Methods for cloning non-human mammals using reprogrammed donor chromatin or donor cells

• US 7,253,334 B2
• Filed: 12/21/2001
• Issued: 08/07/2007
• Est. Priority Date: 12/22/2000
• Status: Expired due to Term

• Current Assignee: SAB LLC (W.P. Carey, Inc. (Real Estate))
• Original Assignee: Aurox, LLC
• Inventors: Robl, James M., Collas, Philippe, Kasinathan, Poothappillai, Sullivan, Eddie
• Primary Examiner(s): WOITACH, JOSEPH T
• Application Number:
  US10/032,191
• Publication Number:
  US 20030046722A1
• Time in Patent Office: 2,055 Days
• Field of Search: 800/3, 800/4, 800/8, 800/21, 800/24, 800/25, 435/325, 435/440
• US Class Current: 800/24
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2227/102 Caprine
• CPC Class Codes: A01K 2227/103 Ovine
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/107 Rabbit
• CPC Class Codes: A01K 2227/108 Swine
• CPC Class Codes: A01K 2267/02 Animal zootechnically ameliorated
• CPC Class Codes: A01K 67/0273 Cloned vertebrates
• CPC Class Codes: C12N 15/873 Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
• CPC Class Codes: C12N 15/8771 Bovine embryos
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2517/10 Conditioning of cells for in vitro fecondation or nuclear transfer

Abstract

The invention provides methods for cloning mammals that allow the donor chromosomes or donor cells to be reprogrammed prior to insertion into an enucleated oocyte. The invention also features methods of inserting chromosomes or nuclei into recipient cells.

Claims

1. A method of cloning a non-human mammal, said method comprising the steps of:

(a) permeabilizing a non-human mammalian cell, thereby generating a permeabilized cell having pores in its plasma membrane or a partial plasma membrane;

(b) incubating said permeabilized cell in an extract from a mitotic cell under conditions that allow chromatin condensation and nuclear envelope breakdown of said permeabilized cell;

(c) inserting said cell formed in step (b) into a nucleated or enucleated oocyte, thereby forming a reconstituted oocyte; and

(d) transferring said reconstituted oocyte or an embryo formed from said reconstituted oocyte into the uterus of a host mammal under conditions that allows said reconstituted oocyte or said embryo to develop into a fetus.

2. The method of claim 1, wherein a chromatin mass is formed from incubation of said permeabilized cell in said extract from said mitotic cell.

3. The method of claim 1, wherein, said cell formed in step (b) is incubated under conditions that allow the plasma membrane of said cell to reseal.

4. The method of claim 1, wherein said cell formed in step (b) is purified from said extract from said mitotic cell prior to insertion into said nucleated or enucleated oocyte.

5. The method of claim 1, wherein said fetus develops into a viable offspring.

6. The method of claim 1, wherein said reconstituted oocyte from step (c) is cultured under conditions that allow cell division and one of the resulting cells is recloned one or more times.

7. The method of claim 1, wherein said permeabilized cell and said nucleated or enucleated oocyte are from the same species.

8. The method of claim 1, wherein said non-human mammal is a cow, sheep, rabbit, pig, mouse, rat, goat, or buffalo.

9. The method of claim 8, wherein said non-human mammal is a cow.

10. The method of claim 1, wherein said permeabilized cell is a fibroblast, epithelial cell, neural cell, epidermal cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, B-lymphocyte, T-lymphocyte, erythrocyte, macrophage, monocyte, muscle cell, embryonic stem cell, embryonic germ cell, fetal cell, placental cell, or embryonic cell.

11. The method of claim 1, wherein said permeabilized cell is a cell of the female reproductive system.

12. The method of claim 11, wherein said permeabilized cell is a mammary gland, ovarian cumulus, granulosa, or oviductal cell.

13. The method of claim 1, wherein said reconstituted oocyte from step (b) expresses lamin A, lamin C, or NuMA protein at a level that is less than 5-fold greater than the corresponding level expressed by a control oocyte from the same species.

14. The method of claim 1, wherein said extract from said mitotic cell is an extract from a mitotic somatic cell.

15. The method of claim 1, wherein said permeabilized cell is generated by incubating a somatic cell from a non-human mammal with streptolysin O.

16. The method of claim 15, wherein said streptolysin O concentration is between 100–

4000 ng/ml.

17. The method of claim 16, wherein said streptolysin O concentrate is 500 ng/ml.

18. The method of claim 15, wherein said incubating with streptolysin O is carried out for 15–

60 minutes.

19. The method of claim 18, wherein said incubating with streptolysin O is carried out for between 25–

30 minutes.

20. The method of claim 15, wherein said incubating with streptolysin O is carried out at between 25–

38°

C.

21. The method of claim 20, wherein said incubating with streptolysin O is carried out at 38°

C.

22. The method of claim 1, wherein said inserting in step (c) is carried out by fusion of said permeabilized cell with said nucleated or enucleated oocyte.

23. The method of claim 1, wherein said oocyte of step (c) is enucleated.

24. The method of claim 1, wherein said reconstituted oocyte is activated prior to transfer into the uterus of said host mammal.