METHODS FOR MODULATING EMBRYONIC STEM CELL DIFFERENTIATION

• US 20120129161A1
• Filed: 12/21/2011
• Published: 05/24/2012
• Est. Priority Date: 03/26/2007
• Status: Active Grant

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America)
• Original Assignee: Government of the United States of America, Department of Health and Human Services (State of Victoria)
• Inventors: Ko, Minoru S.H., Falco, Geppino, Lee, Sung-Lim, Monti, Manuela, Stanghellini, Ilaria
• Granted Patent: US 8,617,813 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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 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.

2. The method of claim 1, wherein the Zscan4 promoter is a Zscan4c promoter.

3. The method of claim 2, wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as nucleotides 1-2540 of SEQ ID NO:

28.

4. The method of claim 2, wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as nucleotides 1-2643 of SEQ ID NO:

28.

5. The method of claim 2, wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as nucleotides 1-3250 of SEQ ID NO:

28.

6. The method of claim 2, wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as nucleotides 1-3347 of SEQ ID NO:

28.

7. The method of claim 1, wherein the expression vector comprises the nucleic acid sequence set forth as SEQ ID NO:

28.

8. The method of claim 1, wherein the reporter gene encodes a marker, enzyme, or fluorescent protein.

9. The method of claim 1, wherein the expression vector is a viral vector.

10. The method of claim 1, wherein the expression vector is a plasmid vector.

11. The method of claim 1, wherein the stem cells are embryonic stem cells.

12. The method of claim 1, wherein the stem cells are mouse stem cells.

13. The method of claim 1, wherein the stem cells are human stem cells.

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

15. The isolated expression vector of claim 14, wherein the Zscan4c promoter comprises the nucleic acid sequence set forth as nucleotides 1-2540 of SEQ ID NO:

28.

16. The isolated expression vector of claim 14, wherein the heterologous polypeptide is a marker, enzyme, or fluorescent protein.

17. The isolated expression vector of claim 14, wherein the vector is a viral vector.

18. The isolated expression vector of claim 14, wherein the vector is a plasmid vector.

19. An isolated embryonic stem cell comprising the expression vector of claim 14.