USE OF HAPLOID GENOMES FOR GENETIC DIAGNOSIS, MODIFICATION AND MULTIPLICATION

• US 20110287429A1
• Filed: 01/28/2011
• Published: 11/24/2011
• Est. Priority Date: 11/02/1999
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Robl, James M., Moreira, Pedro
• Granted Patent: US 8,551,705 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• 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 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/04 Cells produced using nuclear transfer

Abstract

Methods for propagating haploid genomes of male or female origina and genetic screening and modification thereof are provided. These haploid genomes may be used to produce haploid embryos, and embryonic stem-like cells and differentiated cells. Also, these haploid genomes and cells containing, may be used as nuclear transfer donors to produce diploid nuclear transfer units. These diploid NT units e.g., human NT units, may be used to obtain pluripotent cells and differentiated cells and tissues.

Claims

1. A method for selecting a haploid genome containing cell comprising the following steps:

(i) obtaining and amplifying the number of cells containing a haploid genome of male or female origin;

(ii) subjecting the genome of said haploid cells to genetic screening or analysis to determine whether said haploid genome comprises a desired genetic makeup; and

(iii) selecting cells that contain said desired genetic makeup.

10. The method of claim 1, wherein said female- or male-derived haploid cells are amplified by a method selected from the group consisting of (i) allowing a haploid egg cytoplast to undergo cell division;

(ii) allowing a haploid cell to produce a haploid embryo which is then cultured to produce “

propagating haploid”

cells;

(iii) culturing a haploid embryo to produce propagating haploid cells and allowing such embryonic stem-like cells to differentiate; and

(iv) culturing a haploid somatic cell cytoplast under conditions that allow cell division.

2-9. -9. (canceled)

11-37. -37. (canceled)

38. Embryonic stem-like cells or differentiated cells produced from a haploid embryo.

39. (canceled)

40. A method for producing an embryo having a desired genetic makeup, the method comprising:

(i) providing a haploid cell, wherein said haploid cell comprises a haploid genome of male or female origin;

(ii) propagating said haploid cell, thereby producing a cultured population;

(iii) subjecting one or more cells of said cultured population to genetic screening, thereby detecting whether said haploid genome has said desired genetic makeup;

(iv) transferring the nucleus of a haploid cell that contains said desired genetic makeup into an oocyte; and

(v) allowing said oocyte to develop into an embryo having said desired genetic makeup.

41. The method of claim 40, wherein said oocyte is an enucleated oocyte or a haploid oocyte.

42. The method of claim 40, further comprising introducing a second haploid genome into said enucleated oocyte.

43. The method of claim 40, wherein said haploid oocyte comprises a haploid genome having a desired genetic makeup.

46. The method of claim 40, wherein said female-derived haploid cell is produced by activation of an oocyte in which half of the chromosomes are extruded in the polar body.

47. The method of claim 40, wherein said female-derived haploid cells are produced by fertilization of an oocyte and removal of a male pronucleus therefrom.

48. The method of claim 40, wherein said female-derived haploid cells are produced by activation of an oocyte to provide an oocyte containing two female pronuclei, and removal of one of said pronuclei.

49. The method of claim 40, wherein said female-derived haploid cells are produced by insertion of a diploid cell nucleus into an immature oocyte followed by separation of said chromosomes in to two haploid nuclei.

50. The method of claim 40, wherein said female-derived haploid cells are produced by transfer of the nucleus of a parthenogenetic cell.

51. The method of claim 40, wherein said male-derived haploid cell is derived from a fertilized oocyte from which the female pronucleus is removed.

52. The method of claim 40, wherein the male-derived haploid cell is derived by fertilization of an enucleated oocyte.

53. The method of claim 40, wherein the male-derived haploid cell is produced by artificial de-condensation of a sperm nucleus which is then injected into a non-oocyte derived cytoplast.

54. The method of claim 40, wherein said embryo is human.

55. The method of claim 40, wherein said embryo is diploid.

56. The method of claim 40, wherein said haploid genome is genetically modified.

44. The method of claim 44, further comprising (vi) producing embryonic stem cells from said embryo.

45. The method of claim 44, wherein step (vi) comprises culturing inner cell mass cells obtained from said embryo.