JP2003265069A - Method for producing clone animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce a clone embryo comprising many cells to improve a conception rate and thereby enable the increase in the production of a clone animal such as livestock. <P>SOLUTION: This method for producing the clone animal is characterized by subjecting embryos prepared by a nuclear transplantation method to an aggregation culture, namely combinedly using a cell aggregation method originally aiming the production of chimeras with a nuclear transplantation technique, thereby enabling the growth and generation of the embryos. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a cloned animal by using a nuclear transfer technique and a cell assembly (aggregation) method in combination.

[0002]

2. Description of the Related Art In recent years, embryo manipulation technology has made remarkable progress. Among them, nuclear transfer technology has been extensively researched at home and abroad as a clone production technology capable of producing a large number of individuals having the same genetic trait with high economic value, and has made remarkable progress.

That is, first, the first nuclear transfer in livestock was performed by Willadsen using fertilized sheep eggs as donor cells (Nature, 320, 63-.
65, 1986). Then, the group of Roslin in England produced cloned sheep from cultured cells derived from sheep embryos,
(Nature, 380, 64-66, 1996), and further using sheep mammary gland cells to produce cloned sheep (N.
Nature, 385, 810-813, 1997), demonstrating that donor cell undifferentiation is not essential.

Then, in 1998, cloning in bovine and mouse was reported, and it was possible to develop an individual by using somatic cells of a living body by using nuclear transfer technology in mammals. Universal facts are established,
It has reached today.

[0005]

However, in the conventional nuclear transfer technique, it is possible to produce a cloned animal from undifferentiated and differentiated cells, but it is possible to produce a cloned animal from the prepared nuclear transfer embryo. There is a problem that the production efficiency of children is extremely low.

[0006] This is mainly because the conception rate is low and the miscarriage rate is high as compared with the embryo derived from the living body. In particular,
The conception rate is low because the number of cells in the embryos created by the conventional nuclear transfer technique is smaller than that in embryos derived from living organisms of the same age. This low embryo cell number is due to simultaneous removal of not only the nucleus but also the cytoplasm around it during enucleation of the recipient egg, and in vitro culture for a relatively long time after reconstructing the embryo. it is conceivable that.

The present invention has been made in order to solve such a conventional problem, and it is a practical clone which increases the number of embryonic cells per embryo, resulting in an increased conception rate and thus an increased production number of cloned animals. It is intended to provide a method for producing animals.

[0008]

The method for producing cloned animals provided by the present invention is the method described in the following (1) and (2).

(1) A clone characterized in that undifferentiated cells are used as donor cells and a plurality of cloned embryos prepared by nuclear transfer of the same cells to a recipient egg are aggregate-cultured to develop as a single embryo How to produce animals.

(2) A cloned animal characterized by using a differentiated cell as a donor cell and carrying out collective culture of a plurality of cloned embryos prepared by nuclear transfer of the same cells to a recipient egg to develop as a single embryo Production method.

The method for producing a cloned animal provided by the present invention is the application of the conventional cell assembly (aggregation) method for producing a chimera to obtain an embryo (nuclear It is possible to create transplanted embryos).

According to this production method, as shown in FIG. 1, a plurality of embryos produced by the nuclear transfer technique are collected and
Reconstruct the cloned embryo by developing as one embryo,
After development into a transplantable embryo in vitro, the embryo can be transplanted into the uterus of a foster mother to create a cloned animal.

The cell aggregation (aggregation) referred to here
The method merely involves bringing the cytoplasms into close contact with each other, and the operation can be performed relatively easily, and a manipulation device or the like is not required.

According to the nuclear transfer ensemble produced by this method, a high conception rate can be obtained as clarified in Examples. In addition, the genetic traits of the produced individual are
It was also possible to demonstrate that it was not a chimera but a clone.

The animals targeted by the present invention are mammals other than humans.

The donor cells may be undifferentiated cells such as embryonic stem cells derived from early embryos or established from early embryos, or embryonic stem cells or differentiated cells obtained from living organisms, and are not limited to particular types.

Cultivation of donor cells can be carried out by a known method suitable for each cell.

Recipient eggs may also be those used in conventional nuclear transfer techniques. However, it is better to avoid that the produced mitochondrial DNA of the cloned animal shows chimera. It is desirable to use eggs collected from the ovaries of the same individual as much as possible.

As the recipient egg, only one that is confirmed to be enucleated after the enucleation operation is used. Nuclear transfer of a donor cell to a recipient egg is performed by a known method.
That is, after nuclear replacement is performed by cell fusion or nuclear insertion, activation treatment is performed to reconstruct the embryo.

The cloned embryo (nuclear transfer embryo) thus prepared has the zona pellucida removed up to the morula and the cells of a plurality of naked embryos are assembled and brought into close contact with each other. At this time, as embryos used for assembly, only embryos that are in an appropriate developmental stage and have few morphologically good cells and are morphologically good are selected and used.

After confirming that the collected embryos develop into blastocyst stage embryos as one embryo, the embryos are transplanted to a recipient embryo recipient animal to give birth to a cloned animal.

[0022]

DETAILED DESCRIPTION OF THE INVENTION

[0023]

EXAMPLES The method for producing cloned animals of the examples will be described below in the order of steps.

(1) Ovary was promptly removed from the cow after slaughter, put in Ringer's solution kept at 25 to 30 ° C. and brought back to the laboratory, and a diameter of 8 mm was used using a 19G needle.
Unfertilized oocytes were collected from the following small follicles by the aspiration method.

(2) From among the unfertilized oocytes collected,
Only those to which the cumulus cells were tightly attached were selected by morphological observation using a stereoscopic microscope, and 20% inactivated calf serum (C
S; Gibco) -added D-PBS (CSPBS) twice, and then 5% inactivated calf serum-added TCM199 (C
S199) and wash 3 times, and then, about 80 pieces per 600 μl, 35 mm petri dish (FALCO).
N), and in a CO ₂ incubator (3% CO ₂ , 9%
Maturation culture was carried out in 7% air at 38.5 ° C for about 20 to 22 hours.

(3) Oocytes after maturation culture were treated with hyaluronidase (330 IU / ml, Sigma), cumulus cells were removed from them with a Pasteur pipette, and release of the first polar body was confirmed. No. 1 uniform egg was selected and used as a recipient egg.

(4) Oviduct-derived cells were used as donor cells. The cells contained 20% inactivated fetal bovine serum (FBS; BioWh) so that the cell number became 3 × 10 ⁶ cells / ml.
It was adjusted with DMEM containing ittaker), transferred to a 35 mm Petri dish (Falcon), and cultured in a CO ₂ incubator (3% CO ₂ , 97% air, 39.5 ° C.).

(5) When the cells have grown to a confluent state in a petri dish, trypsin solution (Trypsi)
It was isolated from the Petri dish surface using n-EDTA; Gibco), adjusted again with 20% FBS-added DMEM so that the number of cells would be 3 × 10 ⁶ cells / ml, and cultured in the same manner.

After repeating this operation 4 to 6 times, when the cells grow to a confluent state, 0.5% F
Serum starvation culture was performed for 5 days in BS-added DMEM, and this was used for nuclear transfer.

(6) For the nuclear transfer operation, first, a holding pipette and a cutting needle attached to a micromanipulator are operated under an inverted microscope to incise the zona pellucida near the first polar body of the recipient egg that has undergone maturation culture. did.

(7) Next, the incised egg is replaced with CSPB.
Cytochalasin B (5 μg / m
1, sigma), and the cytoplasm in the vicinity of the polar body was pushed out of the zona pellucida for enucleation treatment.

(8) The extruded cytoplasm is Hoechst 3
It was stained with 3342 (Calbiochem) and the presence or absence of the chromosome was confirmed by UV excitation. Only the ova confirmed to be enucleated in this manner were subjected to the subsequent operation.

(9) The enucleated recipient egg was transferred to CS199, and donor cells were inserted into the perivitelline cavity using a holding pipette and an injection pipette.

(10) About 24 hours after the initiation of maturation culture, fusion operation of an egg and a donor cell was performed. Modified ZFM (Zimmerman cell fusion)
medium), the recipient egg and the donor cell are sandwiched by needle type electrodes, and a fusion machine (ECM200
0; BTX) 23-24V-17 μsec × 2 times /
A DC pulse was applied under the condition of 150 μm to reconstruct the embryo.

(11) The fused egg which has been fused is
10 μM Ca Ionophore A23187 (Sigma)
Cycloheximide (10 μg / ml, Sigma
It culture | cultivated for 6 hours in a), and performed the composite activation process.

(12) After the treatment, the presence or absence of fusion and denaturation was confirmed, and the successful fusion embryo was cultured for development. Developmental culture is 5
CR1aa (CSCR1) supplemented with% inactivated calf serum was performed under the conditions of 5% CO2, 95% air and 38.5 ° C. Forty-eight hours after the initiation of developmental culture, a division test was performed, and the culture of only the divided cells was continued.

Table 1 shows the results of nuclear transfer in terms of electrofusion rate and division rate.

[0038]

[Table 1] (13) 72 to 96 hours after the fusion, an embryo divided into 8 cells or more is selected, and this is swollen by immersing it in Actinase E (5 mg / ml, Kaken Pharmaceutical Co., Ltd.) prepared with D-PBS as a medium for about 30 seconds. The blastomer was made naked by removing the transparent zone.

(14) The naked blastomere was lightly pipetted simultaneously with three embryos with a thin Pasteur pipette to bring the blastomere into close contact with each other.

(15) The closely attached embryo was transferred to 20 μl of CSCR1 covered with oil, and the culture was continued for 7 days after fusion.

(16) On day 7, only embryos with confirmed blastocoel cavity were used as developing embryos and transferred to TCM199 (βME199) containing 20% FBS and 100 μM β-mercaptoethanol.
The culture was continued for 24 hours.

Table 2 shows the incidence of embryos based on the presence or absence of sets.

[0043]

[Table 2] Table 2 shows that if the divided nuclear transfer embryos are assembled, it is possible to obtain embryos that reliably form a blastocoel cavity that can be transplanted.

(17) After culturing, the number of inner cell mass and trophoblast cells was measured by double staining.

Table 3 shows the number of cells. For comparison,
The biological embryo, in vitro fertilized embryo, and nuclear transfer embryo are also shown.

[0046]

[Table 3] As is clear from Table 3, the embryos created by the assembly
It can be seen that the number of cells is increased as compared with the conventional nuclear transfer embryo, and it is comparable to the embryo derived from the living body.

FIG. 2 shows a nuclear transfer assembled embryo prepared by the method of the example and a nuclear transfer embryo prepared by the conventional method. As is clear from the figure, the embryos produced by the method of the example have a morphologically large number of cells and a clear inner cell mass, and can be judged to be good quality embryos.

(18) Embryos of good morphology were transplanted to the recieved cows on the 8th day after estrus.

Table 4 shows the transplantation results. As is clear from Table 4, the production rate of surviving individuals also improves as the conception rate increases.

[0050]

[Table 4]

[0051]

As described above, according to the present invention,
By combining the cell aggregation (aggregation) method, which was originally intended to create chimeras, with the nuclear transfer technology, multiple embryos created by nuclear transfer were collectively cultured, so that embryo cells per embryo By increasing the number, the conception rate can be improved, and thus the number of cloned animals produced can be increased.

[Brief description of drawings]

FIG. 1 is a process diagram showing a method for producing a cloned animal according to the present invention.

FIG. 2 is a micrograph showing a nuclear transfer ensemble embryo produced by the method for producing a cloned animal of Example.

FIG. 3 is a micrograph of a nuclear transfer embryo produced by a conventional method for producing a cloned animal.

Claims (2)

[Claims] 1. A cloned animal, wherein undifferentiated cells are used as donor cells, and a plurality of cloned embryos prepared by nuclear transfer of the same cells to a recipient egg are aggregate-cultured to develop as single embryos. Production method. 2. A cloned animal characterized in that a plurality of cloned embryos prepared by nuclear transfer of the differentiated cells as donor cells to recipient ova are developed as single embryos. Production method.