KR20100124891A - Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts - Google Patents

Abstract

PURPOSE: A method for preparing pig embryonic stem cell line is provided to optimize culture condition and to obtain a large amount of pig embryonic stem cells. CONSTITUTION: A method for preparing pig embryonic stem cell line comprises: a step of culturing pig somatic cells to prepare donor nucleus cells; a step of enucleating the donor nucleus cell to prepare recipient oocytes; a step of transplanting the nucleus of the donor nucleus cell and fusing with the recipient oocytes to prepare a nuclear transfer embryo; a step of culturing the embryo in vitro to form blastocyst; and a step of separating inner cell mass from the blastocyst and culturing in immature state.

Description

Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts}

The present invention relates to a pig embryonic stem cell line and a preparation method thereof.

Embryonic stem cells are isolated from the inner cell mass / ectoderm of the blastocyst. These cells have important properties of expressing transcription factors such as Oct-4 in order to maintain their self-renewal and undifferentiated state for a long time. Under certain conditions they can grow into germ cells and primitive trioderm. Gene targeting and clone selection can also produce genetically modified cell clones. For this reason, embryonic stem cells are a good way to study early mammalian development and transformation. After isolation of embryonic stem cells from rat blastocysts (Evans, MJ & Kaufman, MH (1981). Nature 292, 154-6; Martin, GR (1981). Proc. Natl. Acad. Sci. USA 78, 7634 Many researchers have identified embryonic stem cells or embryonic stem-like cells in hamsters (Doetshman, T. et al. (1988). Dev. Biol. 127, 224-7), rats (Ouhibi, N. et al. (1995). Mol.Reprod . Dev. 40, 311-24), cat (Yu, X. et al. (2008) .Mol . Reprod. Dev. 75, 1426-32), cattle (Strelchenko, N. (1996) Theriogenology 45, 131-40), pigs (Li, M. et al. (2004) .Zygote 12, 43-8), monkeys (Thomson, JA et al. (1995) .Proc. Natl. Acad. Sci. USA 92, 7844-8) and humans (Thomson, JA et al. (1998). Science 282, 1145-47). Recently, embryonic stem cells or embryonic stem-like cells were obtained from rats (Wakayama, T. & Yanagimachi, R. (2001). Mol. Reprod. Dev. 58, 376-83), cattle (Wang, L. et al. Biol.Reprod . 73, 149-55) and primates (Byrne, JA et al. (2007). Nature 450, 497-502) have been successfully isolated from embryos before somatic cloning implantation.

In addition to the importance of mammalian embryogenic research, pig embryonic stem cells can be used to produce transgenic animals for xenotransplant donation. Most notable in pigs is the combination of somatic cloning research and stem cell establishment technology, which is of great value in the therapeutic application of xenotransplantation, which may lead to the mass production of immunosuppressive organs for cross-organ transplantation. Porcine embryonic stem cells were isolated from early embryos, but lost pluripotency in cultivation over time (Moore, K. & Piedrahita, JA (1997). In Vitro Cell Dev. Biol. Anim. 33, 62-71 ). Very few attempts have been made to produce pluripotent embryonic stem-like cells derived from porcine in vitro or in vitro fertilized eggs (Miyoshi, K. et al. (2000). Biol. Reprod. 62, 1640-6; Li, M. et al. (2003) .Mol.Reprod . Dev. 65, 429-34). Most of these embryonic stem-like cells did not respond to definitive markers required for embryonic stem cell characterization and did not remain undifferentiated. This problem is due to the fact that most of the cells have not grown for a long time, possibly due to inadequate culture conditions. In addition, examples of embryonic stem cells established from cloned somatic embryos or the birth of cloned litters have only been reported in rodents and cattle (Cibelli, JB et al. (1998). Nat. Biotechnol. 167, 642-6; Wakayama, T. & Yanagimachi, R. (2001) .Mol . Reprod. Dev. 58, 376-83).

The present invention is a porcine embryonic stem cell by optimizing the conditions of the method for separating the inner cell mass from the porcine somatic cloned blastocysts and culturing the isolated inner cell mass in undifferentiated state during the effort to obtain a high yield of porcine embryonic stem cells It was confirmed that it can be obtained in high yield, and came to complete this invention.

One aspect of the invention

To provide a pig embryonic stem cell line having at least one of the following characteristics:

(1) remain undifferentiated for at least 45 passages;

(2) maintain alkaline phosphatase activity;

(3) expresses at least one marker selected from OCT-4, Nanog, Sox-2, Rex-1, SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81;

(4) maintain the normal diploid karyotype; And

(5) Possible to differentiate into ectoderm, endoderm and mesoderm.

Another aspect of the invention

(1) culturing pig somatic cells to prepare donor nuclear cells;

(2) denuclearizing the egg of the pig to prepare a nucleus ovum;

(3) preparing a nuclear transfer embryo by grafting the nucleus of the donor nucleus cell to the nucleus nucleus, and fusing the nucleus of the donor nucleus cell with the nucleus nucleus;

(4) reprogramming, activating and ex vivo culture of the nuclear transfer embryos to form blastocysts; And

(5) to provide a method for producing a pig embryonic stem cell line comprising the step of separating the inner cell mass from the blastocyst and culturing the inner cell mass in an undifferentiated state to establish a pig embryonic stem cell line.

The present invention relates to a pig embryonic stem cell line.

Porcine embryonic stem cell line of the present invention is characterized by having at least one of the following characteristics:

(1) remain undifferentiated for at least 45 passages;

(2) maintain alkaline phosphatase activity;

(3) expresses at least one marker selected from OCT-4, Nanog, Sox-2, Rex-1, SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81;

(4) maintain the normal diploid karyotype; And

(5) Possible to differentiate into ectoderm, endoderm and mesoderm.

Porcine embryonic stem cell lines of the present invention showed the shape of typical embryonic stem cells with a solid appearance and a distinct appearance. It also showed a high ratio of nuclear size to high cytoplasmic inclusions with high distinct nuclei and ribosomes.

Porcine embryonic stem cell line of the present invention is preferably able to maintain an undifferentiated state during passage passages for more than 45 times, in the process maintains alkaline phosphatase activity, embryonic stem cell specific markers OCT-4, SSEA- 1, SSEA-4, TRA-1-60, TRA-1-81 were expressed, and Nanog, Sox-2, and Rex-1 were also expressed.

Porcine embryonic stem cell line of the present invention as a result of karyotype analysis showed that the normal chromosomes of the 38 autosomal and sex chromosomes to maintain the normal diploid karyotype.

Porcine embryonic stem cell line of the present invention expresses the trioderm-specific genes (ectoderm: β-III tubulin, endoderm: amylase and mesoderm: enolase) in the goblet when cultured in a medium excluding LIF (Leukemia Inhibitor Factor) It can be seen that differentiation into ectoderm, endoderm and mesoderm is possible.

The present invention also relates to a method for producing a pig embryonic stem cell line.

The manufacturing method comprises the steps of (1) culturing the somatic cells of the pig to prepare a donor nuclear cell;

(2) denuclearizing the egg of the pig to prepare a nucleus ovum;

(3) preparing a nuclear transfer embryo by grafting the nucleus of the donor nucleus cell to the nucleus nucleus, and fusing the nucleus of the donor nucleus cell with the nucleus nucleus;

(4) reprogramming, activating and ex vivo culture of the nuclear transfer embryos to form blastocysts; And

(5) separating the inner cell mass from the blastocyst and culturing the inner cell mass in an undifferentiated state to establish a pig embryonic stem cell line.

In the production method of the present invention, steps (1) to (4) can be carried out with reference to a conventional mammalian somatic cloning method.

The somatic cells of the pig of step (1) is preferably somatic cells of the somatic cloned pig. The somatic cells are preferably fibroblasts.

In the step (5), the serum antibody and complement were mainly used to separate the inner cell mass from the blastocyst, but in the present invention, it is preferable to physically remove the trophectoderm to separate the inner cell mass. This shortens the time required to separate the inner cell mass, it is possible to minimize the damage of the inner cell mass.

In the step (5), conventionally, the inner cell mass was cultured in an undifferentiated state in a conventional stem cell culture medium containing both a leukemia inhibitor factor (LIF) and basic fibroblast growth factor (bFGF), in the present invention, LIF is It is preferable to exclude and culture in a medium containing only bFGF. This enables the expansion of porcine embryonic stem cell lines with high yield. The stem cell culture medium means a medium capable of maintaining and / or expanding stem cells in an undifferentiated state. The bFGF may be included in the concentration of 10 to 30 ng / ml, preferably 20 ng / ml in the medium.

According to the present invention, it is possible to obtain a high yield of porcine embryonic stem cells by optimizing the method for separating the inner cell mass from the porcine somatic clone blastocyst and the medium for culturing the isolated inner cell mass in an undifferentiated state.

Combining the present invention with a gene targeting method will be an efficient method for producing transgenic pigs for various purposes.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Materials and Methods

1-1. Recovery of Eggs and Culture of Oocyte-Oocyte Complexes

Ovaries were collected from pre-mature sows at slaughterhouses and transported in saline at 30-35 ° C within two hours. The oviduct and oocyte-oval complex were aspirated from a follicle (3-6 mm in diameter) using a 10 cc syringe equipped with an 18G needle. Condensed oocyte-complexed cell complexes were screened with 10 ng / ml EGF (epidermal growth factor), 4 IU / ml PMSG (pregnant mare serum gonadotropin) (Intervet, Boxmeer, The Netherlands) and hCG (human chorionic gonadotropin) (Intervet) Cultured in TCM (Tissue Culture Medium) -199 (Life Technologies, Rockville, MD) containing 10% (v / v) porcine follicle fluid (pFF). Porcine follicle fluid was aspirated from sow follicles (3-7 mm) before maturity. After centrifugation at 500 xg for 30 minutes, the supernatant was taken and passed through a 1.2-µm and 0.45-µm syringe filter (Gelman Sciences, Ann Arbor, MI). The prepared pFF was stored at -20 ° C until use. The prepared follicles were stored at minus 20 degrees. Fifty egg-oocyte complexes were placed in 500 microliters of TCM-199 and incubated in a humidified incubator containing 5% carbon dioxide and 95% air. After 22 hours of incubation, the egg-oocyte complexes were further incubated for 22 hours in TCM-199 without washing with PMSG and hCG after three washes. After maturation, the oocyte- oocyte complexes were incubated for one minute in a HEPES-buffered NCSU-23 culture containing 0.5 mg / ml hyaluronidase (Sigma-Aldrich Corp., St. Louis, MO), The cells were gently pipetted to remove the cumulus cells.

1-2. Preparation of mini pig fibroblasts for donor cells

Mini pig fibroblasts for donor nucleus cells were harvested from tails of cloned pig embryos (CF1) and newborn piglets (CP1) at 30 days of gestation. The head of the fetus was separated by an iris scissor, and the remaining soft tissues containing the liver and intestine were removed by watchmaker's forceps. To separate tail fibroblasts, the outer surface of the tail was washed several times after shaving. After washing twice with PBS (Life Technologies), body tissues were placed in a 100-mm culture dish (Becton Dickinson, Lincoln Park, NJ) and crushed with a surgical knife. Crushed fetuses and tail tissue were ground for 1-2 hours in DMEM (Life Technologies) containing 0.1% (w / v) trypsin and 1 mM EDTA (Life Technologies). Trypsin-treated cells were centrifuged at 300 xg for 10 minutes and then inoculated in a 100 mm culture dish. Inoculated cells were 10% (v / v) FBS (Life Technologies), 1 mM sodium pyruvate, 1% (v / v) non-essential amino acids (Life Technologies) and 10 μg / ml penicillin-streptomycin for 6-8 days. The solution was incubated in a humidified incubator maintained at 39 ° C., 5% CO ₂ and 95% in DMEM containing the solution. After removing the non-stick cells and clumps, the adhered cells were grown until they were full, and were passaged by trypsinization by reacting 0.1% trypsin and 0.02% EDTA for 5 minutes at intervals of 5-7 days. Cryopreserved in liquid nitrogen. The freezing solution consists of 80% (v / v) DMEM, 10% (v / v) DMSO and 10% (v / v) FBS. Before somatic cloning, the cells were cultured for 3-4 days and when 80% of the area became full, serum was removed and starved. Starvation was done by incubation for 3 days in DMEM containing 0.5% FBS. Each cell was trypsinized for 30 seconds in a single cell layer and used for somatic cell nuclear transfer after separation.

1-3. Somatic cell nuclear transfer and fertilized egg culture

After 42 hours of maturation, the eggs were fixed with a fixative pipette and made incisions near the first polar body in the zona pellucida using sharp glass awls. The nearby cytoplasm containing the first and second meiosis mesophase chromosomes was squeezed out and removed. Oocytes were denuclearized in HEPES-buffered NCSU-23 containing 0.3% BSA. After denuclearization, the eggs were stained for 5 minutes with 5 μg / ml bisbenzimide (Hoechst 33342; Sigma), and the presence of residual DNA was observed with a fluorescent inverted microscope. Oocytes carrying DNA were not disclosed. Using a thin pipette, trypsinized fetal fibroblasts with a smooth surface were inserted into the ovulation cavity of denuclearized eggs. The injected eggs were equilibrated in 0.26 M mannitol containing 0.5 mM HEPES, 0.1 mM CaCl ₂ and 0.1 mM MgCl ₂ for 4 minutes and then placed in the space between two electrodes containing mannitol solution. The injected eggs were fused and activated with a single DC pulse of 2.0 kV / cm for 30 seconds using BTX Electro-Cell Manipulator 2001 (BTX Inc., San Diego, Calif.). Activated eggs were washed three times in NCSU-23 containing BSA without 4 mg / ml fatty acid and then at 39 ° C, 5% CO ₂ , 5% O ₂ at 25 microliters of mineral oil. , And incubators maintained at 90% N ₂ . The recombinant embryos were incubated for 168 hours after activation. Incubation and blastocyst formation were observed at 48 and 168 hours, respectively.

1-4. Support Cell Preparation

Rat fetal fibroblasts were isolated from 13.5 day-old fetuses of CF1 mice (Thomson, JA et al. (1998). Science 282, 1145-47). The head was removed using an iris scissor, and the soft tissues of the liver and intestine were removed using watchmaker's forceps. After washing twice in PBS (Life Technologies), the tissues were ground in a 60 mm dish (Becton Dickinson). The ground fetal tissue was pipetted several times, washed by centrifugation at 300 xg for 10 minutes, and inoculated in a 100-mm culture dish. Inoculated cells were 10% (v / v) FBS (Life Technologies), 0.1 mM β-mercaptoethanol, 1% (v / v) non-essential amino acids (Life Technologies) and 10 μg / ml penicillin Incubated for 5-7 days in a humidified incubator maintained at 37 ° C., 5% CO ₂ in DMEM containing streptomycin. Support cells were cultured in DMEM containing 10% FBS (Life Technologies), 0.1 mM β-mercaptoethanol, 1% (v / v) non-essential amino acids (Life Technologies) and 10 μg / ml penicillin-streptomycin. Mice fibroblasts grown to 80% were mitotic by exposure to 10 μg / ml mitocin C (Sigma) for 2 hours. After washing several times with PBS, 0.8 × 10 ⁵ Cultured at the density of cells / spheres.

1-5. Internal Cell Mass Separation and Maintenance of Embryonic Stem-like Cells

After removing the zona pellucida from the cloned blastocyst with 0.5% pronase E (Sigma), the blastocyst with the zona pellucida was washed in DMEM / F12 (Life Technologies) with 20% Knockout serum replacement (Life Technologies). . Internal cell mass was isolated by physical removal of trophectoderm. They were placed on rat fibroblast support cells (Thomson, JA et al. (1998). Science 282, 1145-47), and proliferated internal cell masses were physically fragmented and passaged to new mouse fibroblasts and cultures. In order to maintain porcine embryonic stem-like cells, about 200 undifferentiated cell colonies were physically sliced under a dissecting microscope and then sprinkled on new support cells at 5-7 day intervals. Embryonic stem-like cells contain 20% knockout serum replacement, 0.1 mM β-mercaptoethanol, 0.1 mM non-essential amino acids, 1% (v / v) penicillin-streptomycin solution and 20 ng / ml human recombinant basic human recombination basic FGF; Invitrogen, Carlsbad, Calif.) Was incubated in DMEM / F12.

1-6. Marker Identification

PNTES-1 (embryonic stem-like cells made from CF1-derived blastocysts) and pNTES-2 (embryonic stem-like cells made from CP1-derived blastocysts) at 28 and 24 passages, respectively, were washed with PBS, followed by 4% (v / v) form. After fixing for 10 minutes at room temperature of aldehyde, it was washed several times with PBS and stained with NBT / BCIP solution (Roche Molecular Biochemicals). The color reaction was stopped with PBS. For surface antigen staining, embryonic stem-like cells were fixed several times at 4 ° C. using 4% paraformaldehyde after washing several times with PBS. Anti-Oct-4 Antibody (SC-9081) was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.) And has anti-SSEA-1 (MAB4301), SSEA-4 (MAB4304), TRA-1-60 (MAB4360) and TRA -1-81 (MAB4381) was purchased from Chemicon (Temecula, Calif.). All antibodies were diluted one-to-one and reacted for three hours. After washing three times with PBS, IgM and IgG (SSEA-4) (Chemicon) labeled with FITC were used as secondary antibodies.

1-7. Karyotyping

Karyotypes were analyzed at 28 and 24 passages for pNTES-1 and pNTES-2 cells, respectively. pNTES-1 and pNTES-2 were incubated at 37 ° C. under 5% CO ₂ conditions for one hour with addition of 0.02 μg / ml colcemide (Sigma). After 20 min exposure to unicellularized and hypotonic KCl (0.56%), the cells were placed in stock solution, fixed with a solution of methanol and acetic acid in a 3: 1 ratio, and gently dropped onto a microscope glass slide. After staining with Giemsa (1:20 dilution, Sigma) for 20 minutes, chromosome images were observed at 1,000-fold magnification.

1-8. Hypersatellite analysis

The genomic DNA of pNTES-1, pNTES-2 and individual donor nuclei were extracted using standard phenol extraction methods. Briefly, physically dissected pNTES-1 and pNTES-2 colonies and single-celled donor nucleus cells were lysed buffer containing proteinase K [0.05 M Tris (pH 8.0), 0.05 M EDTA, 0.5% SDS]. It was reacted half day at. Proteins were removed by phenol extraction, and DNA was precipitated with ethanol. Genomic DNA samples were analyzed using nine pig DNA hypersatellite markers (S0005, S0007, S0086, S0230, SW61, SW902, SW980, SW1661, and SWR 1004) FAM, TET, or HEX fluorescently labeled. Changes in fragment length were made on a DNA sequencer (ABI 373; Applied Biosystems, Foster City, Calif.) Using PCR and PAGE with fluorescently labeled region-specific primers. The nucleotide fragment length, an amplification product, was measured using our software (GeneScan and Genotyper; Applied Biosystems).

1-9. Reverse Transcription-Polymerase Chain Reaction

RNA of the cultured pNTES-1 and pNTES-2 embryos was determined by Kim et al. (Kim, S. et al. (2005). Mol. Reprod. Dev. 72, 88-97) using Trizol (Invitrogen). Extraction was carried out using 20 microliters of RNase and dissolved in water without addition. Total RNA was used for reverse transcriptase-polymerase chain reaction. Reverse transcription was performed at 37 degrees for 60 minutes. Each reverse transcription reaction (33 μl each) was performed at 5 mM MgCl ₂ , 1 RT buffer, 2.5 mM oligo (dT), 1 mM dNTP and 50 IU of murine leukemia virus reverse transcriptase (MoMLV; Amersham Pharmacia Biotechnologies, Oakville, ON, Canada) Was done in. Expression of porcine β-actin was used as a control of reverse transcriptase-polymerase chain reaction. The sequence of each primer is listed in Table 1. cDNA (2 μL) was amplified in 50 μL with 250 U Ex-Taq polymerase (Takara, Shiga, Japan), 5 μl of 10 × buffer, 2.5 mM deoxy-NTPs and 50 pmol of up and down primers, respectively. Amplification products were observed under UV wavelengths in 1.5% agarose gel made with 1 × TAE buffer with 1 μl / ml ethidium bromide. Images of each gel were stored using the Gel Doc 2000 Gel Documentation System (Bio-Rad Laboratories, Hercules, CA).

Example 2: Results

In total, 46 and 14 blastocysts were produced from 30-day-old pig fetal fibroblasts (CF1) and neonatal pig fibroblasts (CP1), respectively. As shown in Table 2, there was no difference in recombination, splitting, and blastocyst formation between the two groups. ICM, physically isolated from the blastocyst, from which the zona pellucida was removed, was washed several times and sprayed on the mitotic inactivated mouse fibroblast support cell layer. Colony formation was observed after 5-8 days. Porcine embryonic stem-like cells showed a typical embryonic stem cell morphology with a solid appearance and distinct appearance. Under high magnification phase contrast microscopy, embryonic stem-like cells showed a high ratio of nuclear size to cytoplasm containing high distinct nuclei and ribosomes (Figures 1A and C). However, some colonies maintained embryonic stem-like cell morphology for 2-5 passages and then differentiated or degenerated. Only two cell lines were successfully cultured without differentiation or degeneration. Each cell line was cultured over 52 (pNTES-1) or 48 (pNTES-2) passages. Embryonic stem-like cell line establishment rate was lower than that of CF1 than that of CP1 (2.2% vs. 7.1%; Table 2).

The karyotypes of pNTES-1 and pNTES-2 showed normal chromosomes of 38 autosomal and XX sex chromosomes (Figures 1B and D). Like mouse and human embryonic stem cells, pNTES-1 and pNTES-2 showed high AP activity and Oct-4 expression. Immunostaining of pNTES-1 and pNTES-2 cells was carried out using anti-surface glycochain antibodies, anti-SSEA-1, anti-SSEA-4, anti-TRA-1-60, and anti-TRA-1-81 antibodies. Was performed. Similar expression patterns were shown between pNTES-1 and pNTES-2 (FIG. 2).

The expression levels of Oct-4 , Nanog , Sox-2 and Rex-1 of pNTES-1 and pNTES-2 were measured by RT-PCR. The expression of these markers in pNTES-1 and pNTES-2 was higher than the respective blastocysts (FIG. 3).

After incubating pNTES-1 and pNTES-2 cells in plastic petri dishes for 14 days, embryoid bodies were formed (FIG. 4). Expression of several trigeminal specific genes was measured in embryoid bodies on days 7 and 14. Transcripts of β-III tubulin (ectoderm, neurons), amylase (endoderm, pancreas) and enolase (mesoderm, muscle) were detected.

Microsatellite analysis of pNTES-1 and pNTES-2 cells showed that they are consistent with donor nucleated cells (CF1 and CP1), respectively, from which they originated (Table 3).

a is associated with each hypersatellite marker, genotype determined by size

b is miniature porcine fetal fibroblast used as donor cells in CF1c and CD1d

c is cloned fetal fibroblast and donor cell on day 30 used for pNTES-1

d is the neoplasm of reproductive piglets and donor cells used for pNTES-2

e is negative control: ESCs derived from IVF blastocysts

FIG. 1 is a phase difference and karyotype picture of pNTES-1 and pNTES-2 cells, which are the karyotype images of 28 passages and 24 passages of pNTES-1 (A) and pNTES-2 (C) cells, respectively, pNTES-1 and pNTES- 2 cells have distinct boundaries with typical embryonic stem cell types. Baseline = 200 μm. In addition, the G-banding karyotypes of pNTES-1 (B) and pNTES-2 (D) of the 28 and 24 passages, respectively, both cells show 18 pairs of autosomal and normal XX karyotypes.

2 is a result of analyzing the characteristics of pNTES-1 and pNTES-2, pNTES-1 and pNTES-2 was analyzed using six stem cell specific markers. pNTES-1 (28 passages) has high AP (A), SSEA-4 (B), TRA-1-60 (C), SSEA-1 (D), Oct-4 (E) and TRA-1-81 ( F) showed activity, pNTES-2 (24 passages) also showed AP (G), SSEA-4 (H), TRA-1-60 (I), SSEA-1 (J), Oct-4 (K), and It exhibits TRA-1-81 (L) activity.

Figure 3 shows the reverse transcriptase-polymerase chain reaction of Nanog , Oct-4 , Sox-2 and Rex-1 genes in blastocysts (A), pNTES-1 (B) and pNTES-2 (c). Baseline = 200.

FIG. 4 shows the results of RT-PCR detection of the embryonic body under the light micrograph and the gene expression of the trioderm-specific expression gene. (A) The embryonic body cultured on 7 or 14 days, and (B) pNTES-1 and pNTES- RT-PCR detection results of trioderm specific genes in embryoid bodies derived from 2 cells are shown. Baseline = 100 μm.

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Claims (8)

Porcine embryonic stem cell line having at least one of the following characteristics: (1) remain undifferentiated for at least 45 passages; (2) maintain alkaline phosphatase activity; (3) expresses at least one marker selected from OCT-4, Nanog, Sox-2, Rex-1, SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81; (4) maintain the normal diploid karyotype; And (5) Possible to differentiate into ectoderm, endoderm and mesoderm. The method of claim 1, Porcine embryonic stem cell line characterized in that pNTES-1 or pNTES-2. (1) culturing pig somatic cells to prepare donor nuclear cells; (2) denuclearizing the egg of the pig to prepare a nucleus ovum; (3) preparing a nuclear transfer embryo by grafting the nucleus of the donor nucleus cell to the nucleus nucleus, and fusing the nucleus of the donor nucleus cell with the nucleus nucleus; (4) reprogramming, activating and ex vivo culture of the nuclear transfer embryos to form blastocysts; And (5) A method for producing a pig embryonic stem cell line comprising the step of separating the inner cell mass from the blastocyst and incubating the inner cell mass in an undifferentiated state to establish a pig embryonic stem cell line. The method of claim 3, wherein Pig somatic cells of step (1) is a production method, characterized in that the somatic cells of the somatic cloned pig. The method of claim 4, wherein The somatic cell is a manufacturing method, characterized in that the fibroblasts. The method of claim 3, wherein In the step (5), the inner cell mass is characterized in that the separation by physically removing the trophic ectoderm. The method of claim 3, wherein In step (5), the LIF is excluded from the inner cell mass, the production method, characterized in that the culture in a medium containing bFGF. The method of claim 7, wherein The bFGF production method characterized in that it comprises a concentration of 10 to 30 ng / ml.

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