KR20070024069A - A transgenic pig expressing csf gene in the milk and producing method thereof - Google Patents

Abstract

A method for producing transgenic pig capable of expressing colony stimulating factor(CSF) gene in the mammary gland is provided to be able to easily obtain the transgenic pig, which is able to solve the problems of post-translational modification or mass-production and is used as a supplemental agent of treating leukemia and an immuno-potentiating agent, by expressing hGM-CSF in mammary gland. The method comprises the steps of: (a) isolating somatic cell from pig fetus on days 40-80 pregnancy; (b) after preparing an expression vector including a promoter specific to mammary gland such as alphas1-casein, beta-casein, beta-lactoglobulin and whey acidic protein; and a polynucleotide encoding the CSF such as hGM-CSF, introducing it into the somatic cell; (c) after selecting the expression vector-introduced clone somatic cell, culturing it; (d) removing nucleus of ovum collected from sow and fusing it with the clone somatic cell; and (e) transplanting the fused nuclear transfer embryos to surrogate mother pig and delivering piglets.

Description

A transgenic pig expressing CSF gene in the milk and producing method etc.

Figure 1 shows the pBC1 expression vector u (Milk Expression Vector, Invitrogen, USA ),

Figure 2 shows a transgenic construct for mammary grand specific expression of human GM-CSF,

Figure 3 shows the RT-PCR results of the hGM-CSF transgene introduced in the transformed HC11 cell line,

M; Markers;

One; RT-PCR positive control;

2: G3PDH in untransformed HC11 cell line;

3-6: G3PDH in transformed HC11 cell line;

7: hGM-CSF in untransformed HC11 cell line;

8-11: hGM-CSF in transgenic HC11 cell line,

Figure 4 shows Western blot analysis showing mammary gland specific expression of hGM-CSF in mouse HC11 cell line,

Mock: non-transgenic control HC11 cells;

GM-CSF: transformed HC11 cells,

5 is a GM-CSF and NEO PCR screen result,

 M: marker;

1: positive control;

2: negative control;

3 ~ 10: sample,

6 is a Southern blot result,

M: marker;

P: positive control;

1: clone number 27-4;

2: clone No. 27-28;

3: clone number 27-32,

7 shows PCR results for the offspring of transgenic pigs.

M; Markers;

1: positive control;

2: transformed cell line 27-32;

3: surrogate mother;

4: D.W;

5: transgenic pig 1;

6: transgenic pig 2;

7: transgenic pig 3.

The present invention relates to a transgenic cloned pig expressing human GM-CSF in the mammary gland and a method for producing the same.

Biotransformation technology is a technique for transferring and expressing foreign genes on the chromosome of an organism by artificial methods in order to improve the flora and fauna, to improve productivity and to obtain industrial medically useful products, and to induce and manipulate overexpression of endogenous genes. By deleting the function of a specific gene, a series of techniques such as changing the phenotype according to the expression of the gene and identifying the function of the gene are collectively referred to.

In the case of transgenic animals, the technique of transferring foreign genes to living organisms is pursued by a microinjection method through a microinjector into a fertilized egg, a method using various types of viruses, a method using an electromagnetic field, and a method using a compound such as lipid There are many ways to use it for different purposes. Depending on the promoter that induces the expression of the foreign gene, it is possible to express it in a specific tissue, ie, mammary gland, liver, or blood, or induce the expression of the foreign gene according to the developmental stage of the organism.

The field of transformation in animals has been put to practical use by many research results. In particular, the transgenic cloned animals can improve livestock and create new varieties, improve disease resistance to many diseases in livestock, and obtain by-products from livestock. It is used to improve quality, produce disease model animals, develop long-term production animals that can replace the loss of organs due to human diseases or disasters, and produce useful materials through bioreactor systems.

Cloning refers to the creation of genetically identical organisms. Nuclear transfer is a technique that has attracted new attention due to the recent rapid development of molecular biology as a method of cloning in eukaryotes. Nuclear transfer of early, but using the blastomeres of embryos as a source of nuclei (Prather, et al, B iol Reprod, 1987, 37:...... 856-866; Prather, et al, Biol Reprod, 1989, 41 : 414-418) Recently, somatic nuclei have been used.

Somatic cloning is a technique in which differentiated somatic cells are activated by inserting them into an egg from which a nucleus has been removed and then in the same manner as a normal fertilized egg. Such cloning technology can be widely used in research in basic sciences including developmental biology, and is expected to contribute greatly to various medical and pharmaceutical fields such as production of useful proteins, development of disease models, and transplantation of organs. The usefulness is very large. In other words, the production system using the above technology is very economical, and can not only supply bioactive substances that are currently sold at a low price, but also utilize the bioactive substance production system closest to the human body physiologically. Therefore, it is possible to produce high-quality bioactive substances, and to use the animal protein production system in vivo, so that mass production is possible, and in particular, the development of medicines such as intake vaccines does not require a separate purification process. .

However, in order to realize this, securing of a target gene must be preceded first, and a tissue-specific promoter must be secured to determine the degree and site of expression. In addition, in the case of animals, since the gene should be transplanted in the embryonic development stage, it is essential to culture fertilized eggs and to inject foreign genes into the nucleus. And a technique for transplanting fertilized eggs injected with foreign genes to surrogate mothers is required, and a technique for assaying whether the foreign genes are inserted on the chromosome and the degree of expression of the live births is required.

Since the birth of the first somatic cloning animal, Dollmut (I. et al., Nature , 1997, 385: 810-813), the field of somatic cloning has evolved a great deal (Cibelli, JB. Et al., Science, 1998, 280: 1256-1258; Wells, DN et al, Reprod Fertil Dev, 1998, 10:..... 369-378), mice (Wakayama, T. et al, Nature , 1998, 394:. 369-374), goats (Bagusi, A. et al., Nat. Biotechnol ., 1999, 17: 456-461) have been successfully cloned.

However, compared to other species, pigs have been relatively poorly studied in fertilized eggs, and due to various difficulties, such as physiological characteristics in which at least four fetuses are implanted in the uterus to maintain pregnancy, the pigs have been successfully replicated relatively late among major livestock. (Poleajaeva, IA. Et al., Nature , 2000, 407: 86-90; Onishi, A. et al., Science , 2000, 289: 1188-1190; Betthauser, J. et al., Nat. Biotechnol , 2000, 18: 1055-1059). Subsequently, the gene was successfully cloned by injecting an external gene into pig somatic cells to produce the first transgenic cloned pig (Park, KW. Et al., Anim . Biotechnol ., 2001, 12: 173-181). Cloned pigs knocked out in somatic cells were also successful (Lai, L. et al., Nat. Biotechnol. , 2002 Mar, 20 (3): 251-255). However, despite the rapid development of the porcine somatic cloning field, the efficiency of cloning is still low at 1-5%.

Colony stimulating factors (CSFs) are glycoproteins required for the survival, proliferation and differentiation of hematopoietic stem cells. Cytokines that promote hematopoiesis have been found as protein factors that act on progenitor cells in the bone marrow and promote differentiation and growth of specific cells. When progenitor cells of bone marrow are grown in a medium containing soft agar, and a protein factor is added, a specific population of cells grows in colonies. Examination of the cells in these colonies shows that the proteins put together are involved in the differentiation of those cell types. The cytokines that act on progenitor cells to promote differentiation of specific cell populations are called CSFs.

These CSFs include granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF), interleukin-3 (IL- 3 or multi-CSF), IL-7 and the like.

Monocytes-macrophage CSF (M-CSF) is made by macrophages, endothelial cells, and fibroblasts, acting on cells that become monocytes. IL-3 is a cytokine produced by CD4 T cells and acting on most immature bone marrow cells to produce several types of blood cells (multilineage CSF). IL-7 is produced by bone marrow stromal cells and acts on cells that will become B lymphocytes. G-CSF is made by activated T cells, macrophages, vascular endothelial cells, and fibroblasts, acting on cells that will become granular cells. GM-CSF is made by activated T cells, macrophages, vascular endothelial cells, fibroblasts, etc. to regulate the differentiation of granular and monocyte lineages do.

Globally, only G-CSF and GM-CSF are used as recombinant therapeutic proteins (see Table 1 ).

TABLE 1 Recombinant G- CSF  And GM- CSF Commercially available formulations and approved uses thereof

G-CSF and GM-CSF show various forms of glycosylation in endogenous states (see Table 2 ).

TABLE 2 G- CSF  And GM- CSF Biochemical Properties of

The chromosome location of GM-CSF is linked to IL-3 side by side and is close to IL-4, IL-5, and M-CSF. hGM-CSF is produced by various cells such as T-cells, B-cells, macrophages, mast cells, endothelial cells, and fibroblasts that are stimulated by cytokines, immunity, and infection (see Table 3 ). ).

TABLE 3 G- CSF  And GM- CSF Cell source

While G-CSF has a limited effect on neutrophils and their progenitor cells, GM-CSF shows many actions on monocytes and macrophages. Some of these actions are involved in phagocytosis or phagocytosis that kills intracellular germs. In addition, hematopoietic growth factor (M-CSF & G-CSF) or IL-6, IL-1, TNF, etc., which are directly expressed by endotoxin or other inflammatory reactions, can be enhanced. Or promote the participation in inflammatory and immune responses of cells expressing these. In particular, various actions of GM-CSF show a synergistic effect with inflammatory cytokines (interferon-γ) or hematopoietic factors (M-CSF) (see Table 4 ).

<Table 4> Maturity Effector ( effector ) On the cell  G- CSF  And GM- CSF Biological effects of

Recently, hGM-CSF has been produced by various methods using recombinant DNA technology for the purpose of treatment. Yeast and tissue culture system methods are still the most widely used methods. However, due to the lack of proper post-translational modifications in yeast and the difficulty of mass production in animal cells, the demand cannot be met.

In order to overcome the above problems, the present inventors endeavored to produce recombinant hGM-CSF through pig milk by technology for production of transgenic cloned pigs using adult somatic cells and mammary tissue specific expression, resulting in a goat β-casein promoter (casein). pig fetal cells were transformed with an expression vector containing a promoter) and a polypeptide encoding hGM-CSF, and then the cloned eggs were fused with denuclearized eggs, and the cloned eggs were transplanted into surrogate sows. The present invention was completed by confirming that a cloned pig is produced in which -CSF is expressed in the mammary gland.

It is an object of the present invention to provide a method for producing a transgenic cloned pig which expresses CSF specifically in mammary gland and provides a transgenic pig by the above method.

In order to achieve the above object, the present invention

1) isolating somatic cells from porcine fetuses;

2) preparing an expression vector comprising a mammary gland specific promoter and a polynucleotide encoding a colony stimulating factor (CSF) and introducing the same into the somatic cells;

3) selecting and culturing the cloned somatic cells into which the expression vector is introduced;

4) removing the nucleus of the oocytes taken from the sows and fusing them with the clonal somatic cells; And

5) Provides a method of producing a transgenic pig that expresses the CSF protein comprising the step of transplanting the fused cloned eggs into surrogate sows and giving birth to piglets.

The present invention also provides a transgenic pig that specifically expresses CSF in the mammary gland prepared by the above method.

The present invention also provides a clone cell line of the transgenic pig.

In the present invention, " clone cell line " refers to a cell line having the same position of a vector introduced into the genome.

Also, " gastric cavity " refers to the space between the zona pellucida of the egg and the egg cytoplasm.

In addition, " cloning " refers to the stage at which the ova and somatic cells are fused by electrical stimulation.

<Short description>

SS: aplastic anemia,

AL: acute leukemia,

BMT: bone marrow transplantation,

CIN: chemotherapy-induced neutropenia,

MDS: myelodysplastic syndrome,

PBPC: peripheral blood progenitor cell transplantation,

SCN: severe chronic neutropenia,

HIV: human immunodeficiency virus

ADCC: antibody-dependent cellular cytotoxicity,

sTNFR: soluble tumor necrosis factor receptor,

IL-1 Rap: interleukin-1 receptor antagonist protein,

LTB4R: leukotriene B4 receptor,

LPS: lipopolysaccharide.

Hereinafter, the present invention will be described in detail.

The present invention

1) isolating somatic cells from porcine fetuses;

2) preparing an expression vector comprising a mammary gland specific promoter and a polynucleotide encoding a colony stimulating factor (CSF) and introducing the same into the somatic cells;

3) selecting and culturing the cloned somatic cells into which the expression vector is introduced;

4) removing the nucleus of the oocytes taken from the sows and fusing them with the clonal somatic cells; And

5) Provides a method of producing a transgenic pig that expresses the CSF protein comprising the step of transplanting the fused cloned eggs into surrogate sows and giving birth to piglets. The pig fetus of step 1) is preferably a fetus of 40-80 days of gestation, and the pig fetus of step 1) is more preferably a fetus of 50-70 days of gestation. In a preferred embodiment of the present invention, pig somatic cells were isolated using a 60-day-old fetus. As a method for isolating somatic cells, a conventional method commonly used in the art may be applied to the present invention, and in a preferred embodiment of the present invention, the somatic cells may be obtained by slicing the pig fetus using a razor blade and then passaged with trypsin. Separated.

CSF of step 2) is preferably hGM-CSF, but is not necessarily limited thereto. In addition, the expression vector containing the CSF protein can be used without limitation any known expression vector that can be expressed in mammary cells. In a preferred embodiment of the present invention, a pBC1-neo vector was used. In addition, as promoters for expressing the CSF protein, all promoters known to specifically express in the mammary gland such as regulatory sequences such as bovine αs1-casein, β-casein, β-lactoglobulin, and whey acidic protein are all expressed. Although it can be used without limitation, in the preferred embodiment of the present invention, a β-casein promoter of chlorine was used.

In step 3), the somatic cells into which the expression vector of step 2) is introduced can be easily selected by using an expression vector into which a selection marker is introduced. As such a selection marker, an antibiotic resistance gene may be preferably used. The antibiotic resistance gene and the like, the neo ^r, pac ^{r, r} bsr, hph ^r may be used, according to the present invention was used as the neo ^r. In a preferred embodiment of the present invention, G418 was treated to select somatic cells into which the expression vector containing the CSF gene was introduced. Clonal cell lines finally confirmed by the method were deposited to the Korea Cell Line Research Foundation on July 19, 2005 ( Accession No .: KCLRF - BP -00115 ).

In the present invention, by injecting the hGM-CSF gene as a CSF gene into the porcine somatic cells as described above, the pig clone somatic cell line expressing hGM-CSF is selected, and transplanted into the pig egg from which the nucleus has been removed, mammary glandular A transgenic pig expressing specifically was prepared.

Therefore, the present invention is expected to be able to mass-produce hGM-CSF through the mammary gland of pigs and to obtain a glycosylated protein similar to humans using physiologically close pigs. To this end, pBC1 induced expression vector kit (Milk Expression Vector kit, Invitrogen, USA) was used to induce hGM-CSF expression in the mammary gland of pigs. pBC1 vector was modified to develop a transformed cell line expressing hGM-CSF to prepare pBC1 / neo-hGM-CSF, which was linearized and transfected into Landrace porcine somatic cells. At this time, the ORF (Open reading frame) of the hGM-CSF gene obtained by PCR in the human genome was amplified, and sequencing was used in which the nucleotide sequence was 100% identical to the genomic gene. In addition, transfected mouse mammary gland cells to confirm the efficient expression of the completed pBC1 / neo-hGM-CSF vector, RT-PCR (see Figure 3 ) and Western blot (see Figure 4 ) RNA and protein expression was confirmed through.

HGM-CSF gene was transfected using pig fetal fibroblast cells, and somatic cells selected with G418 were first identified by PCR with hGM-CSF and neo gene (see FIG. 5 ). Cells identified through PCR reconfirmed that hGM-CSF was inserted through Southern blot (see FIG. 6 ). Transformed cloned pigs with hGM-CSF were produced using the somatic cloning method using somatic cells identified for transformation (see FIG. 7 ).

Recently, hGM-CSF has been produced by various methods using recombinant DNA technology for the purpose of treatment. Yeast and tissue culture system methods are still the most widely used methods. However, due to the lack of proper post-translational modification machinery in yeast and the difficulty of mass production in animal cells, the demand cannot be met. Therefore, to overcome these problems, many researchers have put much effort into mass production of therapeutic proteins using transgenic animals. Among these efforts, strategies have been used to express desired genes directly in the mammary gland via major milk protein genes. To date, mice, rabbits or livestock have been used to produce heterologous proteins in the mammary gland using regulatory sequences such as bovine αs1-casein, β-casein, β-lactoglobulin and whey acidic protein. come.

In the present invention, in order to produce human therapeutic protein using pigs, hGM-CSF was efficiently injected into pig somatic cells to establish a transformed cell line and produce a transgenic cloned pig. To this end, the goat β-casein promoter was used to produce a transformed pig to express hGM-CSF in the mammary gland of pigs. Thus, it is believed that the production of recombinant hGM-CSF similar to human hematopoietic growth factor is possible. Therefore, if the amount of hGM-CSF expressed by pigs is scaled up, it is considered to be a good source of recombinant human GM-CSF that has similar characteristics to the original human protein and can be used for treatment.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> Production of Porcine Somatic Cells

After 60 days of gestation, the pig fetus was chopped with a razor blade, and the cells were incubated for 5 minutes in DMEM (bio-whittaka. Inc) to which trysin-EDTA (Gibco. Inc) was added. The supernatant was centrifuged (1000 rpm, 5 minutes), cultured in DMEM containing 10% FBS, and when cells reached 90% confluency, subcultured after trypsin treatment, some were used for nuclear transfer, and the others were cryopreserved.

< Example  2> hGM - CSF  Construction of Expression Vectors

As the hGM-CSF vector, pBC1-neo with neo attached to the pBC1 expression vector (Milk Expression Vector, Invitrogen, see FIG. 1 ) was used. The Neo resistance gene was amplified by PCR using primers containing NotI and cloned into pGEM-T-easy vector. The primers used Neo-Forward-5'-ATG CGG described in SEQ ID NO: 1 CCG CGA GGG CCC CTG CAG GTC AAT-3 ' and SEQ ID NO: 2 Neo-Reverse-5'-GAG is described in CGG CCG CTC GGT ACC CGG GGA TCC TCT-3 '. The pGEM-T-Neo vector and the pBC1 vector were cut and cloned into NotI to prepare pBC1-neo vectors. hGM-CSF was confirmed by sequencing by amplifying the ORF portion of the hGM-CSF gene in human genomic DNA by PCR and cloning it into a pGEM-T-easy vector. The primer was used include XhoⅠ, sequence of the primer is the hGM-CSF-Forward-5'- GAC TCG AGA GGA TGT GGC TGC AGA GCC -3 ' described in SEQ ID NO: 3, hGM- represented by SEQ. ID. NO: 4 CSF- Reverse-5'-GAC TCG AGC ATC TGG CCG GTC TCA CT-3 '. pGEM-T-hGM-CSF and pBC-neo were cut into XhoI and cloned, respectively, to prepare a pBC / neo-hGM-CSF vector (see FIG. 2 ).

< Example  3> hGM - CSF  Recombinant Protein Identification

In order to confirm whether the pBC / neo-hGM-CSF vector expresses hGM-CSF efficiently in the mammary gland, mouse mammary cancer cell line HC11 was used. Using transfection reagent (Lipofectamine ^™ 2000, Invitrogen), transfection was performed in 6 wells for 20 hours at a ratio of 10 μl: 4 μg with DNA. Total RNA was extracted using Trizol (Gibco), and RT-PCR was performed using a One Step RNA PCR kit (TaKaRa, Japan) (see FIG. 3 ). The primers used at this time were hGM-CSF-Forward primers as set out in SEQ ID NO: 3 and Reverse-5'-ATC TGG GTT GCA CAG GAA GT-3 'as set out in SEQ ID NO: 5 . In addition, the ORF (open reading fram) of the hGM-CSF gene obtained by PCR in the human genome was amplified, and sequencing was used in which the sequence was 100% identical to the genomic gene. As a result, as shown in FIG . 3 , the band of hGM-CSF was confirmed by RT-PCR and agarose gel electrophoresis to confirm that the pBC / neo-hGM-CSF vector works properly in the mammary gland.

In addition, Western blotting was performed using hGM-CSF (N-19, sc-1321, Santa Cruz Biotechnology, Inc.) antibody to confirm that the hGM-CSF protein was expressed, thereby mammary gland specific expression of hGM-CSF was observed. It was done (see Figure 4 ).

< Example  4> hGM - CSF  Expression clone cell line construction and hGM - CSF  Check for Gene

<4-1> hGM - CSF  Expression Clone Cell Line Construction

pBC1 / neo-hGM-CSF vectors prepared for hGM-CSF expression were cut with AatII and linearized. The transfection reagent (Lipofectamine ^™ 2000, Invitrogen) was used to inject the DNA thus prepared into porcine somatic cells. After transfection, the cells were cultured for 24 hours, and treated with G418 (Genecitin, Gibco) at 300 µg / ml for about 3 weeks to collect colony-formed clone cells.

<4-2> PCR  Primary confirmation by analysis

Some of the selected somatic cells were isolated genomic DNA to confirm the presence of hGM-CSF gene. The somatic cells were treated with proteinase K (Gibco. Inc) to decompose the protein and then denature the protein with phenol to separate genomic DNA. PCR primers of hGM-CSF in the case SEQ ID NO: 6 pBC1-Forward-5'-GAT TGA CAA GTA ATA CGC TGT TTC CTC-3 ' and SEQ ID NO: pBC1-Reverse-5'-CAT CAG described as 7 described in AAG TTA AAC AGC ACA GTT AG-3 '. Primers for the Neo gene is '5'-ATT CGG CAA GCA GGC ATC GCC-3 is described in SEQ ID NO: 9'5'-GGA TTG CAC GCA GGT TCT CCG-3 represented by SEQ. ID. NO: 8, hGM-CSF PCR conditions for the gene were 94 ° C, 4 minutes and 94 ° C, 1 minute, 59 ° C 1 minute, 72 ° C 1 minute 30 times and 72 ° C 10 minutes, 4 ° C. The PCR conditions for the Neo gene were 94 ° C, 4 Minutes, 94 ° C, 1 minute, 60 ° C 1 minute, 72 ° C 1 minute 30 times, and 72 ° C 10 minutes, 4 ° C.

The PCR method was used to identify GM-CSF and NEO gene bands by agarose gel electrophoresis, indicating the presence of hGM-CSF gene (see FIG. 5 ).

<4-3> Southern Blot (2nd confirmation by Southern blot analysis)

Selected cells were treated with proteinase K (Gibco. Inc) to decompose the protein and then denature the protein with phenol to isolate genomic DNA. The separated genomic DNA was digested with EcoRI and subjected to Southern blot analysis. The probe was used to cut hGM-CSF gene (2 kb) from pBC1 / neo-hGM-CSF into XhoI and labeled using a labeling kit (Gene Image Random Prime Labeling Module, Amersham, INC). As a result, cells identified through PCR reconfirmed that hGM-CSF was inserted through Southern blot (see FIG. 6 ).

Clonal cell lines finally confirmed by the method were deposited to the Korea Cell Line Research Foundation on July 19, 2005 ( Accession No .: KCLRF - BP -00115 ).

< Example  5> using somatic cloning hGM - CSF Production of transgenic cloned pigs

<5-1> culture

Mature cultures were prepared in TCM199 (31100035; Gibco, Grand Island, NY) in 0.1% polyvinylalcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 g / ml LH (Sigma, USA), 0.5 g / ml FSH (Sigma, USA), 10 ng / ml epidermal growth factor (Sigma, USA), 75 g / ml penicillin G (Sigma, USA) and 50 g / ml streptomycin (Sigma, USA) was added.

The micromanipulation culture medium was added 0.3% BSA and 7.5 g / ml cytochalasin (CB) to TCM199. The activated culture was added with 0.3 mM mannitol, 1.0 mM CaCl ₂ H ₂ O, 0.1 mM MgCl ₂ · 6H ₂ O, 0.5 mM HEPES. The embryonated culture of cloned eggs was added 0.4% BSA to NCSU (North Carolina State University) -23 medium.

<5-2> Oocyte collection and micromanipulation ( Micromanipulation )

The ovaries of uncultivated pigs were collected from slaughterhouses and transported to 35-39 ° C and 0.9% physiological saline to the laboratory. Oocytes were collected by inhaling follicular fluid from a 2-6 mm diameter follicle by connecting an 18-gauge needle to a disposable 10-ml syringe. The eggs were washed in mature culture medium and 50-60 cells were placed in 4-well plates containing 500 μl culture medium and incubated for 42-44 hours.

The oocytes were incubated for 5-10 minutes in a microalgae culture medium and then somatic cells were added to the culture solution. A 30 μm diameter glass tube was used to remove the first polar body of the egg and its surrounding cytoplasm, and somatic cells were injected into the egg's gastric cavity using the glass tube.

<5-3> Cell fusion and activation

After micromanipulation, the nuclear transfer embryos were placed between platinum electrical wires 1 mm apart. Cell fusion and activation were induced by supplying two DC pulses 1.0-1.2 kV / cm, 30 μsec to a fusion device (BTX Electro-Cell Manipulation 2001, BTX, USA) and examined the fusion rate after 0.5-1 hour.

<5-4> Cloning  culture

Only fused cloned eggs were selected and 20-30 cloned eggs were incubated for 6 days in a 4-well plate containing 500 μl of culture medium. After incubation, staining with 5μg / ml bisbenzimide (Hoechst 33342) was examined for the nucleus of the cloned eggs under a fluorescence microscope.

<5-5> Transgenic pig production by cloning egg transplant

The fused cloned eggs examined above were incubated for 1 to 2 days in a 4-well plate containing 500 μl of culture solution, and then put the cloned eggs in a freezing tube containing 2 ml of the same culture solution and heated to 39 ° C. ( embryo transfer kit (Minitube, USA) was transferred to the fertilized egg transplant site. Surrogate mothers for cloning embryos are screened and prepared for individuals whose estrus has begun, and then temporarily anesthetized by administering 0.5 g of pentotal sodium (foreign drug) to the vein. The anesthetized surrogate mother was fixed on the operating table and then inhaled anesthesia by supplying 5% isoflurane (Isoflurane). The anesthetized surrogate mother is prepared by making an incision about 5cm along the abdominal line and exposing the uterus and ovaries to the outside. At this time, the cloned eggs are inhaled in a catheter (Tom-Cat catheter, Monoject, USA) and pushed up to the buccal canal to transplant the cloned eggs. A total of 1243 cloned eggs were transplanted into 6 surrogate mothers for the production of GM-CSF transgenic cloned pigs. Surrogate mothers with cloned egg transplants were subjected to a pregnancy test using ultrasound on the 30th day, and those with confirmed pregnancy induced delivery after 114 days. Of these, four surrogate mothers became pregnant, three delivered normally, and produced 10 transgenic cloned pigs. Born cloned pigs were treated with protein and K. protein (Gibco. Inc., USA) to decompose the protein and denature the protein with phenol to isolate genomic DNA. The isolated genomic DNA was transformed using SEQ ID NO: 6 and SEQ ID NO: 7 for the GM-CSF gene, and SEQ ID NO: 8 and SEQ ID NO: 9 for the Neo gene. (See FIG. 7 ).

Human GM-CSF is an important regulator of proliferation, differentiation and survival of hematopoietic and granulocytes, macrophages, erythrocytes, megakaryocytes and precursors of hematopoietic stem cells. If hGM-CSF is expressed in transgenic cloned pigs, the mechanism of hGM-CSF will play a major role in adjuvant therapy for patients with weakened immunity such as leukemia adjuvant and immune enhancer by treatment of cancer or organ transplantation. The development of hGM-CSF transgenic pigs via nuclear transfer of cell lines is expected to be of great potential for research and industrial use in developmental engineering.

<110> MGenbio Inc. <120> A transgenic pig expressing CSF gene in the milk and producing          method <130> 5p-06-02 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Neo-Forward primer 1 <400> 1 atgcggccgc gagggcccct gcaggtcaat 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Neo-Reverse primer 1 <400> 2 gagcggccgc tcggtacccg gggatcctct 30 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hGM-CSF-Forward primer <400> 3 gactcgagag gatgtggctg cagagcc 27 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> hGM-CSF-Reverse primer 1 <400> 4 gactcgagca tctggccggt ctcact 26 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGM-CSF-Reverse primer 2 <400> 5 atctgggttg cacaggaagt 20 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> pBC1-Forward primer <400> 6 gattgacaag taatacgctg tttcctc 27 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> pBC1-Reverse primer <400> 7 catcagaagt taaacagcac agttag 26 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Neo-Forward primer 2 <400> 8 ggattgcacg caggttctcc g 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Neo-Reverse primer 2 <400> 9 attcggcaag caggcatcgc c 21

Claims (9)

1) isolating somatic cells from porcine fetuses; 2) preparing an expression vector comprising a mammary gland specific promoter and a polynucleotide encoding a colony stimulating factor (CSF) and introducing the same into the somatic cells; 3) selecting and culturing cloned somatic cells into which the expression vector of step 2) is introduced; 4) removing the nuclei of the eggs collected from the sows and fusing them with the clonal somatic cells of step 3); And 5) A method of producing a transgenic pig comprising expressing a CSF protein comprising the step of transplanting the fused cloned eggs into surrogate sows and giving birth to piglets. The method of claim 1, wherein the pig fetus of step 1) is a fetus of 40-80 days of gestation. The method of claim 2, wherein the pig fetus of step 1) is a fetus of 60 days of gestation. The method of claim 1, wherein the CSF of step 2) is hGM-CSF. The method of claim 1, wherein the mammary gland specific promoter of step 2) is selected from promoters such as αs1-casein, β-casein, β-lactoglobulin or whey acidic protein. 6. The method of claim 5, which is a promoter of chlorine β-casein. Transgenic pigs expressing CSF specifically in the mammary gland produced by the method of claim 1. Clonal cell line for producing the transgenic pig of claim 7. The clonal cell line according to claim 8, deposited with Accession No .: KCLRF-BP-00115.

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