WO2005032245A1

WO2005032245A1 - Method of producing transgenic pigs expressing green fluorescent protein

Info

Publication number: WO2005032245A1
Application number: PCT/KR2004/001414
Authority: WO
Inventors: Kwang-Wook Park; Jae-Goo Seol; Ki-Nam Heo; Seung-Hyeon Lee; Seung-Pyo Hong; Ki-Myung Choi
Original assignee: Mgenbio, Inc.
Priority date: 2003-10-08
Filing date: 2004-06-14
Publication date: 2005-04-14
Also published as: KR100479704B1

Abstract

The present invention relates to a method of producing a transgenic pig expressing green fluorescent protein (GFP). Particularly, the present invention relates to the method of producing the above transgenic pig including the steps of; (1) separating a somatic cell from a porcine fetus; (2) preparing the expression vector which includes a green fluorescent gene, followed by introducing the above expression vector into the above somatic cell; (3) selecting a cloned somatic cell transduced with the above expression vector and culturing that selected cell; (4) eliminating a nucleus from the oocyte of the donor pig, followed by fusing the resulting oocyte with the somatic cell to form a oocyte-somatic cell complex; and (5) transplanting the fused replicative oocyte into a surrogate pig and delivering piglets of present invention. The transgenic pigs expressing green fluorescent protein produced according to the method of the present invention can be effectively used as a transgenic animal model in the fields of the basic studies of science including cell therapy and in the production of a therapeutic protein by the transgenic animals.

Description

Invention Title

METHOD OF PRODUCING TRANSGENIC PIGS EXPRESSING GREEN FLUORESCENT PROTEIN

Technical Field

The present invention relates to a method of producing a transgenic pig expressing green fluorescent protein (GFP) , more specifically to a method of producing a transgenic pig expressing green fluorescent protein available for the field of producing therapeutic proteins and for the cell therapy.

Background Art

Cloning means producing a number of genetically identical individuals. One of the cloning techniques is an attractive nuclear transfer resulting from a great progress of molecular biology. A blastomere of a fertilized egg was used as a nuclear source in the early nuclear transfer (Prather, et al., Biol . Reprod. , 1987, 37:856-866; Prather, et al . , Biol . Reprod. , 1989, 41:414-418), but it is recently performed by using the nucleus of a somatic cell. A cloning technique of a somatic cell is to introduce an nucleus of a differentiated somatic cell into an enucleated oocyte to develop it in the same manner as that of a general fertilized egg. It is expected that this cloning technique is not only available for the studies of basic science including developmental biology but also for production of a therapeutic protein, especially in the field of medical science, for example developing a disease model and organ transplantation etc, so that it may be greatly useful for the industrial availability.

Since the cloning of the first somatic cell cloned animal "Dolly" (Willmut, I. et al . , Na ture, 1997, 385:810-813), progress has been made in cloning animals, leading to the successful cloning of a cow (Cibelli, JB . et al . , Science, 1998, 280:1256-1258; Wells, DN. et al . , Reprod. Fertil . Dev. , 1998, 10:369-378), a rat (Wakayama, T. et al . , Na ture, 1998, 394:369-374) and a goat (Bagusi, A. et al., Na t . Biotechnol . , 1999, 17:456-461). But in the case of pig cloning, the studies have been nearly made about the fertilized egg of pig and its cloning has succeeded just recently because of difficult physiological characteristics of pigs, for example their pregnancy capable of being maintained only when at least 4 embryos are implanted in their uteruses, etc (Poleajaeva, IA. et al . , Na ture, 2000, 407:86-90; Onishi, A. et al . , Science, 2000, 289:1188-1190; Betthauser, J. et al . , Na t . Biotechnol . , 2000, 18:1055-1059). The first transgenic cloned pig was then produced by using the somatic cells introduced a exogenous gene (Park, KW. et al., Anim . Biotechnol . , 2001, 12:173-181). Then, another cloned pig, in which a specific gene was knockout in a somatic cell, was produced (Lai, L. et al., Na t . Biotechnol . , 2002 Mar, 20 (3 ): 251-255 ) . Although somatic cell cloning with pigs has been rapidly progressed, the cloning efficiency is still very low with 1-5%, and therefore further studies are required to improve the efficiency.

Somatic cell cloning includes the steps of eliminating a nucleus from an oocyte, injecting a somatic cell into the enucleated oocyte, followed by fusing the resulting oocyte with the somatic cell to form a oocyte-somatic cell complex and activating it under electro-stimulation. The conventional method, for example microinjection of DNA has a problem of low productivity of a transgenic animal. Because GFP (green fluorescent protein) derived from fluorescent jellyfish (i.e., Aequorea ictoria ) specifically expresses strong fluorescence when this protein is excited by the ultraviolet radiation, it is widely used as a marker protein in the various fields of animals, plants and microorganisms (Takada T et al., Na t . Biotechnol . , 1997, 15:458-461; Perry AC et al., Science, 1999, 284:1180-1183; Chan AW et al., Mol . Reprod. , 1999, 52:406-413). Recently, it was confirmed that a successful development of a cloned egg was made from a pig somatic cell transfected with GFP gene (Uhm SJ et al . , Mol . Reprod. Dev. , 2000, 57:331-337; Koo D-B et al . , Mol . Reprod. Dev. , 2001, 58:15-21; Park KW et al . , Biol . Reprod. , 2001, 65:1681-1685), and the first transgenic cloned pig expressing GFP gene was produced based on that (Park KW et al . , Anim . Biotech . , 2001, 12:173-181). However, since somatic cells used for the cloning above were not clonal somatic cells, integration sites of GFP gene into chromosome were all different among offsprings. Retrovirus was used to insert GFP gene into cells, which has problems of limitation of size of a gene to be transferred to cells and difficulty in securing stability for a clinical application. Thus, in order to overcome such problems of a conventional method for producing a transgenic cloned pig, the present inventors prepared a clonal somatic cell line transfected with a foreign gene using an lipid-based reagent instead of retrovirus. And the present inventors completed this invention by confirming that a transgenic cloned pig specifically expressing a green fluorescent protein in the muscle tissues may be produced using the nuclear transfer technique of the clonal somatic cells .

Disclosure

Technical Problem It is an object of this invention to provide a method of producing a transgenic pig specifically expressing green fluorescent protein in muscle tissues . It is another object of this invention to provide the transgenic pig produced according to the method of the present invention, wherein the transgenic pig specifically expresses green fluorescent protein in muscle tissues. Technical Solution

In order to achieve the above objects, the present invention provides a method of producing a transgenic pig expressing a green fluorescent protein including the steps of; 1) separating somatic cells from a porcine fetus; 2) preparing an expression vector which includes a green fluorescent gene, followed by introducing the expression vector into the above somatic cells; 3) selecting cloned somatic cells transfected with the above expression vector and culturing those selected cells; 4) eliminating a nucleus from an oocyte of a donor pig, followed by fusing the resulting oocyte with the somatic cell to form a oocyte-somatic cell complex; and 5) transplanting the fused replicative oocyte into a surrogate pig and delivering piglets of present invention. The present invention also provides a transgenic pig specifically expressing a green fluorescent protein in muscle tissues produced by the method of the present invention. The term "cloned somatic cell lines" used herein means cell lines having the identical integration site of a foreign vector. The term "perivitelline space" means the space between zona pellucida and ooplasm of an egg. The term "oocyte-somatic cell complex" means pre-cell fusion status after eliminating a nucleus from an oocyte and injecting a somatic cell into its perivitelline space. And the term "a cloned egg" (i.e., a replicative egg) means a state that the oocyte is fused with the somatic cell by electro- stimulation . Hereinafter, the present invention is described in detail. The present invention provides a method of producing a transgenic pig expressing a green fluorescent protein, comprising the following steps: 1) Establishing somatic cells from a porcine fetus; 2) Preparing an expression vector which includes a green fluorescent gene, followed by introducing the expression vector into the above somatic cells; 3) Selecting cloned somatic cells transfected with the above expression vector and culturing those selected cells; 4) Eliminating a nucleus from an oocyte of a donor pig, followed by fusing the resulting oocyte with the somatic cell to form a oocyte-somatic cell complex; and 5) Transplanting the fused replicative oocyte into a surrogate pig and delivering piglets of present invention.

In the above step 1), a porcine fetus is preferably an embryo at day 20 - 50 after pregnancy, and is more preferably an embryo at day 30 - 40 after pregnancy. In particular, the present inventors established somatic cells from a porcine fetus at day 35 after pregnancy. To establish somatic cells from the porcine fetus, a conventional method was applied equally to this invention and, in the preferred embodiment of the present invention a small portion of a porcine fetus was removed by a razor blade, treated with trypsin, and then sub- cultured to establish somatic cells. In the above step 2), the fluorescent protein is preferably GFP, and enhanced GFP (EGFP) is more preferable, but it is not always limited thereto. All the known expression vectors containing the fluorescent protein may be used as an expression vector only if they can be expressed in pig cells. Especially in the preferred embodiment of this invention, pCX-EGFP/neo was used as an expression vector including a fluorescent protein. All the known promoters may be used without limitation as a promoter to express the fluorescent protein. Particularly in the preferred embodiment of this invention, chicken β-actin promoter, known to induce an specific expression of a fluorescent protein in muscle tissues, was used. In the above step 3), the expression vector containing a selection marker gene was used to select somatic cells transfected with the expression vector of the step 2) . As a selection marker, an antibiotics-resistant gene was preferably used. The example of an antibiotics-resistant gene includes neo^r, pac^r, bsr^r, hph^r, etc and neo^r was preferably used in this invention. In the preferred embodiment of the present invention, somatic cells having an expression vector containing a fluorescent protein gene were selected by the treatment of G418. The present inventors inserted GFP gene into porcine somatic cells in the same manner above and then separated cloned porcine somatic cell lines expressing GFP. Then, the present inventors produced a transgenic pig specifically expressing a green fluorescent protein in muscle tissues by transferring the established somatic cell lines into enucleated oocyte.

Advantageous Effects According to recent reports, oocyte-somatic cell complex was successfully cultured to blstocyst after nuclear transfer with porcine somatic cells containing GFP (Uhm SJ et al . , Mol . Reprod. Dev. , 2000, 57:331-337; Koo D-B et al . , Mol . Reprod. Dev. , 2001, 58:15-21; Park KW et al . , Biol . Reprod. , 2001, 65:1681-1685), and a surrogate pig was transplanted with a replicative egg to give birth to a piglet expressing GFP (Park KW et al . , Anim, Biotech . , 2001, 12:173-181). The problem of the conventional research, though, was that retrovirus was used to transfer GFP into cells. In this case, the size of a gene to be transferred into a cell is limited, causing a problem of stability in clinical use. It was one of the major problems to use a retrovirus so as to produce a transgenic animal. The present inventors have overcome the problem by transferring a fluorescent protein gene into a somatic cell successfully using as a carrier a lipid instead of retrovirus. Thus, in the method of the present invention there is almost not limited to the size of a gene to be tranfered into a somatic cell.

Although earlier studies were successfully made only in introduction of a fluorescent protein gene into the somatic cell, a cloned somatic cell line has not been established. Cloned somatic cell lines have the same integration regions of the expression vector in the genome. If a cloned somatic cell line is established by transfection with the above expression vector, a fluorescent protein may be present only in the same regions of the somatic cells. According to a region on the chromosomes where a fluorescent protein gene is inserted, the expression pattern of the protein differs in each cell. So, when such cell lines was used to produce a transgenic pig, the expression patterns of a fluorescent protein was different in every pigs (Park KW et al . , Anim . Biotech . , 2001, 12:173-181). In the present invention, a cloned somatic cell line was prepared by overcoming the problem of the technique. This technique is expected to enhance production efficiency of a transgenic animal. That is, a gene encoding a protein of interest is randomly inserted into a genome of a somatic cell to obtain various cloned cell lines having different gene insertion regions. Those cell lines were used to produce cloned animals and then the expression patterns of a target protein from the cloned animals were examined to select individuals having high expression rate. It is because that the expression rate may become different in the cell lines having different insertion regions of the expression vector into a genome . In earlier studies, CMV promoter was used to express a foreign gene in whole body. On the contrary, chicken-β actin promoter was used in this method of the present invention to produce a transgenic pig, showing that a fluorescent protein was strongly expressed in muscle-related tissues (i.e., hoofs, nose, myocardium, intestines, etc.) (FIGs. 3 and 4) . The expression of a fluorescent protein was not detected in viscera, bone, and brain except an intestine. A tissue-specific expression technique is highly useful for producing a therapeutic protein by using a transgenic animal. For example, if a gene is controlled to specifically express a target protein in the mammary gland, the target protein can be obtained from milk. A transgenic pig produced by the method of the present invention expresses GFP in muscle tissues of a whole body, so that GFP cells taken from the transgenic pig can aid to understand vital phenomenon. Organs of a pig are similar to those of a human in the physiological aspect. Efforts have been made to transplant an organ of a pig into a human, which is still unsuccessful because of rejection of xenograft. Recently, a transgenic pig, in which a specific gene involved in xenograft rejection was knock out, was produced, promoting possibilities of transplanting a xenograft by using a transgenic pig (Lai L et al . , Science, 2002, 295:1089-1092; Dai Y et al., Na t . Biotechnol . , 2002, 20:251-255). If a xenograft of a pig organ may be successfully transplanted into human, a porcine somatic cell may also be introduced into human. In that case, the development and differentiation of the transplanted somatic cells in a human patient may be easily observed by using the cloned somatic cell line containing the fluorescent protein gene of the present invention.

Description of Drawings

Fig. 1 is a schematic diagram showing the cleavage mapping of pCX-EGFP/neo vector. Fig. 2 is an electrophoresis photograph showing (A) the result of PCR to investigate the presence of GFP gene in geno ic DNA of a transgenic pig, and (B) the result of FISH to analyze chromosome of a cloned pig and localize an insertion region of GFP gene on chromosome of a cloned pig. Fig. 3 is a set of photographs showing the expression patterns of GFP (green fluorescent protein) in a donor cell, an NT embryo and an offspring: A and B) Cultured fetal fibroblast cells; C and D) An NT blastocyst cultured in vi tro for 6 days; and E) a NT piglet. In particular, B, D and E are photographs examining GFP expressions by using fluorescence . Fig. 4 is a set of photographs showing the GFP expressions in the tissues of cloned NT piglets: A to U) Tissues under normal light; and A' to U' ) Tissues under fluorescent light. In Fig 4; A and A' : Hoof, B and B' : nose, C and C : Ribs with skeletal muscle, D and D' : Heart, E and E' :

Tongue, F and F' : Kidney, G and G' : Skeletal muscle (non-transgenic pig, negative control) , H and H' :

Brain, I and I' : Skin, J and J' : Thymus, K and K' :

Ovary, L and L' : Ear, M and M' : Spinal cord, N and N' : Skeletal muscle, O and O' : Umbilical cord, P and

P' : Frontal bone, Q and 0/ : Pancreas, R and R' : Liver, S and S' : Thyroid gland, T and T' : Lung, U and U' : Small intestine. FIG. 5 is a photograph showing two transgenic pigs.

Mode for Invention

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated from those skilled in the art that on consideration of this disclosure, modifications and improvements may be made within the spirit and scope of the present invention, without limiting thereto.

<Example 1> Separation of porcine somatic cells

A porcine fetus at day 35 after pregnancy was cut into small pieces with razor blades. Cells were incubated in DMEM medium (bio-whittaka . Inc) supplemented with trypsin-EDTA for 5 minutes. The cell culture was centrifuged at 1,000 rpm for 5 minutes to obtain supernatant, which was further cultured in DMEM medium supplemented with 10% FCS (Fetal Calf Serum, Hyclone . Inc) . When cells became 90% confluent, they were sub-cultured after trypsin was added. Some of them were used for nuclear transfer and the others were stored in -70 for a future use.

<Example 2> Preparation of cloned porcine cell

lines expressing GFP

To prepare a vector containing GFP gene, pCX- EGFP vector (Kato M et al . , Mol . Reprod. Dev. , 1999, 54:43-48) was connected with neo gene to construct a pCX-EGFP/neo vector (FIG. 1) . The vector pCX- EGFP/neo was constructed by separating neo gene from vector "pPNT" (Tybulewicz VL et al . , Cell . 1991, 65:1153-1163) and attaching the neo gene onto HindiII site of pCX-EGFP. The pCX-EGFP/neo vector was cut with Sail (Roche. Inc) and the target DNA was isolated by phenol-extraction. The DNA was then transfected into somatic cells by the lipid-based method (Fugene 6; Roche. Inc) according to the manufacturer's protocols (Sharon J.H. et al . , Transgenic Research , 2002, 11:143-150). The cell was cultured for 48 hours. Then, G418 (Geneticin; Gibco. Inc) was added thereto to select transfected somatic cells. Their fluorescence was observed under a fluorescent microscope (Nikon) . G418 was added to the cell culture at various concentrations to determine the optimum concentration (250 βg/ l ) . The transfected cells were treated with 250 /_~g/m# of G418 for 2 weeks and then colony-forming cell lines were selected. The resulting cloned cell lines were frozen at -70 ^°C for nuclear transfer. GFP expression in the selected cloned cell lines were observed under a fluorescent microscope. As a result, a cell line PCX-8 expressing over 90% of GFP was selected (FIG. 3) .

The present inventors deposited the cloned cell line PCX-8 in the Korean Cell Line Research Foundation (KCLRF) on May 15, 2003 (Accession No: KCLRF-BP-00079) .

<Example 3> Nuclear transfer using the cloned

cell

<3-l> Preparation and culture of oocytes Ovaries were collected from prepubertal gilts at a local abattoir and stored in 0.9% NaCl solution at 35-39^°C. Cumulus-oocyte complexes (COCs) were inspired from 2-6 mm diameter of antral follicles using an 18-gauge needle fixed to a 10 m# disposable syringe. COCs were washed with tissue culture medium. 50-60 COCs were added to a 500 μJL of culture solution, then cultured for 42-46 hours. At that time, the tissue culture medium was prepared by supplementing TCM 199 (31100035; Gibco, Grand Island, NY) with 0.1% polyvinylalcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 irvM cysteine, 0.5 βg/ϊ l LH (L-5269, Sigma Chemical Co. St. Louis, MO), 0.5 μg/wl FSH (F-2293, Sigma), 10 ng/m£ epidermal growth factor (E-4127, Sigma), 75 βg/mi penicillin G and 50 _~g/ml streptomycin.

<3-2> Micromanipulation COCs obtained in the above Example <3-l> were cultured in micromanipulated medium for 5-10 minutes. The cloned cell line PCX-8, selected in the above Example 2, was added to the micromanipulated medium which was prepared by supplementing TCM 199 with 0.3% BSA and 7.5 βg/wl CB (cytochalasin B) . In order to eliminate a nucleus from an oocyte, the polar body of oocytes were fixed in the direction of 1 o'clock, and a fine glass pipette (30 μrn diameter) having 30-60° beveled grinded point was put into a polar body. The polar body and about 10- 20% of cytoplasm near the polar body were inspired by oil pressure. Most of the nuclei of oocytes were located around polar body, so that they were successfully eliminated by the inspiration above. The porcine somatic cells prepared in the above Example 2 were then inspired through the fine glass pipette, the fine glass pipette was put into the perivitelline space of enucleated oocyte, and then somatic cells were injected into the perivitelline space of the oocyte by the oil pressure.

<3-3> Cell fusion and activation

When micromanipulation was completed in the Example <3-2>, the injected oocyte was transferred into the medium consisting of 0.3 M mannitol, 1.0 mM CaCl₂-H₂0, 0.1 mM MgCl₂-6H₂0 and 0.5 mM HEPES, and the oocyte-somatic cell complex was placed between two platinum electrodes which are 1 mm apart in a medium. Cell fusion/activation was induced simultaneously with two successive DC pulses of 1.1 kV/cm for 30 μsec using a BTX Elector-Cell Manipulator 2001 (BTX, San Diego, CA) . As a control a native oocyte was electrically activated in the same manner described above. The level of fusion was determined at 0.5-1 hr after the DC pulses. <3-4> Cultivation/Development of replicative

embryos

20 to 30 reconstructed embryos prepared in the above Example <3-3> were selected and cultured for 6 days in a 4-well culture dish containing 500 βi of development medium. Then, all embryos were stained with Hoechst 33342 (5

) to determine the number of nuclei by epi-fluorescent microscopy. The development medium was prepared by adding 0.4% BSA to NCSU-23 medium (North Carolina State University- 23; Petters, RM. and Wells, KD., J. Reprod. Fertil . Suppl . , 1993, 48:61-73). PCX-8 cell line was used for nuclear transfer and in vitro development of each stage from external fertilization to blastocysts was investigated. As a result, fusion rate, division rate and each developmental rate up to blastocyst stage of the fused replicative embryos were 78.5%, 67.2%, and 12.9% respectively, and the number of nuclei of blastocysts was 23.8 (Table 1). Strong GFP expression was also observed in blastocysts (FIG. 3),

<Table 1> Development of Nuclear Transfer Embryos

<Example 4> Transplantation of replicative

embryos and production of transgenic piglets

The replicative embryos at day 1 or 2 after the production were transplanted into 20 surrogate pigs in estrus (DO or Dl : the beginning day of estrus cycle or the next day) by surgical operation. Ultrasonography was performed on the 28^th day after the transplantation, confirming early pregnancy. And the stable maintenance of pregnancy was confirmed by using ultrasonography once a week. As a result, eleven healthy female piglets and one stillbirth were delivered from three surrogate sows. The mean birth weight was 1094 g and ranged from 680 to 1580 g (Table 2 and FIG. 5) .

Cloned Piglets Derived from Nuclear Transfer.

<Example 5> Determination of transfected GFP

gene

PCR analysis was performed to investigate the expression of GFP in the piglet above and cloned cell line PCX-8. Particularly, proteinase K (Gibco Inc.) was treated to all piglets' ear skin cells, donor cells, and surrogate skin cells to decompose proteins. Then, genomic DNA was isolated by using phenol-extraction. Two PCR primers represented by SEQ ID No. 1 and No. 2 were used. PCR was performed as follows; predenaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, polymerization at 72°C for 1 minute, 30 cycles from denaturation to polymerization, and final extension at 72°C for 10 minutes. The PCR product was transferred on to agarose-gel for electrophoresis . As a result, it was confirmed that 500 bp of EGFP DNA product was present in both of piglets and PCX-8 cell lines, but not in ear tissues of surrogate pigs (FIG. 2A) . Chromosome analysis of the donor cells and cultured ear cells of piglets suggested that the chromosomes isolated from all the piglets were present in the normal state (FIG. 2B) . FISH analysis showed that the GFP gene was located close to the telomere of chromosome 7 (C7) of the piglet derived from the PCX-8 cell lines, showing that it was present in the same region as in the metaphase spreads (FIG. 2B) . Thus, the result indicated that all the produced piglets were the transgenic cloned pigs derived from the PCX-8 clonal cell line. Next, GFP image was acquired using Las 3000

(FUJI FILM, Tokyo, Japan) equipped with high sensitive cooled CCD camera. After the stillbirth (GFl-1) or the piglet GF2-4 was placed in the dark- box, gray-scale body surface image was taken.

Afterwards, GFP image was taken using blue light (460 nm) for excitation and 510DF10 filter. The piglets were dissected and various organs were removed. Specimens were also placed in the dark box and GFP expression was captured. As a result, strong expression of fluorescence was observed outside body of piglets especially in tongue, eye periphery, foreleg, lumbar region and hoof (FIG. 3). To compare the GFP expression intensity of each organ, twenty-one tissues were separated from two piglets (GFl-1, GF2-4) . GFP expression was mainly detected in muscle related tissues (hoof, nose, heart, tongue, skin, ear, skeletal muscle, and intestine), but was not detected in most internal organs except bone, bone marrow, brain, intestine, etc. (FIG. 4).

Industrial Applicability

As explained hereinbefore, the transgenic pig expressing green fluorescent protein (GFP) produced according to the method of the present invention can be useful in the fields of the basic studies of science including cell therapy and in the production of a therapeutic protein by the transgenic animals.

Sequence List Text

Nucelotide sequences represented by SEQ ID No. 1 and No. 2 are primer sequences used in the PCR reaction of Example 5.

Manifestation om the Deposited Mcxoorgarasm or Otker Biological Materials

A The Mowing manifestation is directed to the deposited microorganism or other biological materials described in the line 21 of page 8 of the present application. B. Identification of tine Microorganism An additional rrfcroorganismte) is described in the next page(s) eκcepl the one D Depository Authority Korean Ceil Line Research Foundation (RCLRP) Address (Zip code and Nationality) Korean Cell Line Research Foundation (KCLRF) Cancer Research Institute, Seoul National University College of Medicine 28 Yeøageon-dong, Jongno-gu, Seoul 110-744, Republic of K rea Date Accession number May 24 2004 IΪCLRF-BP-0007S C, Additional Manifestation (filed as "blank" if there is no disclosure) To be continued to the additional n x page D

D. Characteristic Items directed to the Manifestation

E Any Additional Manifestation (filed as "blank;" if there is no disclosure) Any of the following manifestation will be filed to the International Bureau of the World Intellectual Property Organisation later. (General Properties of the manifestation should be specificaEy described)

Items for a Receiving Authority Items for the International Bureau □ Accepted with the international D Accepted by the International Bureau application Authorised OfficiaKs) Authorized OfficiaKs)

ECX/ftO/134(Iuly 1898)

Claims

Claim 1

A method of producing a transgenic pig expressing green fluorescent protein (GFP) comprising the following steps: 1) Separating somatic cells from a porcine fetus; 2) Preparing an expression vector which includes fluorescent protein gene, followed by introducing the above expression vector into the somatic cell of the step 1); 3) Selecting cloned somatic cells transduced with the above expression vector and culturing those selected cells; 4) Eliminating a nucleus from an oocyte of a donor pig, followed by fusing the resulting oocyte with the somatic cell to form a oocyte-somatic cell complex; and 5) Transplanting the fused replicative oocyte into a surrogate pig and delivering piglets .

Claim 2

The method of producing a transgenic pig as set forth in claim 1 , wherein the porcine fetus of the step 1) is at 30 - 40 days after pregnancy.

Claim 3

The method of producing a transgenic pig as set forth in claim 2, wherein the porcine fetus is at 35 days after pregnancy.

Claim 4

The method of producing a transgenic pig as set forth in claim 1, wherein the fluorescent protein in the step 2) is enhanced green fluorescent protein (EGFP) .

Claim 5 The method of producing a transgenic pig as set forth in claim 1, wherein the expression vector in the step 2) is pCX-EGFP/neo .

Claim 6 The method of producing a transgenic pig as set forth in claim 1, wherein the cloned somatic cell in the step 3) is a PCX-8 cloned cell line (Accession No: KCLRF-BP-00079) . Claim 7

A transgenic pig specifically expressing a green fluorescent protein in muscle tissues produced according to the method of claim 1.