KR100723380B1 - Method of Interclass Nuclear Transfer between Aves and Mammals - Google Patents

Abstract

The present invention comprises the steps of (a) preparing a cell of an avian animal which is a donor nuclear cell; (b) removing the nucleus from the egg of the mammal, which is a hydronucleus; (c) injecting the donor nuclear cell into the denuclearized egg; (d) fusing the donor nucleus cell and the egg to form a nucleated embryo; (e) activating the nucleated embryo; And (f) culturing the activated fertilized egg to produce an embryo that is capable of blastocyst formation and contains a genetic complement of donor nucleus cells. It is about a method.

Nuclear transfer, chickens, cattle, pigs, blastocysts, fertilized eggs, embryos

Description

Method of Interclass Nuclear Transfer between Aves and Mammals             

1A and 1B show nested PCR results of detecting chicken-specific locus LEI0171 in Kraggan embryos obtained by transplanting the nuclei of chicken embryo fibroblasts into mature eggs of denuclearized cattle. Bovine zona pellucida-depleted interclassed embryos developed up to each developmental stage were boiled down and amplified using primer sets for chicken-specific loci LEI0171, followed by CN1 (FIGS. 1A, 271 bp) locus and CN2 (FIG. 1B). , 274 bp) using a primer for the nested PCR. All interclass embryos tested contained the chicken genome. M: 100 bp ladder, lane 1: bovine genome DNA (negative control), 2: chicken genome DNA (positive control), 3: 1-cell phase, 4: 2-cell phase, 5: 4-cell phase, 6: 8-cell stage, 7: 16-cell stage to mortality, 8: no template.

The present invention relates to an interclass nuclear transfer method between birds and mammals.

Advances in gamete manipulation techniques are contributing to the development of biotechnology for therapeutic purposes. Several years ago, interstitial somatic cell transplantation techniques have been tried to preserve endangered animals (Dominko T, et al., Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species. Biol Reprod 1999; 60: 1496-1502; Loi P, et al., Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells.Nat Biotechnol 2001; 19: 962-964; and Lanza RB, . et al, Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer Cloning 2000; 2:. 79-90). The use of this technique developed in animals suggests the possibility of embryonic stem cell lines to be established without the use of human eggs or fertilized eggs, and a series of studies have reported the formation of blastocysts in human-cow species. The human-bovine heterologous embryo thus constructed could occur in vitro up to the blastocyst where the first differentiation was made, and the blastocyst had a human karyotype (Chang KH, et al., Blastocyst formation, karyotype, and mitochondrial DNA of interspecies embryos derived from nuclear transfer of human cord fibroblasts into enucleated bovine oocytes Fertil Steril 2003; 80:... 1380-1387; and KH Chang, et al, Optimized protocol of the interspecies nuclear transfer using human somatic cells and enucleated bovine oocytes Fertil Steril 2003 (submitted)).

Using the developed technology, a groundbreaking method can be established for mass production of human embryonic stem cells with excellent medical therapeutic value without ethical controversy. In addition, in the case of animals, in addition to the existing usefulness, there is an advantage that a mass production system (bioreactor) for producing bioactive substances for the treatment of intractable disease by producing stem cells of various species using somatic cell nuclear transfer.

Algae are the only species capable of producing genetically mixed genes and embryonic stem cell clones with excellent economic value by transplanting embryonic stem cells into the vessels of embryos. Because of these physiological properties (Sang H. Transgenic chickens-methods and potential applications. Trends Biotechnol 1994; 12: 415-420; Harvey AH, et al., Expression of exogenous protein in the egg white of transgenic chickens.Nat Biotechnol 2002; 20; : 396-369; Simkiss K. Embryo manipulation of the germplasm.Poult Sci 1997; 76: 1093-1100; and Echtes RJ.Reproduction in poultry: in Embryonic Development.Cambridge , Cambridge University Press, 1996; 40-49), chicken Embryonic stem (ES) cells can provide an efficient bioreactor system for therapeutic development. Efforts have been made to mass-produce embryonic stem cells of chickens using these advantages, but many attempts have failed until now because embryo manipulation is difficult (Pain B, et al., Long-term in vitro culture). and characterization of avian embryonic stem cells with multiple morphogenetic potentialities Development 1996; 122:. 2339-2348).

If chicken embryonic stem cells can be mass-produced using interstitial somatic cell nuclear transfer technology, not only original techniques to overcome current technical limitations are developed, but also stem cell specific techniques developed in the art. Mass production of transgenic embryonic stem cell mediated bioreactor for the treatment of intractable disease can be successful by using transformation technology.

For these reasons, there is a need for a system that can more easily obtain ES cells of birds, especially chicken.

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made intensive research to develop an alternative system that can more easily obtain birds, particularly chicken ES cells, and have developed a system that enables nuclear transfer between avian cells and mammalian eggs. The interclass embryos produced by the present invention have been completed by confirming that they include the genetic complement of donor nuclear cells.

It is therefore an object of the present invention to provide an interclass nuclear transfer method between a mammal and a bird.                         

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides a method for interclass nuclear transfer between a mammal and a bird, comprising the steps of: (a) preparing a cell of an avian animal which is a donor nuclear cell; (b) removing the nucleus from the egg of the mammal, which is a hydronucleus; (c) injecting the donor nuclear cell into the denuclearized egg; (d) fusing the donor nucleus cell and the egg to form a nucleated embryo; (e) activating the nucleated embryo; And (f) culturing the activated fertilized egg to produce an embryo having blastocyst forming ability and comprising a genetic complement of a donor nucleus cell.

The present invention is the first invention to successfully achieve interclass nuclear transfer. The term "interclass" nuclear transfer, as used herein, is a term used to distinguish between previously successful interspecies nuclear transfers and means nuclear transfer between higher classification levels than species. do. The term "interclass" nuclear transplantation, preferably, interclass nuclear transfer is an animal of the genus or higher, more preferably, an animal of more than or more, even more preferably, an animal of the neck or higher, most preferably different Nuclear transfer between animals in the river.

In the most preferred embodiment of the invention, the partners of nuclear transfer are the avian animal and mammalian cavity. Preferably, the cell derived from an avian animal is a donor nucleus cell and a mammalian oocyte becomes a recipient nucleus. As used herein, the term "oval" refers to a first oocyte, a second oocyte or an egg cell, or a mixture of the cells.

The donor nucleus cells are preferably cells of chickens, quails, turkeys, ducks, geese, pheasants or pigeons, most preferably cells of chickens. On the other hand, the nucleophilic egg is preferably an egg of a cow, a pig, a goat, a sheep, a mouse, a hamster, a dog or a cat, more preferably an egg of a cow or a pig, and most preferably an egg of a cow.

The donor nucleus cell may be of any cell type, including genome or genetic material (eg, nucleic acid), including for example somatic cells, germ cells or stem cells, most preferably somatic cells. Suitable somatic cells include fibroblasts, epithelial cells, muscle cells, cumulus cells, neurons and hepatocytes, most preferably fibroblasts.

For successful interclass nuclear transfer, mammalian eggs as the receptor must be matured before being used for nuclear transfer. For example, this maturation process is performed by Hyun et al. In pigs (Hyun SH et al., Effect of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos. Theriogenology 2003; 59: 1641-1649) If, due to bovine method Cho (Cho JK et al, Development of bovine oocytes Theriogenology 2002 reconstructed with different donor somatic cells with or without serum starvation; 57:.. 1819-1828) and is described in detail in, for example, bovine If so, oocytes mature in TCM-199 (tissue culture medium-199) medium supplemented with follicle stimulating hormone (FSH) and / or fetal bovine serum (FBS). The time required for maturation is generally 10-50 hours, preferably 15-40 hours, more preferably 20-30 hours. This matured mammalian egg is in the mid-II phase.

The matured egg is then enucleated. Nucleation can be performed according to various methods known in the art (see US Pat. No. 4,994,384; Part ES et al., Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with β-mercaptoethanol) and hemoglobin Mol Reprod Dev 2004; 67: 200-206).

Then, the donor nucleus cells are injected into the nucleated eggs. Injection of donor nucleus cells is preferably carried out by inserting into the perivitelline of the nucleus erythematosus.

Then, the donor nucleus cells and the nucleus nucleus are fused. The fusion is preferably carried out by electrofusion. In electrofusion, various media are used, such as mannitol, sucrose or sorbitol, preferably mannitol media. The medium for electrofusion preferably contains calcium and / or magnesium ions.

The fusion is performed by supplying an electric pulse that is capable of temporarily disassembling the cell membrane. It is preferably carried out by treating an electric pulse of 1.0-3.0 kV / cm, more preferably 1.2-2.5 kV / cm, most preferably 1.5-2.0 kV / cm, and the treatment time is preferably 10-40 μ. Seconds, more preferably 12-35 μsec, most preferably 15-30 μsec.

After the fusion treatment, the fused fertilized eggs are selected and activated. Activation is performed according to various methods known in the art. For example, it is carried out by cold culture, electric shock or treatment of chemicals at a temperature lower than physiological temperature. More specifically, it increases divalent cations (eg, calcium ions) in the fertilized egg, reduces phosphorylation of proteins in the fertilized egg, or proteins in the fertilized egg [cell cycle regulatory proteins such as MPF (maturation-promoting) factor) or MAP (microtubule-associated proteins) kinase] synthesis or by a combination of the three methods. The divalent cation includes calcium, magnesium, strontium and barium, most preferably calcium.

The increase in calcium ions in the fertilized egg can be increased by the addition of calcium ions. For example, it can be increased by addition in the form of calcium ionophore or ionomycin. In addition, the increase in calcium ions can be carried out through electric shock or ethanol treatment.

The process of activating the fertilized egg by reducing phosphorylation of the protein in the fertilized egg is carried out by the addition of a kinase inhibitor, in particular a serine-threonine kinase inhibitor. Examples of such inhibitors include 6-dimethylaminopurine, staurosporine, 2-aminopurine and sphingosine, most preferably 6-dimethylaminopurine.

Alternatively, phosphatase such as phosphatase 2A and phosphatase 2B can be injected into the fertilized egg to reduce phosphorylation of the protein.

The process of activating a fertilized egg by inhibiting the synthesis of a protein (cell cycle regulatory protein) in the fertilized egg is carried out using a protein synthesis inhibitor known in the art. Examples of such protein synthesis inhibitors include cycloheximide, cytochalasin D and butyrolactone-1, preferably cycloheximide and cytocalin D.

According to a preferred embodiment of the invention, the activation is carried out by two treatments: an increase in calcium ions in the fertilized egg and the inhibition of the synthesis of the protein in the fertilized egg. Preferably, the increase in calcium ions is carried out by treatment of calcium ionophores or ionomycin, and the inhibition of synthesis of the protein of the fertilized egg is carried out by treatment of cycloheximide and cytocalin D simultaneously.

Finally, the activated fertilized eggs are cultured in a suitable medium to produce embryos that are capable of blastocyst development and contain the genome of donor nucleated cells. Any medium for culturing embryos known in the art can be used. For example, NCSU-23 medium (Petters RM et al., Culture of pig embryos. J Reprod Fertil 1993 (Suppl); 48: 61-73), modified synthetic fallopian fluid (mSOF) medium (Park ES, et al. , Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with -mercaptoethanol and hemoglobin.Mol Reprod Dev 2004; 67: 200- 206), Ham's F-10, TCM-199, Tyrods-Albumin-Rac Tate-pyruvate (TALP), Dulbecos phosphate buffered saline, Eagles and Whites medium, and are preferably NCSU-23 medium and modified synthetic oviduct (mSOF) medium. For example, NCSU-23 medium is suitable for interclass nuclear transfer between chickens and pigs, and modified synthetic fallopian fluid (mSOF) medium is suitable for nuclear transfer between chickens and cows. When using NCSU-23 medium or modified synthetic fallopian fluid (mSOF) medium, it is preferable not to add serum.

By the above-described procedure, a fertilized egg having a blastocyst forming ability and comprising the genome of a donor nucleus cell is produced. The present invention is the first in the world to perform nuclear transfer between animals belonging to different rivers, and also reported pre-implantation, loss of embryonic formation and blastocyst formation of fertilized embryos transplanted between animals belonging to different rivers. The invention is also the first in the world.

Cross-class nuclear transfer between avian and mammals can overcome the limitations of building ES cells in birds, especially chickens. In addition, the methods of the present invention facilitate the production of transgenic birds for the development of a medicament.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example

Experimental Materials and Methods

Preparation of Donor Nuclei Cells

To prepare CEFs (chicken embryonic fibroblast), the skin of 5.5 day old embryos was harvested and digested for 10 minutes at 39 ° C. in a 0.25% (v / v) trypsin-EDTA (Gibco BRL, Grand Island, NY) solution. CEFs were cultured in three passages in DMEM (Dulbecco's Modified Eagle's Medium, Gibco BRL) supplemented with 10% (v / v) fetal bovine serum (FBS, Gibco BRL). Trypsin was treated in CEF monolayers for 5 minutes before icNT (interclass nuclear transfer). Porcine and fetal fiber as a control, according to standard methods in our laboratory (Cho JK, et al., Development of bovine oocytes reconstructed with different donor somatic cells with or without serum starvation. Theriogenology 2002; 57: 1819-1828). The blasts were prepared.

Preparation of Nucleated Eggs

Cumulus-oocytes complexes (COCs) obtained by evaporation from porcine eggs were obtained from 10% (v / v) porcine follicular fluid, 10 ng / ml equine chorionic gonadotropin (eCG, Sigma-Aldrich Corp.). , St. Louis, MO) and 10 IU / ml human chorionic gonadotropin (hCG, Sigma-Aldrich) supplemented for initial 22 hours in tissue culture medium (TCM) -199 (Gibco BRL), followed by eCG - and were further cultured in a medium lacking in hCG- not 39 ℃ 22 periods of time with 5% CO ₂ air atmosphere condition under humidification. Evaporation of COCs from bovine eggs was performed at 39 ° C. for 22 hours in TCM-199 supplemented with 10% (v / v) FBS (Gibco BRL) and 0.005 IU / ml FSH (Antrin, Denka, Kanagawa, Japan). Matured under 5% CO ₂ conditions under a humidified air atmosphere.

After in vitro maturation of oocytes was removed through 0.1% hyaluronidase treatment, only the eggs from which the first polar body was released were selected under a microscope. Selected eggs were treated for 20 minutes in Hepes-mSOF containing 5 μg / ml of cytochalasin B. After fixing the egg so that the first polar body is in the 12 o'clock direction with the fixing pipette, the denuclear pipette was in the 1 o'clock direction, and the transparent zone was cut out to infiltrate the 11 o'clock direction. Thereafter, the fixing pipette was loosened and the fixing pipette was rubbed to make an incision in the transparent zone. Then, a part of the cytoplasm containing the first polar body and the medium substrate was pressurized to release from the egg.

Somatic cell nuclear transfer and In beat  culture

After removing the nucleus, donor nuclei were inserted into the egg's gastric peritelline. For the preparation of chicken-pig class fertilized eggs, CEF and denuclearized pig eggs were treated with a single DC pulse of 2.0 kV / cm in 0.3 M mannitol solution containing 0.5 mM HEPES, 0.1 mM CaCl ₂ and 0.2 mM MgCl _2. Was performed for 30 μsec, using an electric cell manipulator (BTX 2001, San Diego, CA) with a 3.2 mm gap between stainless steel wire electrodes. One hour after treatment, fused fertilized eggs were collected and NC State University (NCSU) -23 medium (Hyun SH, et al., Effect of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos) Theriogenology 2003; 59: 1641-1649) was incubated in a humidified air atmosphere at 39 ° C. for 162 hours under 5% CO ₂ conditions.

For the preparation of chicken-bovine embryos, CEF and denuclearized bovine eggs were subjected to 1.75 kV / cm double DC pulse treatment in a 0.26 M mannitol solution containing 0.5 mM HEPES and 0.2 mM MgCl ₂ for 15 μsec. Was carried out. Four hours after the fusion, the fused fertilized eggs were collected under a light microscope and subjected to one of the following activation treatments: 1) 5 μM ionomycin (I, Sigma-Aldrich) treatment for 5 minutes and then 4 1.9 mM 6-dimethylaminopurine (DMAP, Sigma-Aldrich) treatment for hours; 2) treated with 5 μM calcium ionophore A23187 (CI, Sigma-Alrdich) for 4 minutes followed by 10 μg / ml cycloheximide (CHX, Sigma-Aldrich) and 2.5 μg / ml cytocalin D ( CD, Sigma-Aldrich) treatment.

In each repeated experiment, allogeneic nuclear transfer using porcine or fetal fibroblasts was performed for control purposes.

Interclass embryos prepared by icNT using pig and bovine eggs were serum-free medium, NCSU-23 medium (Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil 1993 (Suppl); 48: 61-73) and Modified synthetic fallopian fluid (mSOF) medium (Park ES, et al., Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with β-mercaptoethanol and hemoglobin.Mol Reprod Dev 2004; 67: 200-206), respectively Incubated at. The development of interclass embryos up to 2-cell, 8-cell, 16-cell, mortality and blastocyst stages was monitored at 42, 66, 90, 114 and 162 hours of culture, respectively.

PCR Analysis of Chicken Specific Sequences in Chicken-Bovine iSCNT Embryos

To confirm the presence of chicken genetic material in the embryo between classes, a primer set for the 363 bp chicken specific microcetalite locus LEI0171 was used (Li HC, et al., Identification of transferred chicken germ cells in quail gonad and semen by amplification of chicken-specific PCR products.J Poultry Sci 2001; 38: 308-316; and Gibbs M, et al., Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats.Animal Genet 1997; 28: 401-417). Zona pellucida was removed with 0.05% (v / w) protease (Sigma) and then interclassed fertilized eggs developed to 1-cell, 2-cell, 4-cell, 8-cell, 16-cell and mortality stages. Were boiled in 30 μl water under the following conditions: 6 cycles at 97 ° C. and 65 ° C. for 5 minutes each.

PCR reactions were 30 μl boiled product, 0.1 mM dNTP (Takara Bio Inc., Shiga, Japan), 5 μl 10 × reaction buffer (100 mM Tris-Cl, 500 mM KCl, 15 mM MgCl ₂ ; Biotools, SA, Spain), 0.4 pM sense and antisense primers (Bioneer, Daejon, Korea) and 0.04 U DNA polymerase (Biotools). The sequence of the LEI017 locus sense primer is 5'-CTCAGGGCACCATTTTCACTG-3 'and the sequence of the antisense primer is 5'-GATACACTACTGT CTACACTC-3', which primers amplify the 363 bp fragment. 4 μl of the PCR product was used as DNA template for subsequent nested PCR and nested PCR was performed using the same components as the PCR reaction mixture. 201 bp and 274 bp fragments of LEI0171, designated CN1 and CN2, were analyzed by nested PCR. The sequences of the sense and antisense primers for analyzing CN1 are 5'-TCTATGTATAAGGAGTACTATGGGG-3 'and 5'-TACATTTTTGCAACACTTAAATTAG-3'. The sequences of the sense and antisense primers for analyzing CN2 are 5'-TAAGCATATCAGCACTTAATCTATG-3 'and 5'-TTAAATTAGCTTAGCATATACAGCC-3'.

10 μl of the PCR product was electrophoresed on a 1.5% (v / w) agarose gel and then visualized using 0.5 g / ml ethidium bromide staining and UV transmission light (Macalaster Bicknell, NJ). As negative and positive controls, 100 ng of genome DNA of cattle and chickens were used, respectively.

Experimental Design and Statistical Analysis

In Experiment 1, CEF or porcine fetal fibroblasts (PFF) were injected into denuclearized porcine eggs, and in Experiment 2, CEF or fetal fibroblasts (BFF) were injected into bovine eggs. In Experiment 2 using the bovine model, two activation methods using I + DMAP or CI + CHX + CD were used for icNT. In Experiment 3, nested PCR amplification of single fertilized eggs prepared in Experiment 2 was performed to detect chicken specific DNA sequences in interclass fertilized eggs derived from chicken-bovine icNTs. The general linear model (PROC-GLM) of the SAS 8.1 program combined with ANOVA and least squares analysis was used for statistical analysis, and the significant difference between treatments was determined when the P value was less than 0.05.

Experiment result

Significant (P <0.0001) model effects were observed for all variables when using denuclearized porcine eggs (see Table 1). This significant difference is due to the lower developmental ability in the class than in the homogeneous group. Although blastocyst formation was not observed in embryos between classes, 13% (21 of 156) of the control (homologous cloned porcine embryos) developed to blastocysts. In interclass embryos, development was observed only up to 4-cell stages. Thus, the 4-cell phase is judged to be a checkpoint at which mitotic ability of the interclass embryo is bestowed.

On the other hand, the fusion rate in chicken-pig icNT was lower than the control (pig-pig). Under the light microscope, the overall shape and diameter of the CEF were similar to the PFF. Therefore, the difference in fusion rate is believed to be due to the difference in the fine membrane structure between the somatic cells of the chicken and the gastric cavity of the porcine egg.

In Vitro Development of Interclass Embryo When Porcine Fetal Fibroblasts (PFF) or Chicken Embryonic Fibroblasts (CEF) Are Transplanted into Nucleated Denuclearized Second Midterm Swine Oocytes Types of Donor Somatic Cells Number of nucleated eggs Number of embryos between developed classes (%) ^b transplantation Successful Convergence (%) ^a Two-cell [24] 4-cell [48] 8-cell [72] 16-cell [96] Lost Belly [144] Fold [168] PFFs 180 156 (87) ^c 119 (76) ^c 89 (57) ^c 57 (37) ^c 49 (32) ^c 33 (21) ^c 21 (13) ^c CEFs 228 158 (69) ^d 51 (32) ^d 20 (13) ^d 0 (0) ^d 0 (0) ^d 0 (0) ^d 0 (0) ^d The numbers in parentheses represent the time after incubation. ^a Percentage of Number of Transplanted Oocytes ^b Percentage of Number of Oocytes Successful in Fusion ^cd The values of the different superscripts in each variable are significantly different (P <0.0001).

On the other hand, in the case of using denuclearized bovine eggs, the significance model effect was confirmed in the part of embryos that developed up to the 2-cell stage (P = 0.0157) and in all embryos of the next stage (P <0.0001). In general, embryonic development rate of icNTs was lower than allogeneic nuclear transfer (59-64% vs. 26-28% at 4-cell stage, 3-13% vs. 49-50% at 8-cell stage, 16-cell stage) 0-5% vs. 37-53%, and 0-3% vs. 23-25% in mortality) (see Table 2). In the production of interclass fertilized eggs, the fusion rate was similar between the two activation methods. However, blastocyst formation of interclass fertilized eggs was observed only when activated with CI + CHX + CD.

The pattern of early embryonic development of birds differs from mammals. In birds, daughter cells are not divided, but are separated by divisions (Echtes RJ. Reproduction in poultry: in Embryonic Development . Cambridge, Cambridge University Press, 1996; 40-49). However, if bovine eggs were used, the mitotic patterns of chicken-bovine embryos were little different from control (bovine-bovine) cloning embryos. Since the lack of the first polar and DNA was observed under UV during nucleation, the mitotic activity described above is believed to be derived from the interaction between the chicken nucleus and the bovine oocyte cytoplasm.

In vitro development of interclass embryos when the nuclei of bovine fetal fibroblasts (BFF) or chicken embryo fibroblasts (CEF) are transplanted into denuclearized mid-II bovine eggs Activation Processing Type Donor Somatic Cell Types Number of nucleated eggs Number of embryos between developed classes (%) ^b transplantation Successful Convergence (%) ^a Two-cell [24] 4-cell [48] 8-cell [72] 16-cell [96] Lost Belly [144] Fold [168] I + DMAP BFFs 143 110 (75) 77 (70) ^cd 65 (59) ^c 55 (50) ^c 41 (37) ^c 27 (25) ^c 16 (15) ^c CEFs 139 90 (65) 56 (62) ^c 25 (28) ^d 3 (3) ^d 0 (0) ^d 0 (0) ^d 0 (0) ^d CI + CHX + CD BFFs 109 77 (70) 62 (80) ^d 45 (64) ^c 34 (49) ^c 24 (53) ^c 16 (23) ^c 12 (16) ^c CEFs 122 77 (63) 45 (58) ^c 20 (26) ^d 10 (13) ^d 4 (5) ^d 2 (3) ^d 2 (3) ^d I: ionomycin (5 μM), DMAP: 6-dimethylaminopurine (1.9 mM), CI: calcium ionophore A23187 (5 μM), CHX: cycloheximide (10 μg / ml), CD: cytosine The treatment model effect at each variable denoted by the calcine D (2.5 μg / ml) P value is 0.0157 for development up to 2-cell, and is characterized by 4-cell, 8-cell, 16-cell, mortality and blastocyst stages. In development, it was less than 0.0001. The numbers in parentheses represent the time after incubation. ^a Percentage of Number of Transplanted Oocytes ^b Percentage of Number of Oocytes Successful in Fusion ^cd The values of the different superscripts in each variable are significantly different (P <0.0001).

Nested PCR results showed that 271 and 274 bp fragments in chicken-specific microcetalite loci LEI0171 were observed at all stages, but no genes for cattle were detected in the same sample (see FIGS. 1A and 1B). These results indicate that genes required for early embryonic development of interclass embryos are faithfully produced from chicken nuclei.

As described in detail above, the present invention provides a method for interclass nuclear transfer between a mammal and a bird. Embryos produced by the nuclear transfer method of the present invention are capable of blastocyst formation and include gene factors of donor nucleus cells, and overcome the limitations of building ES cells of birds, especially chickens.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (11)

Interclass nuclear transfer between mammals and birds, except humans, comprising the following steps: (a) preparing a cell of an avian animal which is a donor nucleus cell; (b) removing the nucleus from the egg of the bovine egg nucleus; (c) injecting the donor nuclear cell into the denuclearized egg; (d) fusing the donor nucleus cell and the egg to form a nucleated embryo; (e) activating the nucleated embryo; And (f) culturing the activated fertilized egg to produce an embryo having blastocyst forming ability and comprising a genetic complement of a donor nucleus cell. The method of claim 1, wherein the donor nucleus cells are cells of chicken, quail, turkey, duck, goose, pheasant or pigeon. The method of claim 2, wherein said donor nucleus cells are chicken cells. delete delete The method of claim 1, wherein said donor nucleus cells are somatic cells. The method of claim 1, wherein said nucleophile is a cell in the second meiosis stage. The method of claim 1, wherein step (c) is performed by inserting a donor nucleus cell into the perivitelline of the hydronucleus. The method of claim 1 wherein said fusing is performed by electrofusion. The method of claim 1, wherein the activation comprises (i) an increase in calcium ions in the fertilized egg; (Ii) inhibition of phosphorylation of proteins in fertilized eggs; (Iii) inhibit the synthesis of protein in the fertilized egg; Or (iii) a combination of the above methods. 10. The method of claim 9, wherein the activation is performed by (i) increasing calcium ions in the fertilized egg and (i) inhibiting the synthesis of the protein of the fertilized egg.

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