JP2002238573A - Method for reconstructing embryo - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for reconstructing an avian clone embryo having genetic information derived from a donor cell. SOLUTION: This method for reconstructing the embryo comprises trans- grafting the donor cell into a bovine egg the nucleus of which is removed to produce a xeno nucleus-grafted embryo and then re-trans-grafting the xeno nucleus-grafted embryo into an avian egg the nucleus of which is removed. On trans-grafting and re-trans-grafting, the embryos are activated by adding an instantaneous electrical stimulation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for reconstructing an embryo. More specifically, the present invention relates to a method for reconstructing an embryo having genetic information of a donor cell which is a bird cell. INDUSTRIAL APPLICABILITY The present invention can be used for producing a clone of a bird (clone bird) or a transgenic bird (transgenic bird).

[0002]

2. Description of the Related Art One fertilized egg repeatedly divides into two cells and four cells to produce an animal. During development, cells follow their fate and become specialized. Cells differentiate and change, but the genome of each cell does not change. Every cell has the same genetic information, no matter how it looks like muscles, bones, or nerves. All cells in the body inherit the same genome from the fertilized egg. Gurdon succeeded in producing tadpoles from frog somatic cells differentiated using nuclear transfer (Gurdon, JB: Journal of Embryology and Experimen
tal Morphology 10: 622-640,1962). This is because in amphibians, even somatic cells that have already differentiated and become specific tissues and organs can be initialized by reinitializing differences in gene expression patterns and having totipotency again. , It is possible to generate a new individual.

[0003] In mammals, cloned bovines have been successfully produced by nuclear transfer of cells from the inner cell mass of a blastocyst (First, NL, Sims, MM, Park, SP and
Kent-First, MJ: Reproduction Fertility, and Deve
lopment 6: 553-62, 1994). This indicated that the differentiation potential of the cells was not limited until a certain early stage of development, that is, they had a totipotency. Later, Campbell and colleagues reported that cloned sheep were successfully produced by nuclear transfer using differentiated cultured cells from the fetus. (Campbell, KH
S., McWhir, J., Ritchie, WA and Wilmut, I .: Nature
380: 64-66, 1996). In a similar manner, Wilmut et al. Succeeded in producing cloned sheep from mammary gland cells, the somatic cells of adult sheep (Wilmut, I. Colman, A. an.
d Campbell, KH: Science 278: 2130-2133, 1997).
Goats, cattle, pigs, and mice have also been reported to produce somatic cell clones using nuclear transfer (Baguisi, A. et.
al .: Nature Biotechnology 17: 456-461, 1999; Cibelli,
BJ et al .: Science 280: 1256-1258, 1998; Wakayam
a, T. et al .: Nature 394: 369-374,1998; Onishi, A. e.
tal .: Science 289: 1188-1190, 2000). Somatic cell cloned animal production by nuclear transfer as described above can be broadly divided
(1) selection of donor cells and genetic manipulation, (2) preparation of recipient enucleated eggs and nuclear transfer by microinjection,
(3) Activation of reconstructed embryos and development in vitro, (4) transfer to foster mother, (5) development in uterus until parturition.

[0004] When a cloned animal is produced, if a foreign gene is incorporated into the nucleus to be transplanted by genetic manipulation, a cloned animal having the foreign gene as a part of genetic information can be produced. That is, the cloning technique can be regarded as one of the gene manipulation techniques. The cloned animal obtained by performing the genetic manipulation is used to prepare a disease model animal,
It can be used for various purposes such as production of organs for human transplantation and use as a useful substance production system. In particular, utilization as a useful substance production system, that is, utilization as an animal bioreactor, has attracted attention. Utilizing an animal individual as a bioreactor has the advantage that a large number of pharmaceuticals and the like that are industrially difficult to produce can be produced. It has been reported that microorganisms and cultured animal cells were used as production systems to produce immune-related substances such as interferons, growth factors, hematopoietic factors, hormones, etc. (Tatsuo Muramatsu (199
6) Animal Production and Biotechnology, pp.6-9, Bunei-do, Tokyo.). Here, when a microorganism is used in a production system, there are problems such as a bacterial toxin possessed by the microorganism itself, and problems such as post-translational modification such as glycosylation, methylation, and SS cross-link formation. On the other hand, when animal cultured cells are used, there is a problem that a huge running cost is required for reagents necessary for culturing the cells and the like, which is not suitable for mass production.

[0005] On the other hand, using an animal individual as a production system means that, once the initial cost for producing an animal suitable for the production of the target substance has been set apart, the animal individual that has been successfully produced once is used for the generation of the animal. The maintenance is excellent in that a target substance can be produced for a long period of time. So far, research has been conducted to produce transgenic animals in which a foreign gene has been introduced into the germ line in cattle, pigs, sheep, goats, chickens, and the like, and to use these as practical systems for producing useful substances.
Sheep and goats have been reported to successfully produce recombinant protein in their milk (Ebert,
KM et al .: Bio / Technology 9: 835-838,1991; Wrigh
t, G. et al .: Bio / Technology 9: 830-834,1991; Wall,
RJ et al .: Transgenic Research 5: 67-72, 1996). However, although transgenic animals have been successfully produced in other animal species (chicken, cattle, etc.), they have not yet been put into practical use due to their inefficiency. In particular, due to the low efficiency of gene transfer into the germ line, there was a problem that the rate of expression of the foreign gene in the resulting offspring was low. So, instead of the existing method, somatic cell cloning technology by nuclear transfer is also expected as a transgenic animal creation method,
In sheep, using human clotting factor IX
Has been successfully secreted into milk (Schnieke, A.
E. et al .: Science 278: 2130-2133, 1997).

When an animal is used as a useful substance producing system (animal bioreactor), it is necessary to consider the sexual maturation period, gestation period or hatching period of livestock and poultry, the number of offspring (eggs), and the like. Chicken has been studied in detail in the development process to date, and is considered to be one of the leading candidates as an animal bioreactor in terms of the establishment of individual breeding management techniques. Chicken eggs contain about 3 g of egg white protein per egg, and if a part of this protein can be used as a recombinant protein, it is possible to mass-produce useful substances simply by collecting eggs laid down every day. It can be a production system. The phenomenal high productivity of chicken eggs is evident in view of their prices.

However, to date, the production of useful substances using chickens has not been put to practical use, and only a few examples of transgenic chickens have been produced. The cause is largely due to bird-specific breeding and spawning systems. Specifically, 1) it is extremely difficult to identify the pronucleus of a fertilized egg because the chicken egg is full of yolk and is opaque. 2) The fertilized egg at the 1-cell stage must be removed from the fallopian tube. 3) At ovulation, about 24 hours after ovulation, the chicken embryo divides to 60,000 to 100,000 cells, which makes genetic manipulation difficult for eggs And so on. However, various methods have been devised for the in vitro operation of fertilized eggs, and it has become possible to hatch chicken embryos by in vitro culture (Perry, MM: Nat
ure 331: 70-72, 1988). Since the development of chick embryos is easy to observe, the development process has been thoroughly investigated so far, and the processes of differentiation and development of germline cells are also known. However, cell division progresses, and immediately after oviposition, it is extremely difficult to create a transgenic chicken by introducing a foreign gene into a chicken embryo consisting of tens of thousands of cells using existing methods developed in mice and the like. Have difficulty. In the case of somatic cell cloning technology using nuclear transfer, gene manipulation can be performed at the stage of cultured cells, and it can be said that this technology can overcome the disadvantages of existing methods such as low gene transfer efficiency. Therefore, application of somatic cell cloning technology to chickens can be expected as a new method for producing transgenic chickens.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of the above technical background, and an object of the present invention is to provide a method for reconstructing an avian embryo in order to establish an avian cloning technique.

[0009]

Means for Solving the Problems With the above object, the inventor of the present invention transplanted a nucleus of a donor cell into a chicken egg and tried to produce a reconstructed embryo having genetic information derived from the donor cell.
As a result, the method of developing a reconstructed embryo by adopting a method in which a nucleus of a donor cell is once transplanted into an ovum of a xenogeneic animal to produce a xenonuclear transfer embryo and then re-transplanted into an enucleated chicken egg Was found, and the present invention was completed.
The configuration of the present invention is as follows. a) nucleating a donor cell consisting of a first avian cell,
Transplanting to enucleated ova of a bird and a heterologous animal to form a xenograft embryo; and b) transplanting the xenograft embryo to an enucleated egg of a second bird. A method for reconstructing an embryo having genetic information derived from the donor cell.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The method for reconstructing an embryo of the present invention comprises the steps of: a) transplanting a nucleus of a donor cell comprising a first bird cell into an enucleated oocyte of an animal different from the first bird; Forming a xenograft embryo, and b) transplanting the xenograft embryo into an enucleated egg of a second bird, wherein the xenograft has genetic information derived from the donor cell. It is a method of reconstructing the embryo.

In the present invention, first, a nucleus of a donor cell composed of cells of a first bird is transplanted into an enucleated ovum of an animal different from the first bird to form a xenotransplanted embryo (step). a)). The species of the first bird is not particularly limited, and is, for example, a chicken, a quail, a duck, a goose, a turkey, or a pigeon. Preferably, it is a chicken or a quail. More preferred are chickens. It is preferable to select birds whose donor cells are readily available.

When a gene is introduced into a donor cell before transplantation, it is preferable to use a donor cell that can easily perform a gene manipulation. Donor cells include, for example, fetal fibroblasts or DT40, a cell derived from chicken B lymphocytes. Fetal fibroblasts can be prepared by a known method. For example,
A method for preparing chicken embryo fibroblasts is described in (Kita et al. (199
6) Asian-AustralasianJ. Anim. Sci., 9: 701-703). DT40 is a cell derived from B lymphoma and has the feature that chromosome 2 is trisomy (Baba, TW, G
iroir, BP and Humphries, EH: Virology 144: 139
-151,1985). DT40 is said to be the only chromosomal target and random integration occurring at the same frequency in higher eukaryotes (Buerstedde, J.
M. and Takeda, S .: Cell 67: 179-188, 1991). Therefore, DT40 can easily knock-in and knock-out a specific gene, and not only can produce a transgenic chicken by insertion mutation of a foreign gene, but also can create a chicken in which a specific gene is knocked-in or knocked out. DT40 can be obtained from Dr. Shunichi Takeda (Kyoto University Graduate School of Medicine). Further, it is preferable to use, as donor cells, cells derived from birds of the same species as the second bird described below. This is because the success rate of embryo reconstruction can be improved by using the same type of birds (derived) as the donor cells and the enucleated eggs whose nuclei are ultimately transplanted.

A foreign gene can be introduced into the nucleus of a donor cell before transplantation, and the foreign gene can be incorporated into chromosomal DNA. By using a donor cell having such a nucleus, a reconstructed embryo having a foreign gene as a part of genetic information can be obtained. If an individual is developed based on this reconstructed embryo, a transgenic animal can be produced.

Here, the foreign gene means a gene other than the gene of the donor cell itself. Therefore, not only genes of different species (eg, genes of humans, cattle, pigs, etc.) are included, but also genes of birds of a different species from donor cells, and cells of the same species but derived from different tissues can be obtained. It is meant to include the gene and the like obtained. Preferably, it is a human gene. This is because by using a human gene as a foreign gene, the present invention can be used for producing a transgenic animal having a gene encoding a protein present in a human body. Such a transgenic animal can be used for producing a protein having a function equivalent to a protein existing in a human body. Human genes include human erythropoietin gene, human fibrinogen gene, human serum albumin gene, human lactoferrin gene, human protein
C, human α-glucosidase gene and the like, but are not limited thereto. When another species has a protein having a function equivalent to that of a human protein, it is also preferable to use a gene encoding a protein of the other species as a foreign gene. Not only the naturally occurring gene, but also a DNA obtained by partially modifying the nucleotide sequence of the natural gene or D synthesized by a well-known or known technique
NA can be used as a foreign gene in the present invention.

The nuclei of the donor cells are transferred to enucleated ova of a xenogeneic animal to produce a xenonuclear transfer embryo. Xenograft embryos refer to embryos from which the nuclei of the donor cells and the enucleated ovum as the recipient cells are derived from different animal species. On the other hand, in the case of the same animal species (which may be a different individual) found in general nuclear transfer, it is a (transgenic) nuclear transfer embryo. The heterologous animal (derived from the enucleated ovum as the recipient cell) here is not particularly limited, but includes, for example, cows, pigs, sheep, goats, and horses. From the viewpoint of ease of operation, it is preferable to employ an animal having a large egg. For example, it is preferable to use enucleated ova of cattle, pig, or sheep. For cow or sheep eggs,
Culture methods, transplantation methods and the like have been well studied, and the use of these methods is particularly preferable from the viewpoint of setting conditions and the like.

The enucleated ova of a heterologous animal can be prepared by removing the first polar body and the female pronucleus from the ova of a heterologous animal by a known method (Toshihiko Asano, Masanobu Hayashi (1)
995) Biomedical Research Manual -Animal Experiment Method- Vol.VII Special Experiment Equipment.pp. 82-118, Yokendo,
Tokyo). For example, the transparent body surrounding the egg is cut with a knife or the like, and then the first polar body and the female pronucleus are extruded by applying external pressure to the egg to extrude the egg cytoplasm including the polar body and the female pronucleus. Can be removed.

The transfer of the nucleus of the donor cell to the enucleated ovum can also be performed by removing the nucleus from the donor cell in advance and introducing the nucleus into the enucleated ovum of a heterologous animal. The transfer is performed by introducing the nucleus of the donor cell into the enucleated egg by introducing the cell membrane of the donor cell and the cell membrane of the enucleated egg into the enucleated egg. The fusion here can be performed by a method of applying an instantaneous voltage. Such a fusion method is generally known as an electroporation method, and can be performed using a known electroporator or the like.
(Kakuta (1992) Nissha Livestock Bulletin, 63: 192-200). As the electrode, a type in which an egg is directly sandwiched or a type in which a parallel electrode is used can be used. In the former case, the electrode is placed in contact with the ovum. On the other hand, in the latter case, the two electrodes are placed in parallel, and the eggs are placed so as not to come into contact between them. Conditions such as the magnitude of applied voltage (pulse voltage), application time (pulse time), number of applications (number of pulses), and pulse interval are appropriately adjusted so that fusion is appropriately performed. For example, using a rectangular DC voltage, an applied voltage of 50 to 300 V /
mm, application time 10 to 100 μsec, application frequency 1 to 5 times, pulse interval 1 sec to 3 hours. As more preferable conditions, for example, using a rectangular DC voltage, an applied voltage of 75 to 250 V / mm, an application time of 15 to 50 μsec, and an application frequency of 1 to 3
The pulse interval is 1 sec to 30 min. The electrical stimulus during this fusion can activate the nucleus after transplantation and initiate division. That is, when the electroporation method is used for the transfer of the nucleus of the donor cell, the nucleus after the transfer is activated simultaneously with the transfer of the nucleus, and the development (division) of the xenogeneic transfer embryo can be started. it can. Donor cell nuclear transfer, Sendai virus method,
In the case of performing by other known methods such as a method using polyethylene glycol (PEG), it is preferable to activate the nucleus after transplantation by giving an electrical stimulation at the time of transplantation or after transplantation. By simply fusing caryoplasts to enucleated eggs by the Sendai virus method, etc., the chromosomes stop in a metaphase-like state and are in the same state as the chromosomes in the second metaphase of meiosis just before fertilization. That is, it is necessary to activate by giving a parthenogenetic stimulus. In addition, when fusion is performed by the Sendai virus method or the like, activation stimulation can be given by ethanol treatment or the like, but it is preferable to give electrical stimulation as described above.

The xenotransplant embryo is reimplanted into a second bird enucleated egg (step b)). As a result, embryos having donor cell-derived genetic information and high developmental activity are reconstructed. That is, a reconstructed embryo is formed. The species of the second bird is not particularly limited, and is, for example, a chicken, a quail, a duck, a goose, a turkey, or a pigeon. An appropriate one can be adopted in consideration of the availability, the ease of nuclear transfer operation, the usefulness in producing a cloned bird or a transgenic bird, and the like. For example, it is preferable to use chicken and quail. In particular,
It is preferable to use a chicken whose development process is well studied. It is an easily available strain among chickens.
For example, it is preferable to use a single crown white leghorn.

The enucleated egg of the second bird is enucleated by a known method (eg, for an appropriate time γ).
Irradiation with a ray). As the eggs of the second bird, a fertilized egg or an unfertilized egg can be used, but according to the study of the present inventors, it was better to use a fertilized egg,
It was observed that development from reconstructed embryos was more advanced. Therefore, it is preferable to use a fertilized egg obtained by enucleation as the enucleated egg in the present invention. Reimplantation can be performed by introducing the xenograft embryo prepared as described above into an enucleated egg of a second bird, and then instantaneously applying a voltage. By this instantaneous application of the voltage, the reconstructed embryo can be activated and the development from the reconstructed embryo can be started.

The method of instantaneously applying a voltage is generally known as an electroporation method, and can be performed using a known electroporator or the like. Conditions such as the magnitude of the applied voltage (pulse voltage), application time (pulse time), number of applications (number of pulses), and pulse interval ensure that reimplantation is performed properly and that the reconstructed embryo is activated And it is adjusted appropriately so that generation can be started. For example, using a rectangular DC voltage,
-100 V, application time 25-200 msec, application frequency 1-10 times, pulse interval 1-1 min. More preferable conditions include, for example, an applied voltage of 20 to 50 V, an application time of 50 to 100 msec, an application frequency of 2 to 4 times, and a pulse interval of 1 to 10 s.
ec. The reimplantation (step b)) is preferably performed as early as possible after the start of the xenograft transfer embryo produced as described above. Thereby, it is expected that the probability of development from the reconstructed embryo can be increased. For example, it is preferable to perform retransplantation using a xenograft embryo of 2 to 8 cell stages, and more preferably 2 to 4 cells.
Reimplantation is performed using the cell-stage embryo.

[0021]

Hereinafter, the present invention will be described in more detail with reference to examples. [Example 1] Preparation of donor cells Donor cells include chicken fetal fibroblasts (hereinafter, "CE
F "), and DT40 derived from B lymphoma caused by chicken leukemia virus. (1) Preparation of chicken embryo fibroblasts The following reagents were prepared. First, the final concentrations are 13
Sodium chloride (special grade, Wako Pure Chemical, Osaka), potassium chloride in distilled water to be 7 mM, 2.7 mM, 4.3 mM, and 1.4 mM
(Special grade, Wako Pure Chemical, Osaka), disodium hydrogen phosphate (special grade, Wako Pure Chemical, Osaka), and potassium dihydrogen phosphate (special grade, Wako Pure Chemical, Osaka) are dissolved, then autoclaved and Phosphate- Buffered Saline (hereinafter “PBS”)
Was prepared. 0.001% Fungizon in PBS (No.15295-017, Gib
co BRL, USA), 50 μg / mL Gentamycin (No. 15750-060, G
ibco BRL, USA) to prepare PBS for washing (hereinafter referred to as “WPBS”). Also, 0.25% Trypsin (No.T80
03, Sigma, USA) and 0.025% EDTA (No. 345-1865, Wako Pure Chemical, Osaka) was added to prepare a dissociation solution. Next, 500mL
Dulbecco's Modified Eagle Medium (No. 11965-092,
Gibco BRL, USA, hereafter referred to as "DMEM"), 50mL Fetal B
ovine Serum, Certified (No.16000-044, GibcoBRL, US
A, hereinafter referred to as “FBS”), 300 μL Penicillin-Strept
mycin-FungizonMixture (No.531-07761, Wako Pure Chemical, Osaka) and 2.95 g / L Tryptose PhosphateBroth (No.0060-
17, Difco Laboratories, USA) and mix with Nalgene Disp.
A culture solution (hereinafter, referred to as “DMEM + 10% FBS + TPB”) was prepared by filtration sterilization with an orsableFilter Unit (No.154-0020, Nalgene, Tokyo). The following operations were performed using these reagents.

Incubator & Heater P-008 (Ashida Sangyo, Okayama)
For 9 days, and then sterilized with 70% ethanol. Next, the chicken embryo was removed from the egg and transferred to a sterile petri dish in a clean bench. All subsequent operations were performed in a clean bench. First, the neck of the fetus was cut off with scissors. The fetus was thoroughly washed with WPBS and transferred to another sterile petri dish. Subsequently, the fetus wings and legs were cut with scissors, and the fetus was minced with scissors. The tissue was transferred to a 50 mL centrifuge tube, 10 mL of the dissociation solution was added, and the mixture was incubated at 37 ° C. for 10 minutes using a shaking incubator. Then 10
The mixture was centrifuged at 00 rpm and 4 ° C for 5 minutes, and the supernatant was discarded. There, 1
0 mL of DMEM + 10% FBS + TPB was added and gently suspended.
Thereafter, the mixture was centrifuged at 1000 rpm at 4 ° C for 5 minutes, and the supernatant was discarded.
10 mL of DMEM + 10% FBS + TPB was added again, and the same centrifugation treatment was performed as described above. After adding and suspending DMEM + 10% FBS + TPB, the cells were plated on a culture dish so that the cell number became 7 × 10 ⁶ / mL. The culture dish was transferred to a 37 ° C. CO ₂ incubator and cultured for 1 hour. DMEM + 10% FBS + TPB was removed from the culture dish after the culture, and 5 mL of DMEM + 10% FBS + TPB was newly added, followed by culturing again in a CO ₂ incubator at 37 ° C.

(2) Preparation of chicken DT40 cells The chicken DT40 cells (hereinafter referred to as "DT40") were provided by Dr. Shunichi Takeda, Graduate School of Medicine, Kyoto University. 500mL DMEM (No. 11965-092, Gibco BRL, US
A), 50mL FBS (No.16000-044, Gibco BRL, USA), 300
μL Penicillin-Streptmycin-Fungizon Mixture (No.53
1-07761, Wako Pure Chemical, Osaka) and mix with Nalgene Dispors
Filtered and sterilized with an able Filter Unit (No.154-0020, Nalgene, Tokyo) was used as a DT40 culture solution (DMEM + 10% FBS). Add 5 mL of this culture solution to a culture dish (No.MS-2005R, Sumil
on, Tokyo) and DT40 was cultured in a CO ₂ incubator at 37 ° C.

[Example 2] Preparation of enucleated bovine egg (1) Collection of bovine ovary First, 8.5 g of sodium chloride was dissolved in 1 L of distilled water and autoclaved. After cooling, 100 IU / mL of penicillin (10000 units) / 1 vial, Meiji Seika, Tokyo), Streptomycin 1
By adding 00 μg / mL (1 g titer / 1 vial, Meiji Seika, Tokyo), 0.85% sterile saline (hereinafter, `` saline '')
Was prepared. Next, tissues such as fallopian tubes and fat were cut off from the ovaries of the cows collected at the slaughterhouse and stored by immersion in physiological saline heated to 37 ° C.

(2) Maturation culture Glucose (special grade, Wako Pure Chemical, Osaka), sodium pyruvate (No. P2256, Sigma, USA), bovine serum albumin in PBS
(No.A4378, Sigma, USA, hereinafter referred to as `` BSA ''), penicillin (No.KOJ-210, Cosmo Bio, Tokyo), and streptomycin (No. , 5.55 mM, 0.33 mM, 0.3%, 100 IU / mL,
And 0.3% bovine serum albumin-added PBS (hereinafter, referred to as “PBS (+)”).

Next, the ovaries were washed three times with physiological saline, then transferred into a beaker containing physiological saline, and immersed in a 37 ° C. constant temperature bath to keep the temperature. Remove the ovaries from the beaker,
Water and blood were wiped off with a Kim towel. The needle of the syringe containing PBS (+) was inserted from the ovarian parenchyma, and the ovum in the small follicle was sucked into the syringe together with the follicular fluid. The aspirated follicular fluid was slowly transferred to a culture dish immersed in a thermostat. Subsequently, the plate is washed once with a culture dish containing PBS (+), and further containing a culture solution of denuded fertilized egg for bovine in vitro fertilization (IFP9651, Functional Peptide Research Institute, Yamagata; hereinafter, referred to as “IVD101”). After washing three times in a culture dish, the cells were transferred into a 50 μL drop of IVD101. Mineral oil (No.0003-0559-61, ERSq
uibb & Sons, USA) and cultured in a CO ₂ incubator at 37 ° C for 24 hours.

(3) Enucleation of Bovine Ovum The following operation was performed using a micromanipulator (micromanipulator M type, Leica, Germany) with a microinjector (LA-205, Leica, Germany) set. First, the cumulus cells attached to the ovum were removed by pipetting. IV made on a sterile petri dish
The D101 drop was covered with mineral oil and the bovine ovum was transferred. Fix the egg so that the polar body is on the top, and use a cutting knife (glass tube (No.2-000-010, Drummond, U
(SA) with a sharpened tip) penetrated the point closest to the junction between the polar body and the egg cytoplasm, and made a slit in the transparent body.
After moving the egg to the top of the holding pipette,
It was pressed from above with a cutting knife to push out the egg cytoplasm, which was thought to contain polar bodies and female pronuclei. The enucleated eggs were cultured with IVD101.

[Example 3] Heteronuclear transplantation and fusion The fetal chicken fibroblasts prepared in Example 1 and DT40 were used as donor cells. The bovine enucleated oocytes prepared in Example 2 were used as recipient eggs. A micromanipulation system was used for the following operations. The IVD101 drop made in a sterile petri dish was covered with mineral oil, and the recipient egg was transferred. Next, donor cells were aspirated one by one using an injection pipette. The recipient egg was fixed with a holding pipette so that the slit of the transparent body was in the horizontal direction.
Donor cells were implanted into recipient eggs using an injection pipette (nuclear transfer of donor cells). Recipient eggs after nuclear transfer were transferred to IVD101 and cultured in a CO ₂ incubator at 37 ° C. Subsequently, a 0.3 M mannitol solution (54.7 g / L mannitol, 5.6 mg / L CaCl ₂ , 24.7 mg / L MgS
O ₄ ) and transfer to an electroporator (Electroporation sy
stem, CUY21, Bex, Tokyo) using 75V / mm, 60μsec, 2
The cell membrane of the donor cell and the cell membrane of the recipient egg were fused by a single electrical stimulation. The reconstructed embryo thus obtained (hereinafter referred to as “heteronuclear transfer embryo”) was transferred to IVD101 and incubated at 37 ° C.
In a CO ₂ incubator.

Example 4 Investigation of Development of Xenograft Transfer Embryos A 40 μL drop of IVD101 was made on a sterile petri dish, covered with mineral oil, and each drop was provided with one xenograft transfer embryo prepared in Example 3. The cells were transferred and cultured in a CO ₂ incubator at 37 ° C. for 12 days. The state of development of the xenotransplantation embryo was observed every 24 hours using a stereoscopic microscope (SZX12, Olympus Optical, Tokyo), and a photograph was taken with a CCD camera (M-3204C, Olympus Optical, Tokyo). FIG. 1 (A) shows the state of development of a xenograft embryo when DT40 is used as a donor cell. FIG. 1 (b) is a graph showing the percentage of cells that have progressed to each developmental stage. The horizontal axis is the development stage, and the vertical axis is the percentage of cells that have progressed to each stage (Percentage of degeneration).
veloping cells). As a comparative control, the case where bovine ES-like cells (CatteleES Like cells) were used as donor cells (left column) is also shown. As shown in FIGS. 1 (A) and (B), when bovine ES-like cells are used as donor cells (bovine / bovine allogeneic nuclear transfer embryo), the division progresses to morula and blastocyst. In contrast, when DT40 cells were used as donor cells (chicken / bovine xenograft embryos), division progressed smoothly at first, but all embryos stopped dividing at the 8-cell stage.

Example 5 Transplantation into enucleated chicken eggs and
In ovo outbreak (1) Enucleation of chicken eggs Chicken eggs and unfertilized eggs purchased from Aichi Chicken Federation (Nagoya) were irradiated with about 2,000 rad of γ-ray, respectively, to enucleate.

(2) Transplantation, fusion and activation to enucleated chicken eggs First, using a transplantation pipette,
Heterogeneous nuclear transfer embryos developed up to the 2-4 cell stage were sucked. On the other hand, the enucleated chicken eggs prepared in the above (1) were disinfected using 70% ethanol with their sharp ends facing up. Next, add a diamond disc to the sharp end of the chicken egg.
(A5131, Nippon Physical and Chemical Instruments, Tokyo), and a hole with a diameter of about 2.5 cm was made in the blunt end side using a diamond bar (A1403, Nippon Physical and Chemical Instruments, Tokyo). Subsequently, the tip of the pipette for injection was carefully pierced into the chicken embryo, and the xenonuclear transfer embryo prepared in Example 3 was injected so as to be placed on the yolk.

Next, a 20G injection needle electrode (NN
-2038R, Terumo, Tokyo) and an eye spot electrode was placed on the chicken embryo on the acute end side. In this state, electrical stimulation (applied voltage 30
V, an application time of 100 μsec, and a pulse number of 2 times). After the electroporation, the hole on the blunt end of the chicken egg was closed with an adhesive, and the hole on the sharp end was closed with an adhesive and a polyvinylidene chloride film. After that, the incubator continued to incubate the eggs at an angle of 90 degrees every three hours in an incubator. A reconstructed embryo was prepared as described above. As a control, the CEF or DT40 prepared in Example 1 was used instead of the xenograft embryo.
Was directly transplanted to enucleated chicken eggs.

Example 6 Investigation of Development of Reconstructed Embryo Embryo development was observed through a polyvinylidene chloride film through a window opened on the acute end side of the chicken eggs transplanted in Example 5. Eggs that have stopped developing are removed to a 100 mm sterile petri dish and the size of chicken embryo (Diameter of blastodermal dis
c) was measured to estimate how many days had survived.
As a result, it was found that when reconstructed embryos were produced by directly introducing donor cells into enucleated chicken eggs, development hardly progressed even if hatching was continued. On the other hand, when the donor cells are once transplanted into a bovine enucleated non-fertilized egg to construct a xenogenic nuclear transfer embryo, and when the xenonuclear transfer embryo is re-transplanted into an enucleated chicken egg, the fertilized egg is regarded as an enucleated chicken egg 4 to 5 when using
Embryonic development was observed in day embryos, and about one day embryo was observed even when unfertilized eggs were used as enucleated chicken eggs.

The table in FIG. 2 summarizes the results of observation of embryo development. The upper row in the table shows donor cells (DT40 or CE
F) is once transplanted into a bovine ovum (bOocyte) as a recipient to construct a xenogenic nuclear transfer embryo, and thereafter, a chicken egg (fertilized egg: cFertilized egg or unfertilized egg: cUnfertilized egg) obtained by enucleating the xenogenic nuclear transferred embryo ) Is the result of development when re-implanted to produce a reconstructed embryo, lower row (below the dotted line)
Is a chicken egg (fertilized egg: cFertilized egg or unfertilized egg: cUnfertiliz) enucleated from donor cells (DT40 or CEF)
This is the result of development when a reconstructed embryo was produced by direct transplantation into an ed egg). Diameter of blastoderm
al disc), the number of embryos (N
o. of developing embryos) is indicated. In the table, development of the vascular system (blood islands) was observed in the embryos marked *.

Example 7 Investigation of the Origin of the Embryo Embryo The origin of the genome of the most advanced embryo in Example 6 (in the table of FIG. 2, the blastoderm diameter is 10 mm or more) is as follows. I looked up like that. (1) Extraction of genomic DNA from embryos Wizard Genomic DNA Purification Kit (A1120, Promeg
a, USA) to extract genomic DNA from the embryos that had developed.

(2) Extraction of Genomic DNA from Donor Cells Genomic DNA was extracted from DT40, a donor cell, as follows. First, after washing DT40 with PBS, cells were collected by centrifugation, and subsequently, a 5% Chelex100 solution (Chelex100 solution).
(124-2822, Bio-Rad, USA) dissolved in sterile distilled water to a concentration of 5% (W / V)) and incubated at 100 ° C. for 8 minutes. Next, after stirring for 15 seconds, 5000 rpm 4 ° C
And centrifuged for 5 minutes. Then, it was stored at -70 ° C.

(3) Investigation of the origin of genomic DNA by PCR method PCR was performed using the genomic DNA extracted in (1) and (2). The PCR method was performed in two steps using two primer sets designed to amplify a sequence specific to DT40. FIG. 3 shows the sequences of the primers. The upper (F1 (SEQ ID NO: 1) and R1 (SEQ ID NO: 2)) primer sets used in the first-stage reaction, the lower (F2 (SEQ ID NO: 3) and
R2 (SEQ ID NO: 4)) is the primer set used in the second-stage reaction.

First, the DNA (template DN) obtained in (1) or (2)
A) 1.0 μL, upstream primer (F1) 0.5 μL, downstream primer (R1) 0.5 μL, dNTP mixture (No.R001A, Takara Shuzo,
Kyoto) 8 μL, 10 × PCR buffer (No.R001A, Takara Shuzo, Kyoto) 10 μL, Taq DNA Polymerase (No.R001A, Takara Shuzo,
(Kyoto) 0.5 μL and sterilized water 79.5 μL were dispensed into a 0.5 mL tube, and several drops of minerala oil (No. 538-2113, Wako Pure Chemical, Osaka) were dropped from above. PCR reaction is 94 ° C for 30 seconds, 60 ° C for 90 seconds.
Second cycle and 72 ° C. for 60 seconds were defined as one cycle, and this cycle was repeated for 20 cycles.

Next, PCR was performed again using the amplified DNA as a template.
Was done. At this time, F2 (upstream primer) and R2 (downstream primer) in FIG. 3 were used. Other conditions were the same as above. After the above two-step PCR reaction, the amplified band was confirmed by electrophoresis.

FIG. 4 is a photograph of the gel after electrophoresis.
In the figure, the arrow indicates a band (243 bps) specific to DT40.
Lane 9 is a lane in which a sample obtained by amplifying the DNA extracted in the above (1) (DNA extracted from the most developed embryo) by PCR was run. Lane M is a molecular weight marker, P is a lane (positive control) in which a sample obtained by PCR-amplifying the extracted DNA (DNA extracted from DT40) was run (positive control), and N1 was a PCR without using template DNA. Lane (negative control) in which the sample was electrophoresed, N2 was a lane in which plasmid DNA (pRL-SV40) was flown (negative control), lanes 1 to 8 were DNAs extracted from embryos normally developed from normal chicken eggs, This is a lane in which a DNA sample subjected to PCR in the same manner as described above was electrophoresed.

As can be seen from FIG. 4, no DT40-specific band was amplified at all from DNA derived from normal embryonic development (lanes 1 to 8), but the most reconstructed reconstructed in Example 6. A band specific to DT40 was detected from the embryo DNA (lane 9). Therefore, this reconstructed embryo
It is presumed to have DNA from DT40, a donor cell. That is, it was estimated that the embryo having the donor cell-derived genetic information was reconstructed.

The present invention is not at all limited to the description of the above-described embodiments and examples. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art.

[0043]

According to the method of the present invention, a reconstructed avian embryo having genetic information derived from a donor cell can be obtained. That is, cloned birds derived from donor cells (clone bird)
Provided is a method for producing a reconstructed embryo for producing E. coli. In addition, a method for producing a reconstructed embryo for producing a transgenic animal (transgenic bird) into which a desired foreign gene has been incorporated by subjecting the donor cell to genetic manipulation in advance and incorporating the foreign gene is provided. Thus, the method of the present invention provides a basic technology for producing cloned birds or transgenic birds.

[0044]

[Sequence List] SEQUENCE LISTING <110> Muramatsu, Tatsuo <120> A method for reconstituting embryo <130> C01002 <140> <141> <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer designed for amplifying DT40 specific sequence <400> 1 tctctcctct ccctctccag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer designed for amplifying DT40 specific sequence <400> 2 ctcgcacagt aatagacagc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer designed for amplifying DT40 specific sequence <400> 3 tctccaggtt ccctggtgca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer designed for amplifying DT40 specific sequence <400> 4 gaccccagtg atggttaatg 20

[Brief description of the drawings]

FIG. 1 is a photograph and a graph showing a state of development of a heterologous nuclear transfer embryo in Example 4. FIG. 1 (A) shows the state of development of a xenograft embryo when DT40 is used as a donor cell. FIG. 1 (b) is a graph showing the percentage of cells that have progressed to each developmental stage. The horizontal axis is the development stage, and the vertical axis is the percentage of cells that have progressed to each stage (Percentage of degeneration).
veloping cells). As a comparative control, a case where bovine ES-like cells (Cattele ES Like cells) were used as donor cells (left column) is also shown.

FIG. 2 is a table summarizing the results of observation of embryo development in Example 6. In the upper row of the table, the donor cells (DT40 or CEF) were transformed into bovine ovum (bOo
cyte) to construct a xenogeneic embryo, and then
Chicken eggs enucleated from xenograft embryos (fertilized eggs: cFertili
This is the result of development when a reconstructed embryo was produced by reimplantation into a zed egg or an unfertilized egg: cUnfertilized egg. (Fertilized egg: cFertilized egg or unfertilized egg: cUnfertilized egg). Blastoderm diameter (Diamet
For each er of blastodermal disc, the number of embryos that have developed to that diameter (No. of developing embryos) is shown.

FIG. 3 is a diagram showing the sequences of PCR primers used in Example 7. Each primer set is designed to amplify a sequence specific to DT40. The upper row (F1 and R1) is the primer set used for the first-stage reaction, and the lower row is the primer set used for the second-stage reaction.

FIG. 4 is a photograph of a gel obtained by electrophoresing a sample after a PCR reaction in Example 7. In the figure, the arrow is DT
A band specific to 40 (243 bps) is shown. Lane 9 shows the DNA extracted in (1) above (extracted from the most advanced embryo).
This is a lane in which a sample obtained by amplifying DNA) by PCR was passed.
Lane M is a molecular weight marker, and P is the D (2) extracted above.
Lane (positive control) in which a sample obtained by amplifying NA (DNA extracted from DT40) by PCR was run (N1) was the template DN
A lane (negative control) in which a sample subjected to PCR without A was run (negative control), N2 was a plasmid DNA
In the lanes (negative control) and lanes 1 to 8 in which 0) was flown, DNA was extracted from embryos that normally developed from normal chicken eggs and subjected to PCR in the same manner as described above.
This is a lane in which a DNA sample was subjected to electrophoresis.

Claims (14)

[Claims] A) transplanting a nucleus of a donor cell comprising a first avian cell to an enucleated oocyte of an animal heterologous to the first avian to form a xenograft embryo; A) transplanting the heterologous nuclear transfer embryo into an enucleated egg of a second bird, the method for reconstructing an embryo having genetic information derived from the donor cell. 2. The method according to claim 1, wherein the step (a) comprises a step of introducing the donor cell into the enucleated ovum and a step of fusing the cell membrane of the donor cell with the cell membrane of the enucleated ovum. The method of claim 1, wherein 3. The method of claim 2, wherein the fusing step is performed by applying a voltage momentarily. 4. The method according to claim 1, wherein the step (b) comprises the steps of: introducing the xenograft embryo into the enucleated egg of the second bird; and applying a voltage instantaneously. The method according to claim 1. 5. The method according to claim 1, wherein the step b) is carried out as early as possible after the heterologous nuclear transfer embryo has started to develop. 6. The method according to claim 1, wherein the heterozygous nuclear transfer embryo is 2 to 4 times.
6. The method of claim 5, wherein the method is in the cell phase. 7. The method according to claim 1, wherein the enucleated egg of the second bird is an enucleated fertilized egg. 8. The method according to claim 1, wherein the first bird is a chicken. 9. The method of claim 8, wherein said donor cells are DT40. 10. The method according to claim 1, wherein the second bird is a chicken. 11. The animal according to claim 1, wherein the heterologous animal is an animal selected from the group consisting of cows, pigs, sheep, goats, mice, rats, and rabbits.
0. The method according to any one of 0. 12. The method according to claim 1, wherein the heterogeneous animal is a cow. 13. The chromosomal DNA of the donor cell has a foreign gene integrated therein.
13. The method according to any of the above items 12. 14. A reconstructed embryo obtained by the method according to claim 1.