgenic nonhuman mammals producing phosphorylated lysosomal proteins in their milk, and methods of generating the same. Phosphorylation occurs at the 6' position of a mannose side chain residue. Also provided are methods of purifying lysosomal proteins from milk, and incorporating the proteins into pharmaceutical compositions for use in enzyme replacement therapy.

Lysosomal proteins produced in the milk of transgenic animals

Transgenes for producing recombinant polypeptides transgenic bovine species are described. A transgene for producing recombinant polypeptides in the milk of transgenic bovine species comprises at least one expression regulation sequence, a secretory DNA sequence encoding a secretory signal sequence which is functional in mammary secretory cells of the bovine species and a recombinant DNA sequence encoding the recombinant polypeptide. Also included are methods for producing transgenic bovine species. The method includes introducing the above transgene into an embryonal target cell of a bovine species, transplanting the transgenic embryonic target cell formed thereby into a recipient bovine parent and identifying at least one female offspring which is capable of producing the recombinant polypeptide in its milk. The invention also includes transgenic bovine species capable of producing recombinant polypeptides in transgenic milk as well as the milk from such transgenic bovine species and food formulations containing one or more recombinant polypeptide.

Transgenic bovines and milk from transgenic bovines

A transgenic bovine is disclosed whose somatic and germ cells contain a transgene, wherein the transgene comprising a mammary gland specific promoter, a mammary gland specific enhancer, a DNA sequence encoding a signal sequence functional in bovine mammary gland secretory cells and a DNA sequence encoding a heterologous polypeptide of interest wherein the transgenic bovine expresses the transgene such that the polypeptide of interest is detectable in milk produced by the transgenic bovine.

Transgenic bovine

The invention relates to methods for intracellularly producing DNA segments by homologous recombination of smaller overlapping DNA fragments and transgenic mammalian cells and transgenic non-human mammals produced by such methods.

Homologous recombination in mammalian cells

A method is disclosed for the production of a transgenic bovine or a transgenic bovine embryo comprising obtaining an ovum from bovine ovaries, maturing the ovum in vitro, fertilizing the mature ovum or ova in vitro to form a zygote, introducing a transgene into the zygote in vitro and maturing the zygote to a preimplantation stage embryo in vitro. To produce the transgenic bovine, the embryo is transplanted into a recipient female bovine, wherein the female bovine gestates the embryo to produce a transgenic bovine.

Method of producing a transgenic bovine or transgenic bovine embryo

Growing community of inventors

Utrecht, Netherlands

Frank R Pieper