METHODS FOR CONTROLLING STEM CELL DIFFERENTIATION

• US 20100203632A1
• Filed: 01/24/2008
• Published: 08/12/2010
• Est. Priority Date: 01/25/2007
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Lee, Jeannie T.
• Granted Patent: US 8,975,068 B2
• Time in Patent Office: Days
• US Class Current: 435/354
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2330/10 naturally occurring
• CPC Class Codes: C12N 2501/065 Modulators of histone acetylation
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/60 Transcription factors
• 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
• CPC Class Codes: C12N 5/16 Animal cells

Abstract

Disclosed herein are methods for controlling stem cell differentiation through the introduction of transgenes having Xic, Tsix, Xite, or Xic flanking region sequences to block differentiation and the removal of the transgenes to allow differentiation. Also disclosed are small RNA molecules and methods for using the small RNA molecules to control stem cell differentiation. Also disclosed are stem cells genetically modified by the introduction of Xic, Tsix, XUe, or Xic flanking region sequences.

Claims

1-42. -42. (canceled)

43. A stem cell comprising an Xic flanking region transgene substantially identical to a sequence selected from the group consisting of SEQ ID NOs:

46-52 or a fragment thereof.

44. The cell of claim 43, wherein said Xic flanking region transgene is expressed in said stem cell.

45. The cell of claim 43, wherein said stem cell is an embryonic stem cell.

46. The cell of claim 45, wherein said embryonic stem cell is female or male.

47. The cell of claim 45, wherein said embryonic stem cell is mammalian.

48. The cell of claim 45, wherein said embryonic stem cell is human.

49. The cell of claim 45, wherein said embryonic stem cell is mouse.

50. The cell of claim 45, wherein said embryonic stem cell is a blastocyst stage stem cell.

51. The cell of claim 45, wherein said embryonic stem cell is from an agricultural animal.

52. The cell of claim 43, wherein said Xic flanking region transgene further comprises a selectable marker.

53. The cell of claim 43, wherein said Xic flanking region transgene is flanked by LoxP or FRT sequences.

54. The cell of claim 43, wherein said stem cell further comprises a recombinase.

55. The cell of claim 43, wherein said stem cell further comprises a second transgene.

56. A isolated small RNA molecule comprising a nucleic acid sequence substantially identical to or complementary to at least 15 nucleotides of an Xic flanking region transgene or a fragment thereof.

57. The isolated small RNA molecule of claim 56, wherein said small RNA is a double stranded RNA.

58. The isolated small RNA molecule of claim 56, wherein said small RNA is 15 to 32 nucleotides in length.

59. The isolated small RNA molecule of claim 56, wherein said small RNA is 32 to 50 nt in length.

60. The isolated small RNA molecule of claim 56, wherein said small RNA is an siRNA.

61. The isolated small RNA molecule of claim 56, wherein said Xic flanking region transgene comprises a nucleic acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs:

46-52, or a fragment thereof.

62. A composition comprising the isolated small RNA molecule of claim 56, formulated to facilitate entry of the small RNA molecule into a cell.

63. (canceled)

64. (canceled)