Cells and methods for the generation of transgenic pigs
AssigneeThe Texas A&M University System
InventorsJorge A. Piedrahita, Fuller W. Bazer
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Abstract
Disclosed are methods for the isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, methods for transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated by the present invention is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.
Claims69
A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor.
A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on STO feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said STO feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor.
A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor.
A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added leukemia inhibitory factor.
A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor or leukemia inhibitory factor.
A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells other than S1/S14 or S1-m220 for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor.
A method of growing porcine primordial germ cells, comprising growing a cell culture comprising porcine primordial germ cells from an embryo of a pig on feeder cells other than S1/S14 or S1-m220 for a time sufficient to obtain undifferentiated porcine primordial germ cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, said culture medium including no exogenously added soluble stem cell factor or leukemia inhibitory factor.
A method of preparing a porcine primordial germ cell-derived cell line, comprising:
- a) plating a cell culture comprising porcine primordial germ cells on feeder cells, said feeder cells at a density of between about 2.5×10 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor;
- and
- b) culturing the plated porcine primordial germ cells for a period of time effective to provide a porcine primordial germ cell-derived cell line.
A method of preparing porcine primordial germ cells that contain a selected DNA segment, comprising:
- a) introducing said selected DNA segment into a composition comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;
- and
- b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain said porcine primordial germ cells that contain said selected DNA segment.
A method of producing a transgenic pig, comprising:
- a) introducing a selected DNA segment into a cell culture comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;
- b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain undifferentiated porcine primordial germ cells that contain said selected DNA segment;
- and
- c) generating a transgenic pig from said undifferentiated porcine primordial germ cells that contain said selected DNA segment, wherein said selected DNA segment is contained and expressed in somatic and germ cells of said transgenic pig.
A cell culture comprising:
- (a) porcine primordial germ cells;
- (b) feeder cells sufficient to achieve a density of between about 2.5×105 cells/cm2 and about 106 feeder cells/cm2;
- and
- (c) basic fibroblast growth factor in an amount effective to promote the growth and continued proliferation of said porcine primordial germ cells.
A kit comprising, in suitable container means:
- (a) porcine primordial germ cells;
- (b) feeder cells sufficient to achieve a density of between about 2.5×105 cells/cm2 and about 106 feeder cells/cm2;
- and
- (c) basic fibroblast growth factor in an amount effective to promote the growth and continued proliferation of said porcine primordial germ cells.
A method of preparing a porcine blastocyst that contains a selected DNA segment, comprising:
- a) introducing said selected DNA segment into a cell culture comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;
- b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain undifferentiated porcine primordial germ cells that contain said selected DNA segment;
- and
- c) injecting said undifferentiated porcine primordial germ cells that contain said selected DNA segment into a porcine blastocyst, thereby preparing a porcine blastocyst that contains said selected DNA segment.
A method of preparing a porcine oocyte that contains a selected DNA segment, comprising:
- a) introducing said selected DNA segment into a cell culture comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;
- b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cells/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain undifferentiated porcine primordial germ cells that contain said selected DNA segment;
- c) isolating a nucleus from said undifferentiated porcine primordial germ cells that contain said selected DNA segment;
- and
- d) injecting said nucleus into an enucleated porcine oocyte, thereby preparing a porcine oocyte that contains said selected L)NA segment.
A method of preparing an early stage porcine embryo that contains a selected DNA segment, comprising:
- a) introducing a selected DNA segment into a cell culture comprising porcine primordial germ cells to obtain candidate porcine primordial germ cells that contain said selected DNA segment;
- b) plating said candidate porcine primordial germ cells that contain said selected DNA segment on feeder cells, said feeder cells at a density of between about 2.5×105 cells/cm2 and about 106 cell/cm2, in a culture medium comprising an effective amount of basic fibroblast growth factor, to obtain undifferentiated porcine primordial germ cells that contain said selected DNA segment;
- and
- c) aggregating said undifferentiated porcine primordial germ cells that contain said selected DNA segment with an early stage porcine embryo, thereby preparing an early stage porcine embryo that contains said selected DNA segment.
Dependencies are inferred from the claim text.