Double nuclear transfer method and results thereof

• US 7,531,715 B1
• Filed: 01/13/2000
• Issued: 05/12/2009
• Est. Priority Date: 01/13/1999
• Status: Expired due to Fees

• Current Assignee: Revivicor, Inc (United Therapeutics Corporation)
• Original Assignee: PPL Therapeutics
• Inventors: Campbell, Keith H. S.
• Primary Examiner(s): BERTOGLIO, VALARIE E
• Application Number:
  US09/600,130
• Time in Patent Office: 3,407 Days
• Field of Search: 800/3, 800/21
• US Class Current: 800/24
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2227/108 Swine
• CPC Class Codes: A01K 67/0273 Cloned vertebrates
• CPC Class Codes: C12N 15/8773 Ovine embryos
• CPC Class Codes: C12N 15/8778 Swine embryos

Abstract

A method is described for the production of an animal (offspring) by the process of nuclear transfer. The animal may be produced using donor genetic material in cells taken directly from any stage of an animal, such as an embryo, foetus or adult or from cell cultures established from material from any stage of an animal, such as embryonic, foetal or adult material. The process may be used to introduce genetic modifications into the resultant offspring by genetic manipulation and selection of the cells to act as nuclear donors prior to embryo reconstruction.

The present invention provides a method of reconstituting an animal embryo, the process comprising transferring a nucleus into a first oocyte followed by removing and transferring the nucleus from the oocyte to a further oocyte or to an enucleated fertilized zygote.

Claims

1. A method of reconstituting a non-primate mammalian embryo, comprising

(i) transferring a non-primate mammalian donor nucleus from a diploid somatic cell into a first recipient oocyte;

(ii) removing the donor nucleus from the first recipient oocyte;

(iii) providing a second recipient oocyte or enucleated fertilized zygote; and

(iv) transferring the donor nucleus from the first recipient oocyte into the second recipient oocyte or the enucleated fertilized zygote to obtain a reconstituted non-primate mammalian embryo, wherein the second recipient oocyte is activated prior to, concomitant with or following transfer of the donor nucleus.

2. The method as claimed in claim 1, wherein the first oocyte is a mature metaphase II oocyte or an activated metaphase II oocyte.

3. The method as claimed in claim 1, wherein the second oocyte is an enucleated metaphase II oocyte.

4. The method as claimed in claim 1, in which a reconstructed embryo obtained thereby is cultured in vitro or in vivo to a stage suitable for transfer to a final surrogate recipient for development to term.

5. The method as claimed in claim 1, in which a reconstructed embryo obtained thereby is transferred to a final surrogate recipient to support embryo development and development to term.

6. The method as claimed in claim 1, in which the donor nucleus is genetically modified.

7. The method as claimed in claim 1, wherein the donor nucleus is from a G1 cell.

8. The method as claimed in claim 1, wherein the diploid cell is arrested at the G1/S-phase border.

9. The method as claimed in claim 1, wherein the donor nucleus is donated by a diploid cell arrested by any point in the cell cycle.

10. The method as claimed in claim 1, wherein the first recipient oocyte is enucleated prior to transfer of the donor nucleus into said first recipient oocyte.

11. The method as claimed in claim 1, wherein the donor nucleus is transferred into the first recipient oocyte by cell fusion, or by cell or nuclear injection.

12. The method as claimed in claim 1, in which the non-primate mammalian embryo is an ungulate species embryo.

13. The method as claimed in claim 12, wherein the non-primate mammalian embryo is a cow or bull, pig, sheep, goat, camel, or water buffalo embryo.

14. The method as claimed in claim 1, wherein the non-primate mammalian embryo is a mouse, rat, or other rodent embryo.

15. The method as claimed in claim 1, wherein the non-primate mammalian embryo is a lagomorph embryo.

16. The method as claimed in claim 15, wherein the non-primate lagomorph embryo is a rabbit embryo.

17. The method as claimed in claim 1, wherein the donor nucleus is transferred from the first recipient oocyte to a fertilized zygote.

18. The method as claimed in claim 1, wherein the second recipient oocyte is activated by chemical or physical means.

19. The method as claimed in claim 1, wherein the second recipient oocyte is enucleated.

24. The method as claimed in claim 1, further comprising isolating a cell line or cell population from the reconstituted embryo.

28. The method as claimed in claim 18, wherein the chemical or physical activation is by a treatment that induces calcium entry into the oocyte or release of internal calcium stores.

29. The method as claimed in claim 18, wherein the chemical activation is by treatment with ethanol, ionomycin, inositol tris-phosphate or calcium ionophore A23187.

30. The method as claimed in claim 18, wherein the chemical activation is by treatment with extracts of sperm.

31. The method as claimed in claim 18, wherein the physical activation is by application of a DC electrical stimulus.

32. The method as claimed in any one of claims 28-31, wherein the chemical or physical activation further comprises treatment with inhibitors of protein synthesis or inhibitors of serine threonine protein kinases.

33. The method as claimed in claim 1, wherein the non-primate mammalian embryo is a pig embryo.

34. The method as claimed in claim 12, wherein the non-primate mammalian embryo is a cow embryo.

35. The method as claimed in claim 12, wherein the non-primate mammalian embryo is a sheep embryo.

20. A method of preparing a non-primate mammal, the method comprising:

(a) transferring a non-primate mammalian donor nucleus from a diploid somatic cell into a first recipient oocyte;

(b) removing the donor nucleus from the first recipient oocyte;

(c) either enucleating a second recipient oocyte or enucleating a fertilized zygote of the donor species; and

(d) transferring the donor nucleus from the first recipient oocyte into the second recipient oocyte or zygote of the same species as the donor to obtain a reconstituted non-primate mammalian embryo, wherein the second recipient oocyte is activated prior to, concomitant with or following transfer of the donor nucleus;

(e) causing a fetus to develop from the embryo, thereby obtaining a non-primate mammalian fetus; and

(f) causing a non-primate mammal to develop to term from the non-primate mammalian fetus, thereby obtaining a non-primate mammal.

21. The method as claimed in claim 20, further comprising:

(g) breeding the non-primate mammal.

22. A method as claimed in claim 20, wherein the non-primate mammalian embryo is further manipulated prior to full development of the embryo.

23. A method as claimed in claim 20, wherein the non-primate mammalian fetus is further manipulated prior to full development of the fetus.

25. The method as claimed in claim 20, further comprising isolating a cell line or cell population from the fetus.

26. The method as claimed in claim 20, wherein a new cell line or cell population is derived from the non-primate mammal.

27. The method as claimed in claim 20, wherein more than one non-primate mammal is derived from the reconstituted embryo.