In vivo methods for effecting tissue specific differentiation of embryonic stem cells

• US 20060008451A1
• Filed: 07/06/2005
• Published: 01/12/2006
• Est. Priority Date: 07/06/2004
• Status: Abandoned Application

• Current Assignee: Michigan State University
• Original Assignee: Michigan State University
• Inventors: Cibelli, Jose, Tsunekawa, Naoki, Yabuuchi, Akiko
• Application Number:
  US11/174,603
• Publication Number:
  US 20060008451A1
• Time in Patent Office: Days
• US Class Current: 424/93.210
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2517/02 Cells from transgenic animals

Abstract

Methods for producing derived differentiated cells from chimeric animals, fetuses and embryos derived from non-human chimeric embryos are provided. These non-human chimeric embryos are obtained by introduction of at least one transgenic ES or ES-like cell into a recipient non-human embryo, e.g., blastocyst stage embryo, wherein said ES or ES-like cell comprises DNA constructs that contain markers and promoters which allow for selective survival of a desired differentiated cell, tissue organ and ablation of other cells that originate from said transgenic ES or ES-like cells.

Claims

1. A transgenic embryonic stem (ES) or embryonic stem-like (ES-like) cell that comprises at least the following DNA constructs:

i. a first DNA construct that contains a regulatory (promoter) sequence selectively or preferentially expressed in pluripotent cells that regulates the expression of at least one selectable and/or detectable marker gene;

ii. a second DNA construct that contains a regulatory (promoter) sequence selectively or preferentially expressed by cells that differentiate into a desired cell type, which sequence regulates the expression of at least one selectable and/or detectable marker gene; and

iii. a third DNA construct that contains a selectable or detectable marker gene that is expressed under the control of a constitutive promoter.

5. The transgenic ES or ES-like cell of claim 1 which is obtained from an embryo.

6. The transgenic ES or ES-like cell of claim 5 wherein said embryo is obtained by nuclear transfer cloning methods.

7. The transgenic ES or ES-like cell of claim 5 wherein said embryo is obtained by parthenogenic methods.

8. The transgenic ES or ES-like cell of claim 1 which is further genetically engineered such that it is incapable of differentiating into a specific cell lineage.

15. A method of producing a chimeric non-human embryo comprising introducing at least one transgenic ES or ES-like cell according to claim 1 into a recipient non-human embryo.

16. The method of claim 15 wherein said embryo is a blastocyst-stage embryo.

17. The method of claim 15 wherein the resultant chimeric non-human embryo is implanted into the uterus of a non-human female recipient animal.

18. The method of claim 17 wherein said animal is a mouse.

19. The method of claim 18 wherein said mouse is immunocompromised.

20. The method of claim 17 wherein said non-human chimeric embryo is allowed to develop into a fetus.

21. The method of claim 17 wherein said recipient animal is contacted with a selection agent that permits survival of cells that express the selectable marker contained on the first DNA construct which are preferentially or selectively expressed by pluripotent (primordial germ or ES cells).

22. The method of claim 21 wherein said contact is maintained until about 10 day post-conception.

23. The method of claim 22 wherein the chimeric embryo is thereafter contacted with a selection agent that permits survival of cells that express a selectable or detectable marker expressed by the second DNA construct which is only expressed by non-recipient transgenic cells which differentiate into a particular differentiated cell, tissue and/or organ.

24. The method of claim 23 wherein said differentiated cell, tissue or organ is a monkey differentiated cell, tissue or organ.

25. The method of claim 24 wherein said differentiated cell, tissue or organ is a monkey oocyte.

2. The transgenic ES or ES-like cell which is selected from the group consisting of primate, equine, murine, canine, feline, bovine, goat, sheep, bear guinea pig, panda, and rabbit ES or ES-like cells.

3. The transgenic ES or ES-like cell which is primate.

4. The transgenic ES or ES-like cell of claim 3 which is a monkey ES-or ES-like cell.

9. A chimeric non-human embryo or fetus of claim 9 which is murine.

10. The chimeric non-human embryo or fetus of claim 9 which is murine.

11. A non-human recipient female that comprises a chimeric non-human embryo according to claim 9.

12. A differentiated cell, tissue or organ derived from the chimeric non-human embryo, fetus or animal of claim 9.

13. The differentiated cell, tissue or organ of claim 12 which comprises a cell selected from the group consisting of an oocyte, neural cell, hematopoietic cell, digestive cell, respiratory cell, organ cell, eye cell, ear cell, olfactory organ cell, skin cell, circulatory organ cell, reproductive organ cell, urinary organ cell, taste organ cell, and immune cell.

14. The differentiated cell, tissue or organ of claim 13 which is a monkey oocyte.