WO1999053025A1

WO1999053025A1 - Method of nuclear transfer

Info

Publication number: WO1999053025A1
Application number: PCT/AU1999/000275
Authority: WO
Inventors: Orly Lacham-Kaplan; Alan Osborne Trounson
Original assignee: Monash University
Priority date: 1998-04-15
Filing date: 1999-04-15
Publication date: 1999-10-21
Also published as: AUPP294898A0

Abstract

The invention relates to a method of nuclear transferring an isolated nucleus into an enucleated cell. In particular the donor nucleus is isolated by rupturing the cell membrane and aspiration using a tube having an internal diameter less than that of the donor cell and approximately the same or greater than that of the nucleus. The isolated nucleus is then transferred into an enucleated recipient oocyte by means of microinjection. The reconstituted cell thus obtained is transplanted as an embryo to produce an animal.

Description

1

METHOD OF NUCLEAR TRANSFER

The present invention relates to a method of nuclear transfer, and more particularly to a method of transferring an isolated nucleus into an enucleated cell. The present invention also relates to a method of isolating a nucleus from a donor cell. The present invention also relates to reconstituted cells, enucleated cells, nuclei, embryos and animals produced by the said methods.

The process of nuclear transplantation is described as the transfer of an intact nucleus from one cell to another which has had its nuclear DNA removed or destroyed. More particularly, the process involves the introduction of a foreign nucleus into the cytoplasm of an enucleated recipient oocyte. Hence the process may be applied to a recipient oocyte which will receive genetic material from a donor nucleus. Foreign genetic material is generally introduced via fusion which reconstitutes the genome of the oocyte. Fusion with the oocyte then results in a reconstituted, transplanted oocyte of known genetic constitution which has the potential of developing into an embryo whose cells may be used in time for nuclear transplantation or the production of embryonic stem cells thereby increasing the potential number of genetically identical embryos. Resulting embryos can be transferred to recipient females to enable the production of primordial germ cells or embryo development to term. The methodology of the prior art relies on four main elements:

(1) Enucleation of mature oocytes to produce recipient cytoplasts needed to support early embryonic development

(2) Production and separation of advanced stage embryonic cells serving as a source of clonal material (3) Fusion of enucleated oocyte and embryonic cell together to create a new, reconstituted embryo (4) Culture of these embryo clones up to transferable embryo stage.

These techniques are highly skilled, technically demanding, expensive and time consuming. The overall efficiencies of the techniques are relatively low, preventing an effective transfer of technology from experimental laboratories to more commercial environment.

It is an object of the present invention to overcome, or at least alleviate, one or 2 more of the difficulties or deficiencies associated with the prior art.

Accordingly, in a first aspect of the present invention there is provided a method of transferring an isolated nucleus to an enucleated cell, said method including providing a donor cell, and an enucleated recipient cell; isolating a nucleus from the donor cell; and transferring the nucleus to the enucleated recipient cell.

As used herein the term "isolated nucleus" and variations thereof such as "isolating a nucleus" refers to a nucleus which is no longer contained within the plasma membrane of a cell, but which may still be associated with cytoplasmic material from the cell.

The donor cell may be of any suitable type. Preferably the donor cell is a primary or cultured somatic cell, more preferably a fibroblast, most preferably a foetal fibroblast. Preferably the donor cell is mammalian, more preferably it is bovine.

The enucleated recipient cell may be of any suitable type. Preferably the enucleated recipient cell is an oocyte. Preferably the enucleated recipient cell is mammalian, more preferably it is bovine. The recipient cell may be enucleated by techniques known to those skilled in the art. For example, the recipient cell may be enucleated by microsurgery. Alternatively, the chromosomes may be inactivated by microsurgery or microaspiration, for example using an enucleation pipette.

The nucleus may be isolated from the donor cell by rupturing the plasma membrane of the donor cell and separating the intact nucleus from the plasma membrane and at least some of the cytoplasmic material. Following this isolation step the nucleus may still be associated with some of the cytoplasmic material of the donor cell. Preferably the nucleus is isolated from the donor cell by drawing the donor cell into a tube, whereby the plasma membrane of the cell ruptures and the intact nucleus is drawn into the tube, optionally together with some of the cytoplasmic material of the donor cell. The tube may have an internal diameter substantially less than that of the donor cell and approximately the same or greater than that of the nucleus. Preferably the tube has an internal diameter of between approximately 4 and 8 μM, more preferably between approximately 4 and 5 μM. Preferably the tube is a pipette, more 3 preferably a micromanipulation pipette, most preferably a glass micromanipulation pipette.

Preferably the donor cell is placed in a viscous solution, for example methyl- cellulose, for the nucleus isolation procedure. The nucleus may be transferred to the enucleated recipient cell by any suitable technique. However, it is preferred that the isolated nucleus is transferred to the enucleated recipient cell by injection, more preferably using the same tube (eg. micromanipulation pipette) as that used to isolate the nucleus. In this manner the nucleus may be isolated from the donor cell by drawing it into the tube, and then transferred from the tube to the enucleated cell in the one operation and without the need to remove the nucleus from the tube prior to transfer.

Thus, in a preferred form of this aspect of the invention there is provided a method of transferring an isolated nucleus to an enucleated cell, said method including providing a donor cell having a plasma membrane and a nucleus, a tube having an internal diameter substantially less than that of the donor cell and approximately the same or greater than that of the nucleus, and an enucleated recipient cell; rupturing the plasma membrane of the donor cell with the tube; drawing the nucleus into the tube; inserting the tube into the enucleated recipient cell; expelling the nucleus from the tube into the enucleated recipient cell; and removing the tube from the enucleated recipient cell.

In a further aspect of the present invention, there is provided a method of isolating a nucleus from a donor cell, said method including providing a donor cell having a plasma membrane and a nucleus, and a tube having an internal diameter substantially less than that of the donor cell and approximately the same or greater than that of the nucleus; rupturing the plasma membrane of the donor cell with the tube; and drawing the nucleus into the tube. The donor cell may be of any suitable type. Preferably the donor cell is a 4

primary or cultured somatic cell, more preferably a fibroblast, most preferably a foetal fibroblast. Preferably the donor cell is mammalian, more preferably it is bovine.

The tube may be of any suitable type. Preferably the tube has an internal diameter of between approximately 4 and 8 μM, more preferably between approximately 4 and 5 μM. Preferably the tube is a pipette, more preferably a glass micromanipulation pipette, most preferably a micromanipulation pipette.

Preferably the donor cell is placed in a viscous solution, for example methyl- cellulose, for the nucleus isolation procedure. Following this isolation step the nucleus may still be associated with some of the cytoplasmic material of the donor cell. In a still further aspect of the invention the donor cell is treated to facilitate isolation of the nucleus therefrom.

Accordingly, the present invention provides a method of transferring an isolated nucleus to an enucleated cell, said method including providing a donor cell, a calcium releasing agent, and an enucleated recipient cell; contacting the donor cell with the calcium releasing agent for a time sufficient to cause a morphological change in the donor cell; isolating the nucleus from the donor cell; and transferring the nucleus to the enucleated recipient cell.

In a preferred form of this aspect of the invention there is provided a method of transferring an isolated nucleus to an enucleated cell, said method including providing a donor cell having a plasma membrane and a nucleus, a calcium releasing agent, and an enucleated recipient cell; contacting the donor cell with the calcium releasing agent for a time sufficient to cause a morphological change in the donor cell; rupturing the plasma membrane to isolate the nucleus from the donor cell; and transferring the nucleus to the enucleated recipient cell. The present invention also provides a method of isolating a nucleus from a 5

donor cell, said method including providing a donor cell, and a calcium releasing agent; contacting the donor cell with the calcium releasing agent for a time sufficient to cause a morphological change in the donor cell; and isolating the nucleus from the donor cell.

In a preferred form of this aspect of the invention there is provided a method of isolating a nucleus from a donor cell, said method including providing a donor cell having a plasma membrane and a nucleus, and a calcium releasing agent; contacting the donor cell with the calcium releasing agent for a time sufficient to cause a morphological change in the donor cell; and rupturing the plasma membrane to isolate the nucleus from the donor cell.

The calcium releasing agent may be of any suitable type. Preferably the calcium releasing agent is a calcium ionophore, more preferably the calcium ionophore A23187.

Preferably the donor cell is cultured in vitro prior to treatment with the calcium releasing agent. Preferably the donor cells are synchronised prior to treatment with the calcium releasing agent. Methods of synchronising cells are known to those skilled in the art. For example, the cells may be cultured in a serum-containing medium, transferred to a serum-reduced medium to achieve synchronisation, and then returned to a serum containing medium. The cells may be contacted with the calcium releasing agent in the serum-containing medium.

Preferably, treatment of the donor cells with the calcium releasing agent is performed approximately 30 min to 3 hours, more preferably approximately 1-2 hours prior to isolation of the nucleus therefrom.

The donor cells are contacted with the calcium releasing agent for a time sufficient to cause a morphological change in at least some of the cells. The morphological change may be observed as a change to a more rounded morphology, a more obvious nucleus and a less opaque cytoplasm than the cells prior to treatment. 6

Whilst applicant does not wish to be restricted by theory it is thought that this change in morphology reflects a reduction in the elasticity of the plasma membrane, making it less resistant to mechanical disruption.

Preferably the donor cells are contacted with the calcium releasing agent for approximately 50 to 180 min, more preferably for approximately 60 to 90 min.

The concentration of the calcium releasing agent should be sufficient to cause a morphological change in at least some of the donor cells. Preferably the donor cell is contacted with the calcium releasing agent at a concentration of approximately 5 to 50 μg/ml, more preferably approximately 10 to 20 μg/ml. In a still further aspect of the present invention there are provided reconstituted cells, enucleated cells and nuclei prepared by the methods of the present invention.

In a still further aspect of the present invention there is provided a method of preparing a transplantation embryo, said method including providing a reconstituted cell prepared by the methods of the present invention; and culturing the reconstituted cell to a transferable embryo stage.

Culturing of the reconstituted cell may be conducted by any appropriate method. Such methods are known to the skilled addressee.

In a preferred form of this aspect of the invention the reconstituted cell is activated by treatment with a calcium releasing agent prior to or during the culture step.

Preferably the reconstituted cells are contacted with the calcium releasing agent for approximately 5 to 180 min, more preferably for approximately 60 to 90 min.

Preferably the reconstituted cell is contacted with the calcium releasing agent at a concentration of approximately 5 to 50 μg/ml, more preferably approximately 10 to 20 μg/ml.

The reconstituted cell may also be treated with a phosphorylation inhibiting agent prior to or during the culture step. Whilst applicant does not wish to be restricted by theory, it is thought that this treatment reduces the levels of maturation promoting and cytostatic factors, thereby initiating cleavage and maintaining the cells in a diploid 7

state.

The phosphorylation inhibiting agent may be of any suitable type. Di-methylaminopurine (DMAP) has been found to be particularly suitable.

In a still further aspect of the present invention there is provided a transplantation embryo produced by the methods of the present invention. The transplantation embryo is preferably mammalian, more preferably it is bovine. The cells of such a nuclear transplantation embryo may be recycled to provide donor cells for further cycles of nuclear transfer, as described in Australian patent 687422 to the present applicant, the entire disclosure of which is incorporated herein by reference. The transplantation embryos produced by the methods of the present invention may be used to produce genetically identical or similar animals by transplantation into a recipient female, preferably a synchronised female. Preferably, the recipient female is synchronised using fertility drugs, steroids or prostaglandins. Methods for transfer of embryos to recipient females are known to those skilled in the art. Accordingly in a further aspect of the present invention there is provided an animal produced by the methods of the present invention.

Preferably the animal is a mammal, more preferably it is bovine.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

In the Figures:

Figure 1 shows (arrow) cells which have responded to the calcium-ionophore treatment (A and B) and an untreated control (C). Figure 2 shows a preferred procedure according to the present invention for isolating nuclei from donor cells. Example 1 Preparation of the recipient oocvtes (eggs)

Oocytes are placed in calcium/magnesium-free solution. This solution is used to disturb the junctions of the oocyte and the polar body. The genomic material of mature bovine oocytes is mechanically removed by a glass instrument, which is inserted under the shell of the egg, the zone pellucida. A portion of the egg containing the 8 chromosomes, which is located under the first polar body, is removed. This procedure is commonly used to enucleate mammalian oocytes (1).

Example 2 Preparation of the donor cells (somatic cells)

Somatic cells, preferably foetal fibroblasts, are isolated from bovine foetuses and cultured in vitro in serum containing medium. The cultured cells are replicating, providing a "cell line". Each replicate in culture is termed "passage". Cell lines are cultured until they reach the desired passage. The culture medium is then replaced with serum-reduced medium. This treatment presumably synchronizes the cell population. The cells are put back into serum containing medium 48hr before injection to restart cleavage cycles. About 1 to 2 hr before the injection the cells are exposed to calcium releasing agent (calcium-ionophore). This treatment induces some of the cells to change morphologically. The cytoplasm becomes transparent, the cells become round and the nucleus is very obvious (Figure 1A and B). These cells can be morphologically distinguished from other cells within the cell-line population (Figure 1C).

Example 3 Chemical Separation of Donor Cell Nuclei - Preparation of Microiniection Somatic cells (fetal fibroblasts) treated with or without calcium ionophore are exposed to chilled NP-40 lysis buffer (100 μl) to induce digestion of the cell membrane. The lysis buffer includes 0.5% Nonidet P-40, 2mM MgCI_2, 2mM KCI and 10mM TrisCI (pH 8.0). The treated cells are washed 3 times with the lysis buffer. Using this procedure, nuclei are separated from the cell cytoplast by centrifugation at approximately 5000 rpm for 30 seconds. The selected nuclei are further suspended in 100 μl lysis buffer and then centrifuged at 5000 rpm for 30 seconds. Isolated nuclei are then available for nuclear transfer by microinjection. They may also be frozen by resuspending in 200 μl Glycerol storage buffer and storing at 0°C. Following injection and oocyte activation as described below, nuclear transfer embryos develop blastocysts in rates of 10 to 15%. The use of chemically isolated nuclei reduces the time of injection and the number of eggs destroyed by injection. Example 4 9

Nuclei injection procedure

The injection procedure takes place on a glass chamber. The oocytes are placed in handling calcium/magnesium-containing medium. The cells are diluted in a viscous solution (2) to prevent the cells from sticking to the glass chamber or the glass pipette during the injection. Cell nuclei are mechanically removed by a glass micromanipulation pipette (3). The cell membrane of cells with the morphology described before is less resistant to mechanical disruption than other cells. The somatic cell nucleus is separated from the cytoplasm (Figure 2). The glass pipette containing the nucleus is inserted under the zone pellucida. The membrane of the egg is drawn into the pipette until is broken. Gently the solution in the pipette containing the nucleus is expelled into the egg. The glass pipette is slowly withdrawn. Example 5 Induction of embryonic cleavage

After injection, oocytes are chemically activated by calcium releasing agent (calcium ionophore). After that treatment the levels of maturation promoting and cytostatic factors (MPF and CF) are reduced using other chemical (DMAP). This combined treatment allows the initiation of cleavage cycles as well as keeping the reconstituted egg with two sets of chromosomes (diploid). The treated eggs are placed in culture medium for 7 days. The proportion of oocytes that cleave is 50 to 80% and these developing to blastocysts 20 to 30%.

References

(1) Collas, P. and Barnes, F.L. (1994) Molecular Reproduction and Development 38:264-267. (2) Lacham, O. and Trounson, A. (1991) Molecular Reproduction and Development 29:85-93. (3) Kimura, Y. and Yanagimachi, R. (1995) Biology of Reproduction 53:855-862.

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

Claims

10CLAIMS:

1. A method of transferring an isolated nucleus to an enucleated cell, said method including providing a donor cell, and an enucleated recipient cell; isolating a nucleus from the donor cell; and transferring the nucleus to the enucleated recipient cell.

2. A method of transferring an isolated nucleus to an enucleated cell, said method including providing a donor cell having a plasma membrane and a nucleus; a tube having an internal diameter substantially less than that of the donor cell and approximately the same or greater than that of the nucleus; and an enucleated recipient cell; rupturing the plasma membrane of the donor cell with the tube; drawing the nucleus into the tube; inserting the tube into the enucleated recipient cell; expelling the nucleus from the tube into the enucleated recipient cell; and removing the tube from the enucleated recipient cell.

3. A method of isolating a nucleus from a donor cell, said method including providing a donor cell having a plasma membrane and a nucleus; and a tube having an internal diameter substantially less than that of the donor cell and approximately the same or greater than that of the nucleus; rupturing the plasma membrane of the donor cell with the tube; and drawing the nucleus into the tube. 11

4. A method of transferring an isolated nucleus to an enucleated cell, said method including providing a donor cell; a calcium releasing agent; and an enucleated recipient cell; contacting the donor cell with the calcium releasing agent for a time sufficient to cause a morphological change in the donor cell; isolating the nucleus from the donor cell; and transferring the nucleus to the enucleated recipient cell.

5. A method according to claim 4 including providing a donor cell having a plasma membrane and a nucleus; a calcium releasing agent; and an enucleated recipient cell; contacting the donor cell with the calcium releasing agent for a time sufficient to cause a morphological change in the donor cell; rupturing the plasma membrane to isolate the nucleus from the donor cell; and transferring the nucleus to the enucleated recipient cell.

6. A method according to claim 4 or 5 wherein the calcium releasing agent is a calcium ionophore.

7. A method according to claim 6 wherein the calcium ionophore is A23187.

8. A method according to any one of claims 4 to 7 wherein the donor cell is cultured in vitro prior to treatment with the calcium releasing agent.

9. A method according to any one of claims 4 to 8 wherein the donor cell is synchronized prior to treatment with the calcium releasing agent. 12

10. A method according to any one of claims 4 to 9 wherein the donor cell is treated with the calcium releasing agent approximately 30 minutes to 3 hours prior to isolation of the nucleus.

11. A method according to any one of claims 4 to 10 wherein the donor cell is contacted with the calcium releasing agent at a concentration of approximately 5 to 50 ╬╝g/ml.

12. A method of isolating a nucleus from a donor cell, said method including providing a donor cell; and a calcium releasing agent; contacting the donor cell with the calcium releasing agent for a time sufficient to cause a morphological change in the donor cell; and isolating the nucleus from the donor cell.

13. A method according to claim 12 including providing a donor cell having a plasma membrane and a nucleus; and a calcium releasing agent; contacting the donor cell with the calcium releasing agent for a time sufficient to cause a morphological change in the donor cell; and rupturing the plasma membrane to isolate the nucleus from the donor cell.

14. A method according to claim 12 or 13 wherein the calcium releasing agent is a calcium ionophore.

15. A method according to claim 14 wherein the calcium ionophore is A23187.

16. A method according to any one of claims 12 to 15 wherein the donor cell is cultured in vitro prior to treatment with the calcium releasing agent. 13

17. A method according to claim 16 wherein the donor cell is synchronized prior to treatment with the calcium releasing agent.

18. A method according to any one of claims 12 to 17 wherein the donor cell is treated with the calcium releasing agent approximately 30 minutes to 3 hours prior to isolation of the nucleus.

19. A method according to any one of claims 12 to 18 wherein the donor cell is contacted with the calcium releasing agent at a concentration of approximately 5 to 50 ╬╝g/ml.

20. A reconstituted cell prepared by transferring an isolated nucleus to an enucleated cell by the method according to any one of claims 1 , 2, or 4 to 11.

21. An isolated nucleus prepared by the method according to any one of claims 3 or 12 to 19.

22. A method of preparing a transplantation embryo, said method including providing a reconstituted cell according to claim 20; and culturing the reconstituted cell to a transferable embryo stage.

23. A method according to claim 22 wherein the reconstituted cell is activated by treatment with a calcium releasing agent prior to or during the culture step.

24. A method according to claim 23 wherein the calcium releasing agent is a calcium ionophore.

25. A method according to claim 24 wherein the calcium ionophore is A23187.

26. A method according to any one of claims 23 to 25 wherein the reconstituted cell is contacted with the calcium releasing agent for approximately 5 to 180 minutes. 14

27. A method according to any one of claims 23 to 26 wherein the reconstituted cell is contacted with the calcium releasing agent at a concentration of approximately 5 to 50 ╬╝g/ml.

28. A method according to any one of claims 22 to 27 further including treating the reconstituted cell with a phosphorylation inhibiting agent prior to or during the culture step.

29. A method according to claim 28 wherein the phosphorylation inhibiting agent is Di-methylaminopurine (DMAP).

30. A transplantation embryo prepared by the method according to any one of claims 22 to 29.

31. An animal produced from a transplantation embryo according to claim 30.