METHODS FOR MODULATING EMBRYONIC STEM CELL DIFFERENTIATION

• US 20100105043A1
• Filed: 03/26/2008
• Published: 04/29/2010
• Est. Priority Date: 03/26/2007
• Status: Abandoned Application

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America)
• Original Assignee: United States Department Of Health And Human Services (Government of the United States of America)
• Inventors: Lee, Sung-Lim, Monti, Manuela, Ko, Minoru S. H., Falco, Geppino, Stanghellini, Ilaria
• Application Number:
  US12/529,004
• Publication Number:
  US 20100105043A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/052 inducing gain of function
• CPC Class Codes: A01K 2217/15 Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: C07K 14/47 from mammals
• 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 15/85 for animal cells
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/85 mammalian
• 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 5/0611 Primordial germ cells, e.g. embryonic germ cells [EG]

Abstract

Described herein is Zscan4, a gene exhibiting 2-cell embryonic stage and embryonic stem cell specific expression. Identification of nine Zscan4 co-expressed genes is also described Inhibition of Zscan4 expression inhibits the 2-cell to 4-cell embryonic transition and prevents blastocyst implantation, expansion and outgrowth. Provided herein are methods of inhibiting differentiation of a stem cell, promoting blastocyst outgrowth of embryonic stem cells and identifying a subpopulation of stem cells expressing Zscan4. Further described is the identification of Trim43 as a gene exhibiting morula-specific expression. Also provided are isolated expression vectors comprising a Zscan4 promoter, or a Trim43 promoter operably linked to a heterologous polypeptide and uses thereof. Further provided are transgenic animals comprising transgenes encoding marker proteins operably linked to Zscan4 and Trim43 promoters.

Claims

1. A method of inhibiting differentiation of a stem cell, comprising increasing the expression of Zscan4 in the stem cell, thereby inhibiting differentiation of the stem cell.

2. The method of claim 1 wherein increasing the expression of Zscan4 comprises increasing the expression of Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e, Zscan4f, human ZSCAN4 or a combination thereof.

3. The method of claim 1, wherein increasing the expression of Zscan4 in the cell comprises transfecting the stem cells with a nucleic acid encoding Zscan4 operably linked to a promoter.

4. The method of claim 3, wherein the nucleic acid encoding Zscan4 comprises SEQ ID NO:

60.

5. The method of claim 3, wherein the nucleic acid encoding Zscan4 is at least 95% identical to Zscan4c (SEQ ID NO:

19), Zscn4d (SEQ ID NO;

     21) or Zscan4f (SEQ ID NO;

     25).

8. The method of claim 3, comprising transfecting the cells with a vector comprising the nucleic acid encoding Zscan 4.

10. The method of claim 1, wherein inhibiting the differentiation of the stem cell increases the viability of the stem cell.

11. The method of claim 1, wherein inhibiting the differentiation of the stem cell prevents senescence of the stem cell.

12. The method of claim 1, wherein the stem cell is an embryonic stem cell, an embryonic germ cell, a germline stem cell or a multipotent adult progenitor cell.

6-7. -7. (canceled)

9. (canceled)

13. A method of promoting blastocyst outgrowth of embryonic stem cells, comprising increasing the expression of Zscan4 in the embryonic stem cells, thereby promoting blastocyst outgrowth of the embryonic stem cells.

14. The method of claim 13, wherein increasing the expression of Zscan4 comprises increasing the expression of Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e, Zscan4f, human ZSCAN4 or a combination thereof.

15. The method of claim 13, wherein increasing the expression of Zscan4 in the cell comprises transfecting the stem cells with a nucleic acid encoding Zscan4 operably linked to a promoter.

16. The method of claim 15, wherein the nucleic acid encoding Zscan4 comprises SEQ ID NO:

6064.

17. The method of claim 15, wherein the nucleic acid encoding Zscan4 is at least 95% identical to Zscan4c (SEQ ID NO:

19), Zscn4d (SEQ ID NO;

     21) or Zscan4f (SEQ ID NO;

     25).

20. The method of claim 15, comprising transfecting the cells with a vector comprising the nucleic acid encoding Zscan 4.

18-19. -19. (canceled)

21. (canceled)

22. A method of identifying a subpopulation of stem cells expressing Zscan4, comprising transfecting the cells with an expression vector comprising a Zscan4 promoter and a reporter gene, wherein expression of the reporter gene indicates Zscan4 is expressed in the subpopulation of stem cells.

23. The method of claim 22, wherein the Zscan4 promoter is a Zscan4c promoter.

24. The method of claim 23, wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as:

nucleotides 1-2540 of SEQ ID NO;

28,

nucleotides 1-2643 of SEQ ID NO;

28,

nucleotides 1-3250 of SEQ ID NO;

28,

nucleotides 1-3347 of SEQ ID NO;

28, or SEQ ID NO;

28.

25-28. -28. (canceled)

29. An isolated expression vector comprising a Zscan4c promoter operably linked to a nucleic acid encoding a heterologous polypeptide.

30. The isolated expression vector of claim 29, wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as

nucleotides 1-2540 of SEQ ID NO:

28,

nucleotides 1-2643 of SEQ ID NO;

28,

nucleotides 1-3250 of SEQ ID NO;

28, or

nucleotides 1-3347 of SEQ ID NO;

28.

34. The isolated expression vector of claim 29, wherein the polypeptide is a marker, enzyme, or fluorescent protein.

37. An isolated embryonic stem cell comprising the expression vector of claim 29.

31-33. -33. (canceled)

35-36. -36. (canceled)

38. An isolated expression vector comprising a Trim43 promoter operably linked to a nucleic acid encoding a heterologous polypeptide.

39. The isolated expression vector of claim 38, wherein the Trim43 promoter comprises the nucleic acid sequence set forth as SEQ ID NO:

31.

40. The isolated expression vector of claim 38, wherein the polypeptide is a marker, enzyme, or fluorescent protein.

43. An isolated embryonic stem cell comprising the expression vector of claim 38.

45. An isolated stem cell identified according to the method of claim 40.

41-42. -42. (canceled)

44. A method of identifying a subpopulation of stem cells, wherein the stem cells express Zscan4, comprising detecting expression of one or more of AF067063, Testv1/Tcstv3, Tho4, Arginase II, BC061212 and Gm428, Eif1a, EG668777 and Pif1.