Transgenic ungulate compositions and methods

• US 7,071,373 B1
• Filed: 03/24/1995
• Issued: 07/04/2006
• Est. Priority Date: 05/14/1993
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Board of Trustees of The University of Illinois
• Inventors: Wheeler, Matthew B.
• Primary Examiner(s): Falk, Anne-Marie
• Assistant Examiner(s): Ton, Thaian N.
• Application Number:
  US08/410,539
• Time in Patent Office: 4,120 Days
• Field of Search: 800/2, 800/DIG.1, 800/DIG.4, 800/24, 800/8, 800/14, 800/4, 435/172.3, 435/325, 435/378, 935/63, 935/70
• US Class Current: 800/24
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2227/102 Caprine
• CPC Class Codes: A01K 2227/103 Ovine
• CPC Class Codes: A01K 2227/108 Swine
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 2502/14 hepatocytes
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696

Abstract

Transgenic ungulates and compositions and methods for making and using same, are provided. Central to the invention are porcine, bovine, ovine and caprine embryonic stem cell lines and methods for establishing them. Cells of such lines are transformed with exogenous genetic material of interest and then used to provide chimeric ungulates confirmed by genetic markers which have germ cells comprising the exogenous genetic material. The chimeric ungulates are bred to provide transgenic ungulates. The transgenic animals of the invention may show improved qualities and can be used to provide human proteins or peptide hormones or can be used as xenograft donors.

Claims

1. A method for making a chimeric ungulate comprising:

(a) introducing at least one a cultured ungulate embryonic stem cell from a cultured ungulate embryonic stem cell line, wherein said cells grow in multilayers and wherein said embryonic stem cells have a rounded shape as observed with the light microscope, a diameter of approximately 8–

15 microns, and a cytoplasmic to nuclear diameter ratio of approximately 10–

25;

75–

90 as show in FIG. 2, wherein the ratio is approximated by the cytoplasmic diameter from the periphery of the nucleus to the end of the cell, to the diameter of the nucleus, and said cell has a first genetic complement, into a recipient embryo of the same species as the embryonic stem cell, said recipient embryo having a second genetic complement which differs from the first genetic complement, in at least one gene, to form a chimeric ungulate embryo; and

(b) placing the chimeric ungulate embryo in an environment suitable for the completion of development to form a chimeric ungulate.

2. The method of claim 1, within the ungulate embryonic stem cell is pluripotent.

3. The method of claim 1, wherein the embryonic stem cell is introduced into the embryo at a pre-implantation stage.

4. The method of claim 3, wherein the pre-implantation stage is the blastocyst stage.

5. The method of claim 1, wherein the embryonic stem cell is derived from a first breed of ungulate and the recipient embryo is derived from a second breed of the same species as the first breed.

6. The method of claim 1, wherein the first genetic complement comprises an exogenous nucleotide sequence stably integrated into the genetic complement of the embryonic stem cell by cell line transformation.

7. The method of claim 6, wherein the first genetic complement comprises a nucleotide sequence capable of being expressed to provide human Factor IX in recoverable form from the chimeric ungulate.

8. The method of claim 6, wherein the first genetic complement comprises a nucleotide sequence encoding a protein selected from the group consisting of human blood proteins, human hormones, human growth factors, human cytokines, human enzymes human hormone receptors, human binding proteins, antigens, translation factors, transcription factors, onco-proteins, protooncoproteins, human milk proteins, and human muscle proteins.