Transgenic swine compositions and methods

• US 5,942,435 A
• Filed: 06/06/1995
• Issued: 08/24/1999
• Est. Priority Date: 05/14/1993
• Status: Expired due to Term

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Board of Trustees of The University of Illinois, Biotechnology Research And Development Corporation (Dow, Inc.)
• Inventors: Wheeler, Matthew B.
• Primary Examiner(s): Campell, Bruce R.
• Application Number:
  US08/473,030
• Time in Patent Office: 1,540 Days
• Field of Search: 800/2, 800/DIG. 1, 800/DIG. 4, 435/172.3, 435/172.1, 435/325, 435/378, 435/380, 435/381, 435/384, 935/63
• US Class Current: 435/325
• 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 swine, and compositions and methods for making and using same, are provided. Central to the invention are porcine (Sus scrofa) 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 swine, which have germ cells comprising the exogenous genetic material. The chimeric swine are bred to provide transgenic swine. Transgenic swine of the invention can be used to provide human proteins or peptide hormones or can be used as xenograft donors.

Claims

1. A method of isolating and purifying an embryonic stem cell culture, said method comprising:

(a) culturing dissociated cells from a swine embryo in conditioned stem cell medium in the absence of a feeder layer, wherein the dissociated cells are obtained from a swine embryo which was developed in vitro in stem cell medium (SCM) on a feeder layer; and

(b) subculturing the culture until a stable culture with morphological features and growth parameters characteristic of an embryonic stem cell culture is established.

2. The method of claim 1, wherein the medium is conditioned by Buffalo Rat Liver Cells, growth factors, vitamins, amino acids and antibiotics.

3. The method of claim 2, wherein the stem cell conditioned medium (CSCM) comprises approximately 40% of stem cell medium (SCM) and approximately 60% of Buffalo Rat Liver Cell conditioned medium (BRL/CM).

4. An isolated swine embryonic stem cell.

5. The isolated swine cell of claim 4 which is totipotent.

6. The embryonic stem cell of claim 4, which has 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 shown 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.

7. The embryonic stem cell of claim 4, wherein the source of the cell is an embryo of a swine line selected from the group consisting of Meishan, Duroc and Yorkshire.

8. The embryonic stem cell of claim 4, wherein the cell is capable of producing a chimeric swine when said cell is introduced into a host embryo.

9. A cell line comprising undifferentiated cells derived from the embryonic stem cell of claim 4.

10. A stable cell line derived from a culture of undifferentiated swine embryonic stem cells.

11. A method for making a chimeric swine comprising:

(a) introducing a cultured swine embryonic stem cell that has a first genetic complement into a swine host embryo that has a second genetic complement, to form a chimeric swine embryo; and

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

12. The method of claim 11, wherein the cultured embryonic stem cell is derived from a first breed of swine, and the host embryo is derived from a second breed of swine.

13. The method of claim 12, wherein the first breed of swine is the Meishan breed, and the second breed of swine is the Duroc breed.