KR20190052563A - Knock-in vector using whey acid protein and use thereof - Google Patents

Abstract

The present invention relates to a knock-in vector using a whey acid protein (WAP) gene, capable of efficiently producing foreign proteins, and a use thereof. The knock-in vector has the base sequence of SEQ ID NO: 1 and the base sequence of SEQ ID NO: 2.

Description

Knock-in vector using whey protein gene and its use {Knock-in vector using whey acid protein and use thereof}

The present invention relates to knock-in vectors using whey protein genes and their uses. More specifically, the present invention relates to a knock-in vector using a swine whey protein gene capable of efficiently producing an exogenous protein using a transgenic animal, a transformed cell line containing the same, and a method for producing an exogenous protein using the same .

In general, milk, urine, and the like of transgenic animals have been attracting attention as a bioreactor for mass production of useful recombinant proteins (Houdebine, 2000), and many studies have been conducted on mammals secreted from mammary glands (Reichenstein et al. , 2001; Takahashi, 2001).

The production of transgenic animals producing mammalian exogenous proteins in cattle, sheep, sheep, pigs, and goats, including mice, has been attempted using the promoter region of a major milk protein present in milk. Among these, the promoters of beta-lactoglobulin, goat beta-casein and small alpha-S1-casein in sheep were most efficiently known for the expression of external proteins in the mammary gland of transgenic mice. Using genomic DNA sequences instead of cDNA, It has also been reported that expression of a transgene is increased when a detoxified exon or intron is used in a vector (Murray and Maga, 1999).

Milk protein expression and milk secretion in the mammary gland are regulated by tissue- and time-specific gene expression, respectively (Malewski and Zwierzchowski, 1995; Zhao et al., 2002). The Cis-active base sequence or CoREs (composite response elements) are composed of a group of transcription factor binding sites and contain all of the transcriptional activity / repression factors controlling the timing and location of the mammalian lipoprotein gene Et al., 1999; Wyszomierski and Rosen, 2001).

Expression vectors using the promoter of the pig gene so far revealed various patterns of mammary gland-specific expression patterns. However, there is always a need for the development of expression vectors with more useful expression of the target protein and a greater possibility of regulation.

As a background of the present invention, Korean Patent Registration No. 10-1236724 discloses a gene targeting knock-in vector comprising a protein over-expression cassette, a method for producing the same, and a transgenic animal for transplantation into which the vector has been introduced.

A number of patent documents are referenced and cited throughout the present invention. The disclosures of the cited patent documents are hereby incorporated by reference into the present invention in its entirety to better illustrate the state of the art to which the present invention pertains and the content of the present invention.

According to an embodiment of the present invention, a knock-in vector using a swine whey protein gene capable of efficiently producing an exogenous protein using a transgenic animal is provided.

According to one embodiment of the present invention, there is provided a transformed cell line comprising a knock-in vector using a whey protein gene of swine.

According to one embodiment of the present invention, there is provided a method for producing an exogenous protein using a transgenic animal comprising a knock-in vector using a whey protein gene of swine.

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

According to one aspect of the present invention, a knock-in vector using a whey protein gene is provided, having the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2.

According to an embodiment of the present invention, the nucleotide sequence of SEQ ID NO: 1 may be a promoter region of a whey acid protein (WAP) gene of a pig and a pWAP 5'-arm derived from exon 1.

According to one embodiment of the present invention, the nucleotide sequence of SEQ ID NO: 2 may be a pWAP 3'-arm derived from exon 3 of the whey acid protein (WAP) gene of pigs.

According to one embodiment of the present invention, the knock-in vector using the whey protein gene is DT-A, the pWAP 5'-arm of the nucleotide sequence of SEQ ID NO: 1, htPA-bGHpA, EF1-neo / GFP- The pWAP 3'-arm of the nucleotide sequence of SEQ ID NO: 2 may be operatively sequenced.

According to an embodiment of the present invention, a knock-in vector using whey protein gene comprises DT-A, pWAP 5'-arm of the nucleotide sequence of SEQ ID NO: 1, hG-CSF-bGHpA, EF1-neo / GFP-bGHpA , And the pWAP 3'-arm of the nucleotide sequence of SEQ ID NO: 2, in sequence.

According to another aspect of the present invention, there is provided a transformed cell line into which a knock-in vector is introduced using a whey protein gene.

According to another aspect of the present invention, there is provided an embryo produced by nuclear transfer of the transformed cell line.

According to another aspect of the present invention, there is provided a transgenic cloned animal produced by nuclear transfer of the above transformed cell line.

According to one embodiment of the present invention, the transgenic cloned animal may be a pig.

According to another aspect of the present invention, there is provided a method for producing an exogenous protein, comprising the step of producing a foreign protein in mammalian cells and a transgenic cloned animal using a knock-in vector using a whey protein gene.

In accordance with one embodiment of the present invention, the exogenous protein is selected from the group consisting of erythropoietin (EPO), tissue plasminogen activator (tPA), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor ), Thrombopoietin (TPO), interleukin, interferon, growth factor, insulin or an insulin-like growth factor.

According to one embodiment of the present invention, the exogenous protein may be a human tissue plasminogen activator (tPA).

According to one embodiment of the present invention, the exogenous protein may be a human granulocyte colony stimulating factor (G-CSF).

According to one embodiment of the present invention, a knock-in vector using a whey protein gene capable of efficiently producing a foreign protein can be provided using the transgenic animal.

According to one embodiment of the present invention, the transformed cell line capable of producing an exogenous protein and the transgenic cloned animal can be efficiently produced using the knock-in vector.

According to one embodiment of the present invention, foreign protein such as tissue plasminogen activator (tPA) and granulocyte colony stimulating factor (G-CSF) can be efficiently produced using the transgenic cloned animal.

FIG. 1 is a graph showing the structure of a porcine WAP gene genome to produce a transgenic animal according to an embodiment of the present invention, the structure of tPA and hG-CSF knock-in vector constructed using the same, FIG.
2 is a schematic diagram of a knock-in vector constructed in the first embodiment of the present invention.
FIG. 3 is a photograph showing a result of identifying a gene inserted using a restriction enzyme for a knock-in vector constructed in Example 1 of the present invention. FIG.
4 is a photograph of a transformed somatic cell in culture selected according to Example 3 of the present invention.
FIG. 5 is a photograph of a PCR result showing that a knock-in vector was introduced into the transformed somatic cells selected according to Example 3 of the present invention.
FIG. 6 is a photograph of a PCR result of a foreign gene inserted between whey protein gene loci in transgenic somatic cells selected according to Example 3 of the present invention. FIG.
FIG. 7 is a karyotype image of transformed somatic cells tPA # 3, tPA # 4 and tPA # 5 selected from porcine ear cell derived somatic cells (pEF) prepared according to an embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, a knock-in vector using a whey protein gene is provided, having the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2.

In the present invention, " whey protein " means a casein-free milk protein and accounts for about 20% of the milk protein. Approximately 80% of all whey proteins are lactoalbumin and lactoglobulin, which are not precipitated by acids, but are also known as thermostable proteins in milk because they coagulate and settle by heat. It also contains some proteases and peptones which are not solidified by acid or heat. These are not homogeneous according to the electrophoresis experiment, and the classification of lactoalbumin is made into? -Lactoglobulin,? -Lactoalbumin, and serum albumin, and the classification of lactoglobulin becomes similar to intrinsic globulin.

In the present invention, " knock-in vector " is a vector capable of inserting a desired gene at a specific gene position in the genome, and includes a homologous sequence to a specific gene to be targeted so that homologous recombination occurs do. The knock-in vector of the present invention is a whey protein targeting vector in which a nucleic acid sequence encoding an exogenous protein is inserted into a whey protein gene on the genome.

The whey protein gene or promoter of the present invention may contain one or more disruption, deletion, insertion, point, substitution, non-sense, or deletion in the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: a nonsense, a misinse, a polymorphism, and a rearrangement mutation can result in a selected one of functional equivalents.

According to one embodiment of the present invention, the nucleotide sequence of SEQ ID NO: 1 is the pWAP 5'-arm of the whey acid protein (WAP) gene of the pig and the 4.8 kb region derived from exon 1 (Fig. 1).

According to one embodiment of the present invention, the nucleotide sequence of SEQ ID NO: 2 may be a pWAP 3'-arm of about 3.2 kb derived from exon 3 of the whey acid protein (WAP) gene of pigs ).

The knock-in vector of the present invention may comprise, but is not limited to, a promoter, an enhancer, a selective marker, a 5'-untranslated region (UTR) , A 3'-UTR, a polyadenylation signal, a ribosome binding sequence, a base sequence that can be inserted into a specific part of the genome, an intron, or a woodchuck hepatitus virus posttranscriptional regulatory element (WPRE) Facilitating the construction of the transformed cell line, maximizing the expression amount of the exogenous protein, and stabilizing the expression.

The knock-in vector of the present invention can be used to select transformed cells using positive and / or negative selection genes, if necessary. The selection gene is a gene for selecting a cell transformed with a vector and can be selected from genes which give selectable phenotypes such as drug resistance, nutritional requirement, tolerance to cytotoxic agents or expression of surface proteins, and a positive selection gene And negative selection genes.

Neomycin (Neo) resistance gene, Hygromycin (Hyg) resistance gene and the like, which enable positive selection by allowing only the cells expressing the selected gene to survive in the environment treated with the selective agent, have.

The negative selection gene is a gene that enables negative selection to selectively remove cells that have undergone random insertion, and is a gene encoding a herpes simplex virus-thymidine kinase (HSV-tk) gene, hypoxanthine phospholibosyl A hypoxanthine phosphoribosyl transferase (Hprt) gene, a cytosine deaminase gene, and a diphtheria toxin gene. The negative selection gene is located at the 5 ' end of the promoter region or at the 3 ' end of the 3 'arm.

The positive selection gene and the negative selection gene may have separate promoters, poly A, and the promoter used may be the monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, HIV long terminal repeat (LTR) promoter , The Moloney virus, the cytomegalovirus (CMV) promoter, the Epstein Barr virus (EBV) promoter, the rosaceum virus (RSV) promoter, or the phosphoglycerate kinase (PGK) promoter.

When the knock-in vector of the present invention and the whey protein gene on the genome are homologous recombination, the nucleic acid encoding the exogenous protein on the vector is integrated into the whey protein genome gene of the host cell and expressed instead of the whey protein of the host cell.

The vector production of the present invention can be carried out using gene recombinant techniques well known in the art, and site-specific DNA cleavage and linkage are performed using enzymes generally known in the art.

In the present invention, the term " exogenous protein " or " target protein " is a useful protein industrially available. Examples of the protein include all proteins used as active ingredients of drugs, erythropoietin (EPO), tissue plasminogen activator (GM-CSF), thrombopoietin (TPO), interleukin, interferon, growth factor, insulin or insulin-like growth factor .

The exogenous protein may suitably be a human tissue plasminogen activator (tPA) or a human granulocyte colony stimulating factor (G-CSF).

According to one embodiment of the present invention, the knock-in vector using the whey protein gene is DT-A, the pWAP 5'-arm of the nucleotide sequence of SEQ ID NO: 1, htPA-bGHpA, EF1-neo / GFP- The pWAP 3'-arm of the nucleotide sequence of SEQ ID NO: 2 may be operatively sequenced (Figs. 1 and 2).

In general, human tissue plasminogen activator (htPA), which has a thrombolytic action in a biological blood stream, is produced by hydrolysis of a single bond of arginine and valine in the plasminogen amino acid sequence It has a zymogen function that converts plasminogen to plasmin and is a plasmin mediated proteolytic enzyme that is used for tissue remodeling and degradation and cell migration and various other physiological functions It plays an important role in physiopathological events.

The human tissue-type plasminogen activator (htPA) is a protein of about 68 to 72 kDa, consisting of 562 amino acids, of which 527 amino acids are glycoproteins (htPA) constituting the histopathologic plasminogen activator (htPA) glycoprotein. These histoplasic plasminogen activator (htPA) exhibit a highly complex molecular structure, which is formed by a disulfide bond to form a heterodimer form of the A chain and the B chain (chain B). It binds to fibrin in the blood due to its high affinity, and the interaction of these two chains increases enzyme catalytic efficiency by 100-1,000 times by increasing affinity for plasminogen in the blood. This shows similar results as heparin binding increases the activity of plasminogen in the blood. The synthesis of htPA is known to be produced mainly in vascular epithelial cells and is also synthesized in various tissues including tumors and can be expressed in plasma, uterine fluid, saliva saliva, gingival crevicular fluid, tears, seminal fluid, and milk.

Such human tissue plasminogen activator (htPA) activates plasminogen protein in the bloodstream with fibrin clots-degrading plasmin, which causes fibrin, (Thromboembolism) such as acute myocardial infarction, pulmonary embolism, paralysis, cerebral apoplexy, and arteriosclerosis by dissolving thromboembolism while dissolving thromboembolism, do.

However, until now, the above-mentioned therapeutic agent has been used to purify the recombinant tPA produced by culturing microorganisms and animal cells, but the secretion amount is too small, and the physiological activity is not the same as that of natural type, and htPA produced in cells is produced And the activity was different.

According to the present invention, a large amount of human tissue-type plasminogen activator (htPA) is produced through the mammary gland of a transgenic cloned animal, whereby a safe and continuous substance can be continuously supplied in the animal's living body, And the effect of producing a biologic drug as a therapeutic agent in the thrombus.

According to an embodiment of the present invention, a knock-in vector using whey protein gene comprises DT-A, pWAP 5'-arm of the nucleotide sequence of SEQ ID NO: 1, hG-CSF-bGHpA, EF1-neo / GFP-bGHpA , And the pWAP 3'-arm of the nucleotide sequence of SEQ ID NO: 2, in sequence.

Human granulocyte colony stimulating factor (hG-CSF) is a glycoprotein with a size of 19.6 kDa that regulates proliferation and differentiation of hematopoietic progenitor cells (Bober et al., 1995, Immunopharmacology 29: 111-119) (Kaushansky, 2006, N. Engl. J. Med. 354: 2034-2045) in the treatment of patients with chemotherapy, in the case of aplastic anemia, in the development of myelodysplastic syndrome, and in hematopoietic stem cell transplantation. . Most of the currently used hG-CSFs are produced in recombinant protein form in E. coli. However, since the protein produced in Escherichia coli has many problems in the post-translational modification process after glycoprotein translation such as glycosylation, its use is limited in many ways. The most ideal way to solve this problem is to harvest the glycoprotein directly from the animal in which the hG-CSF gene is introduced and expresses the gene, that is, the transformation.

Using the knock-in vector of the present invention, the hG-CSF gene of a human can be efficiently transfected, and transgenic cells or cloned animals of various animals can be efficiently transformed to express the hG-CSF gene. In addition, hG-CSF can be economically produced in large quantities using transgenic animal cells or cloned animals as a bioreactor.

According to another aspect of the present invention, there is provided a transformed cell line into which a knock-in vector is introduced using a whey protein gene.

The cell line may be somatic cells of an animal, and somatic cells of an animal into which the vector of the present invention is introduced may be primary, secondary, or permanent cells derived from pigs and the like.

The method of introducing the vector of the present invention into the cell may be any of the methods of introducing the nucleic acid into the cell, and it is possible to use techniques known in the art, such as electroporation, calcium phosphate co- precipitation, retroviral infection, microinjection, DEAE-diethylaminoethyl-dextran, cationic liposome, and the like can be used. In this case, the vector may be introduced into a vector form of a linear shape or a vector form of a linear form with the plasmid removed, by cutting a circular vector into an appropriate restriction enzyme.

According to another aspect of the present invention, there is provided an embryo produced by nuclear transfer of the transformed cell line.

That is, the present invention also provides an animal embryo prepared by nuclear transfer of a nucleus of an somatic cell of an animal transformed by introducing the knock-in vector of the present invention into an enucleated oocyte.

In the present invention, " nuclear transfer " refers to implantation of a nucleus of a cell into a nucleus-removed oocyte, wherein the born organism is implanted with a nucleus-transferred cytoplasm Because they are delivered as they are, they become genetically identical duplicate objects.

The present invention also provides an animal embryo implanted with a knock-in vector of the present invention. Specifically, microinjection technique for injecting a gene as a micro manipulation technique into the male pronucleus of the conjugate immediately after fertilization immediately after fertilization, gene insertion into embryonic stem cells, A retroviral insertion technique is used to inject a gene into a male's testis using a stem cell insertion technique and a retroviral vector to insert the gene into a sperm, A sperm-mediated gene transfer technique, or the like. A preferred example is a microinjection method.

According to another aspect of the present invention, there is provided a transgenic cloned animal produced by transplanting said animal embryo.

An animal that can be transformed with a knock-in vector of the present invention is any animal that secretes milk, such as a pig, a mouse, a cow, a sheep, or a goat, and the like, have.

The production method of the transgenic animal using the knock-in vector of the present invention is by a conventional method.

That is, the embryos are collected from healthy individuals such as rats in the animals to be transformed, the expression vectors of the present invention are introduced into fertilized eggs, and the pregnant mice and the like are obtained by using a vasculature ligation mouse or the like, , And then screening the transformed individuals among the offspring obtained from the surrogate mother.

In addition, ovarian follicles are collected from healthy individuals such as pigs and cultured in a culture medium for in vitro maturation. The expression vector of the present invention is introduced into donor somatic cells collected and cultured from embryos such as pigs, and somatic cells into which the vector has been introduced are selected and cultured. The nucleus is removed from the in vitro matured oocyte, the donor cells are injected into the space, and the donor cells and the cytoplasm of the oocyte that has undergone the nuclear transfer through the fusion are fused and in vitro cultured. This cloned fertilized embryo is transplanted into a superovulation-induced parasite and then screened for transgenic individuals from offspring derived from the parasite.

The thus produced transgenic animal of the present invention can express an exogenous protein in milk.

According to still another aspect of the present invention, there is provided a method for producing an exogenous protein, comprising culturing a knock-in vector using a whey protein gene and producing an exogenous protein in a mammary gland of a transgenic cloned animal.

After collecting the juice from the individuals identified as being transformed, the exogenous protein is separated and purified to produce an exogenous protein (A. Gokana, JJ Winchenn, A. Ben-Ghanem, A. Ahaded, JP Cartron, Lambin (1997) Chromatographic separation of recombinant human erythropoietin isoforms, Journal of chromatography, 791, 109-118).

In the method for producing an exogenous protein according to the present invention, a conventional method may be used for the separation and purification, and specifically, a filtration method or a chromatography method may be used.

Therefore, the swine whey protein gene, promoter, knock-in vector and transgenic animal using the same of the present invention can be usefully used in the field of production of exogenous protein.

The issues related to genetic engineering techniques in the present invention are described in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 and Frederick M Ausubel et al., Current Protocols in Molecular Biology volume 1, 2, 3, John Wiley & Sons, Inc., 1994).

Hereinafter, the present invention will be described in detail with reference to examples.

Example

Example  1. Construction of knock-in vector using the porcine WAP gene region

Example  1-1. The 5 'region of the knock-in vector Cloning

PCR cloning was performed to use about 4.8 kb region containing the promoter region and exon 1 portion of the whey acid protein (WAP) gene as the 5 'region from the pig genomic DNA.

Ear cells of Landrace cultivated at the National Livestock Academy were collected and cultured to construct ear cell - derived fibroblasts (porcine Ear Fibroblast: pEF). Genomic DNA was extracted from the constructed cells, and the pWAP-LongArm-F primer (SEQ ID NO: 3) and pWAP-LongArm-R primer (SEQ ID NO: 4) (TaKaRa). After PCR, the amplified product was confirmed by electrophoresis and purified by gel elution kit (Qiagen) for the product of desired size.

The purified amplified product was TA cloned into pSTBlue-1 vector (Novagen), transformed into E. coli, and subjected to blue / white selection and colonization with ampicillin drug resistance. The selected colonies were inoculated on LB medium, cultured in O / N, plasmid excised and cut with EcoRI to confirm gene insertion.

The inserted clone DNA was finally identified as a clone into which the desired gene was inserted by analyzing the base sequence. The plasmid was digested with restriction enzymes SalI and ClaI. The digested product was electrophoresed on agarose gel, and pWAP-LongArm (4788 bp) was purified and purified using gel elution kit.

Example  1-2. The 3 'region of the knock-in vector Cloning

PCR cloning was performed to use about 3.2 kb of exon 3 of the pig WAP gene as a 3 'region. Genomic DNA was extracted from the cultured fibroblast (pEF) and used as a template. Using pWAP-ShortArm-F primer (SEQ ID NO: 5) and pWAP-ShortArm-R primer (SEQ ID NO: 6) Polymerase was used for PCR cloning. PCR amplification products were purified using a gel elution kit and TA cloning was performed on the pSTBlue-1 vector. The plasmid was extracted from the colonies selected for resistance to ampicillin drug, and the DNA was cut with EcoRI to confirm whether or not the gene was inserted. The inserted clone DNA was subjected to sequencing to select the clone into which the desired gene was inserted, and restriction enzymes EcoRV and NotI were used The cleaved product was electrophoresed on an agarose gel, and pWAP-ShortArm (3262 bp) was isolated from the gel.

Example  1-3. DT-A and Neo / GFP  Cassette gene

The gene cassette expressing the diphtheria toxin gene as a negative selection marker uses the MC1 promoter, diphtheria toxin gene, SV40 T antigen intronic regulatory sequence and rabbit β-globin polyA sequence, and a positive selection marker to select transformed cells Neomycin and Green Fluorescence protein (GFP) were linked to T2A using a gene cassette. References The selection marker gene cassette described in PLOS ONE 9 (2), E88549 (2014) was used for gene synthesis ).

Example  1-4. Human G- CSF and  tPA gene Cloning

Human G-CSF and tPA genes for gene expression are amplified by PCR using cDNAs prepared from human-derived HEK293 and MCF7 cell mRNA as templates, and the amplified genes are analyzed by TA cloning and sequencing of selected clones Respectively.

Example  1-5. Knock-in vector construction

The above genes were cloned in order to construct the final knock-in vector used in the present invention.

First, pBluesript II SK (+) was cleaved with KpnI and XhoI, followed by DT-A cassette cloning, and the inserted gene was confirmed. Next, the pWAP-LongArm region was inserted, followed by the hG-CSF or tPA gene region, followed by the GFP / neo selection marker, and finally the pWAP-ShortArm region to construct a knock-in vector. The constructed knock - in vectors were identified by restriction enzyme mapping and confirmed the inserted nucleotide sequence by the primer walking method.

Example  2. Porcine somatic cell culture

As a host cell for transducing the knock-in vector constructed in Example 1, land cell-derived somatic cells derived from Landrace were used. Porcine somatic cells were cultured using culture medium (M106 and DMEM).

Example  3. Knock-in vector transduction and selection of transformed somatic cells

The knock-in vector prepared in Example 1 was transfected into the host cell pEF cells cultured in Example 2 to select transformed cells.

Specifically, the host cell pEF cells cultured in Example 2 were washed with 1X HBSS, and trypsin was continuously treated to recover somatic cells. The trypsin was inactivated with 1X HBSS buffer containing 2% FBS Cells were collected by centrifugation at 800 rpm for 5 minutes at room temperature. The recovered cells were resuspended in 1X HBSS buffer, and the cell number and viability were checked using a cell counter device. The number of cells was adjusted to 1 × 10 ⁶ / ml and centrifuged under the same conditions to recover the cells.

The recovered cells were resuspended in the buffer contained in the kit, knocked-in vector prepared in Example 1 was added thereto, and the cells were subjected to electroporation to transfect somatic cells. The transfected somatic cells were allowed to stand for 10 minutes on ice, and the cells were cultured in a 6 well plate containing the culture solution for about 24 hours. In order to select only the transformed cells in the cultured cells, the culture medium containing 600 ug / mL G418 was replaced with the culture medium containing G418 at intervals of 3 days, and the culture was further cultured for about 14 days. Respectively.

Finally, in order to select the transformed somatic clones, the first selected transformed somatic cells were diluted by extreme dilution method and cultured on a 96-well plate. The cells were sequentially transferred into 24 wells, 12 wells and 6 wells, Respectively.

Example  4. Transformant cells into which a knock-in vector was introduced PCR  analysis

Transformation of the transformant cells selected in Example 3 was confirmed by PCR using genomic DNA at a stage from 12 wells to 6 wells. Genomic DNA was isolated from some of the recovered cells for subculture and used as a template, and pWAP-KI-372-F (SEQ ID NO: 7) and pWAP-KI-372-R (SEQ ID NO: 8) PCR was performed. PCR products were confirmed by electrophoresis on 1% agarose gel. As shown in FIG. 5, the specific band was amplified only in the cell transfected with the foreign gene and not in the control cell pEF somatic cell.

PCR was carried out for the analysis to confirm the transfected form of whey protein gene using pWAP-KI-372-F (SEQ ID NO: 7) and pWAP-KI-257-R (SEQ ID NO: 9) primers PCR was performed. The PCR products were confirmed by electrophoresis on 1% agarose gel. As a result, as shown in FIG. 6, the band type shown in the control group pEF somatic cell showed a chromosome form that did not contain a foreign gene, and amplified products were detected in the gene inserted into the whey protein gene by the knock-in vector . This indicates that the inserted form in the genome by the knock-in vector is contained only on one side of the chromosome.

Example  5. Screening transformant cell karyotype

In order to use recombinant somatic cells selected in Example 3 for the production of transgenic animals, a karyotyping analysis was carried out to confirm the chromosome abnormality. FIG. 7 is a photograph showing the karyotypes of tPA # 3, # 4 and # 5 cells in which knock-in pEF somatic cells and knock-in cells were confirmed. As shown in FIG. 7, it was confirmed that all 38 and XX normal chromosomes were present. Therefore, the selected transgenic somatic cells were analyzed to be usable for pig replication.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Knock-in vector using whey acid protein and use thereof <130> NPF31379 <160> 9 <170> PatentIn version 3.2 <210> 1 <211> 4787 <212> DNA <213> Sus scrofa <400> 1 gggtgctgtt ccccaacaga agagattctg ggcacccagg aaccagtgcc caggctgtgc 60 ccatcccgga aaagacggtc actcttggga ggggagaaac cagggcagtg aggccgaggg 120 gcaagtgtag atgtgacccc aaaatgggca gagcaggagg agcagcggcg gcggagggac 180 cactgggcag ggccagggga ggagtcagga ccgaggaggg aacggaaagg aggtgaggcg 240 ggttgagggg cacctttcca tggaggccag actgtgcaga gggggagacc atggcagccc 300 aaagcggagg gaagaggact cagaaagagg cccgctcttt taattttttg ttgttgttgt 360 ctttttaggg ccacacccgt ctccaggcca ggcgtcgaat cagagctgca gccgttagcc 420 tacgccacag ccacgccaga tccaagccat gactgggacc tacgcaacag ctcacggcaa 480 cgccagatct ttaacccact gagcgaggcc agggatcaaa cctgtgtcct catggatgct 540 aggcagattc atttccactg agccaccatg cgaactctga aagaggcctt ctagcacaca 600 aagttcattt gtcttgccca gaaagaagat gcaacggact cccaaacccc tagaattcca 660 tatgtatgaa agtgtcccca agctctctaa cttcatactc aacagaacat tttttttctc 720 tttttacagc catacctgca cacatggaag gccccgtgct aagggtcaaa tcggagctgc 780 agctgcagct gccgcctccg ccagagcact cggcaacatc agatccttaa cccgctgagc 840 aaggccaggc gtcaaaccca catcctcatg ggagatgagt caggttctta acccactgag 900 ccacaatggg aactcctcga caggacactc gatacgatgt ggagccttgc gagggcgggg 960 caaggcatgt gacatgtgtg tccacccttc ctggccaccc caagtgtacc cggcattcgt 1020 gatgtcccat gtgaccccaa ctggagagga acggacatcc tgggtcccta ggagacttca 1080 gacacaggga ggaggcaggg gcgtctaaga accccaaggg acccagaacg cccctccccc 1140 ttccaatgag gcttcttcct gtaggaccag ccagttacac agaaaatgag gacaaagaga 1200 aagggacaga cagaaagagc cctccgctcc ttctctttca gccttgcaga ctccgggccg 1260 gctgggtgga gtgcagacag actgtgcagg taggcagcga aaccaggaat accctcagca 1320 gcactactca gcaacagccc gagcagccgt cggtacagga agggcgaaca agacgtggtc 1380 cagccgctcg gccgagcagt gtccggccat aaacagaaag gaagcgctga gcacgctccc 1440 ctgggacgcc ccctgaggac tcgatgctca ctgagggcca gacacaaaag gacgcacagg 1500 ccacggttcc gtctacatgc aaagtccaag acgggcaaac ccacagagac aggaagcaga 1560 ctggaggttg ccaggcgctg ggaggcggcg agcagtcact gctaacagac acagggcttc 1620 ctgggagagg aatgaaatgc tctaaaatta aagagttagt tgcagggagt tccggtcatg 1680 gctcagcaga aatgaacctg actagtatcc tcagggatgc ggattcgacc cctggcctcc 1740 ctcagtgggt taaggatcca gcattgccgt gagccgtggt gtaggtcgca ggcatggttc 1800 agatcccaca ttgctatagc tgtggtgtag gccggcaaat gcagctccaa ctggacccct 1860 agcctgggaa cctctacatg ctgagaaaag acaaataagg agttcccatt gtggtgtggt 1920 ggaaacaaat ctgactagga accatgaggt tgtgggttcg atccctggcc tcgctcagtg 1980 ggttaaggat ccagtgttgt tgtgagcaat ggagtaggtt gcaggcgcag cttggatcct 2040 gcattgctgg ggctgtggtg tcggctggca gctgtagcac cgaatcaacc tctagtctgg 2100 gaatgtctac atgtcgtggg tacagcccta aaaagcaaaa aaaaaaaaaa aaaaaagaca 2160 aagagttagt tgcacagttc tgcgaactaa aaaccactga cttgtacacc agtgaacttt 2220 atgacatgga aatcatatct cagtaaaaaa gaaaaataag ataaaatcaa gaatgcaaat 2280 tttaccctct tcaacataga aacacaccta gaagtgtgcg gccgtgaatg aagaccagac 2340 cttcaggcag tgtctggcct cacccttcgg gcacaggcag gaccatggag ggttaaaggg 2400 gttttgttga cgggagaggc ccgtctgaca aaagtcaatc agttcattgt atcaggaaaa 2460 ataaaagtgc ttccgaggac acagactcct cagaacaaga atgcgtctga aaataaaagg 2520 aaaagaggtg aagagaaagc tgctgggtga gttttgtgca gaatgaaata cacgtgcctg 2580 tcccagccag ggatgtgaag tgggtgattt cggtgattct gcataagagt taaatgctcc 2640 tgagtttgca tttcaagcgg cattgcacaa cataaagaca aacggtaata atagaaacat 2700 ttaatttttt tttttttcat ttttggccac cctgcagcaa tgtggaggtt cccaggctag 2760 gggtctagtt agagctgcag ctgccggcct acaccacaac cagagcaaca caagatccaa 2820 gctgcgtctg tgacctacac cacagtgctt ggcaataccg gatccttaac ccactgagtg 2880 cggccaggga tcgaacccgc atccacatgg ttatagttag ttggattcgt ttccactgca 2940 ccacaagggt aactctaaca catttttttt ttaaacaccg tttttaaaaa caaaagagag 3000 acctggaaaa gaggaaaacc tttgtattta gtctatttgc ccactttttt cccacttgtt 3060 gaacaaagaa ccccacaaag tacatcgcca gccctctgtc agcgaggaag ggaccaggct 3120 ctgtgaccct gtgactgcag gccccagagg ccaagggctg tgaccgtggg agccagagac 3180 tggagactca gaatgaggcc acagcccagt ggtctgagtt cctggtgtgg tcaagccagg 3240 ccttggacct tgcatctgag taggggggac cctgagacct cccggccatg gtgtaaggga 3300 agcgcagaga atagggagag aaaggccagc tgggccagac agagagtgat gatggcctgg 3360 ccgcccctcg cttctccacc ctcagtgtct ggcgtaggta ccgcctcttt tccaggctgc 3420 aggacgggaa acaaccaaag gcgtggcatt gtgccaacta ccagcacgga aaggcaccaa 3480 gagaaagcaa gtcacccttg gccaggacct ccagacccca atccccgaag gccagccctg 3540 gaggggtctg ggctgaggcc ctagaatgat gctgaggggt tttctgggcc cagacggtgc 3600 tgcgtggggt ccagggcggg gagagccagg ggcgttggag ccacctatct gaatgcaagt 3660 gtgttctggg ctagaacatc tggaaaggtc tgtagcacaa gctgagggcc cgatgggaag 3720 aagtgagggg ctctgtgccc aaggcctcga ggccgcaggc ccagcagagg ggtgcggcct 3780 ggaccagagc ggggtactga ctatcggcac atgtggtcgc cctggggcca ccttccctgt 3840 ccatgccctc agggcacaag gatcaaggaa gtctccccag agaagaaggg gcagagtctg 3900 agcattcatc ctgcccccca cccctgcagc ctcccagggc acaagcgggg tccttctggg 3960 aaggggccct cccagtgccc acactgcacg gaaacagccc cgacactgaa cctgcctgcc 4020 cctccccttc tcagacacct gacattctct gcacagagca cacagctcat aaactcaaga 4080 tggccattct tgctgcaggt tcggggcggg gggcagaggg gagaaaacca ctgcaggaga 4140 aataaaatgc cctccacagt aacccctgct ccacctcctc tgctacctgt gggccatgtt 4200 ctgaactgga catgtcactc cctgtccctg aagggccctc catgaccaca gaaaaggttg 4260 gctatcgtgg aacttgtttc ttcctttgtc tgccctaatt ttcttaattc tcatcatcag 4320 catacattac tttcaaatgt atgcttattg tgggaaaaaa gtccacctta ataggaattc 4380 ttttattttt tcctttaaaa atctggccca gggtgctcct gttgtggctc agcgaaaatg 4440 aatccgacta atatccatga ggatgcaggt tcaatccctg gccctgctca gtgagttaag 4500 gatctggcat tgctgtgagc tgaggagtag atggcagatg cagctcggat cctgtgttgc 4560 tgtggctgtg gctgtggcac aggccagcag ctctagctcc aattcaaccc ctagccctag 4620 cctgagaact gcagatgcag ccctaaaaaa gcaggaaaaa aaaaaaaaag tggcccaagg 4680 cagcaactac cccatctggg gtctatccaa gccggaagcc cctaggccct gctggggcag 4740 cgccagccac ccgccctccc tctaggttcc tgtacccgct gcctcct 4787 <210> 2 <211> 3268 <212> DNA <213> Sus scrofa <400> 2 gtcactggga tacagagggt ctccgtctag cagagggggc ccagaggagc cctgacttcc 60 gggtgggact tccagccagg cccgcctgcc acgcttgtgc ctttgccact caatctgggg 120 aggagacctg ggctaggagc ccagagatgc ggttcctgga taaccagtcc ctgccaccct 180 tgaccttgcc ggcccaccta cggcaatgat gagggcccag gagggatggg tgtggacggc 240 tcgctggctt gcccacacag cccctccctc cccgccccca catggctggg ctccacctcc 300 tctgaggcct ccgtgtcccc tacagaggcc cctcttcagt gagggacatc cctgggagcc 360 cccggctgcc aggagtgacc agcccgagtc cactcagcaa gaaccttctc tctcggatcc 420 agagaccaca cgatgcctcc tatctgctgc taataaaaac ctactcggct tcatgtctct 480 ccgccctctg tctgtctgtc cctggaacct gccagagggc acgagacaca caggccttgc 540 ctctgagagc tccccagcaa gcctggaagc ccctggaaac cggcagtccc agctgaggtg 600 tagtgagacc aggcaggatg taggtagaca ggacgggtcc tgggtgagcc agaggaaagg 660 ccaccacctc ttctctccca gggtctgggg gagctatggg ccccaggtgg gcacaggctt 720 gacctggggt cagtcctgtc tgggccatca gccagcactt aaggtcaagg gggccaccca 780 catcatcaca ctggctgaga tgaaggtggg acatggggac gtgtctcacc tccctccatc 840 cccgccctgg ggagaagacc cttcccagag cttggaaggt agggtgacag ggccagtcat 900 gggaagccca ggcccatggc acatgaggtc actcctccct ggctttggtg cccacgaggc 960 ctggccccca ttatacggag tcagtgaaga ggaaaaaagc attctcgact gcctattagc 1020 ccctgtgtac cagccctccc caggctactg gctttgggcg tacacaaggt cccccacaaa 1080 agagagcctg ctgtgttgcc atatgcagga gaagagagga gccctcctaa atgaaagtgg 1140 ccctagaact gctggagaag gggagcggtg gggcccaggg ccatctcaga aggcaccagg 1200 cactgagagt gggcaactgc ttgagaccct gggcctctgt gtctcctcca ccttcctgtc 1260 cagattcact cgataattgg ccacatttca gtctctatca tgtgccagtt gatgagcggg 1320 aattggagag acaggacgaa ttgtcccaaa tgaaaagggc ttcaaccaat ggctgaattc 1380 tgtttctgaa ccactaaagg aatcagaatt ccttggagaa acagccaagc tgaagaacta 1440 tcaagctagt gtgggcatct tgcatcataa agcatggagc cagcctgagt ccccaccagc 1500 caaacttggc accaccaggg tatccatggt aaagcatgac attggagcaa agggaaagct 1560 cttcttcaca gaagatgcta gttaacacat ggagaaggaa cgactgagtt agaaaagcca 1620 ccattttgca acacccaaaa taatcgttaa tctgagcaag aatcattaga aggcgaaaac 1680 ccaggtgcag agattgccca ggattcacaa agggggaagc tgcttccaca acaggcagtg 1740 aggagaggga ggaggaggaa aacagagact agggggagaa cagctgaaga aattcgagtg 1800 tggactagac actagatgac ctgagtgaat cgatgtgaat cttctcggtg aaataacgac 1860 gtggtggtca cgtggtgaac atacactgct gaattactga gagttcacga tgtctgcaac 1920 tcactttcac aggatatatc gcactaacca atacacacac aggcttacat ctatctatgg 1980 aatgcctacc gacatgtgcg cgtgcgcgcg cacacacaca cacacacaca cacacacaca 2040 cacacacaca cacacgtgtg ccgtggggag gagagaggga gaaaacaaag gcacgagagc 2100 aactgctacc ggctgaagcc aagtaaaggg gagaggatgg tcactgtgct gtgctctgag 2160 cttttcggaa aggtggccga ttttcacaat aaaaagggaa agaaggtcaa tgtccatgag 2220 ccgaccttcc aggcctagag agtgctgggt cacccgatgg gaggcctgtg caggctgcca 2280 tgtttggaag gtcctgaagt gggtaacgcg tggctgggaa cccagcacca gagaggaggc 2340 gctgctcgag gacacacagg agggaggcct ggatgcccag caggcaaaag ggcactccag 2400 ggggcgagga agttgggtct gcaggtctgg agggagcccc agccaaccaa cacgcaaagg 2460 gactctggac tttggtcaag gacacacaga gctaccacac ctccaggaag ccccaaggct 2520 ggaagagaga aggcacccct tcccccaccc ccacctgtgc tggacaggtg ccggtagagc 2580 ttctggggag acaggtgagt ctgagatgga gctgaacccc tggacggggc tgtcactcat 2640 gctggggccc aggcccagga aagacccctg gctggcccta atcccagcct gggcagctgg 2700 aaccacccag cccaccctgc cctgagctca taatgaatgg tgtggaagat gcagcagccc 2760 tgctcactcc catgctgggg atccttctcc tgcagagaac acccatcccg gcatcgacca 2820 catgccagct tctctactgt gatgctctta acatctaccc tacatgcata tcctggacgc 2880 ctcaatgaca cgcccgtccc accctgctcg gcttcccgcc tcaggtgtgc aaatcaacca 2940 acccagagac cacaccccca ccacctcctc tatggggtct tatactccag gtcactgtcc 3000 taccacaggg ccagggacag acaactaggg gcagccccta ccccccagag ccctctgcca 3060 ttactcacac tagccagtcc taagcctgct catcctgact ctcccactcc ttccacggaa 3120 accacagtaa aggctcctgc ccgccaaccc cacccctcct ggctgaccct gtgcttcccc 3180 cggtggccct tcatgaggga ggtgcctctt tctcgtggga actgttagta ataaaccatc 3240 ctcactggca gtcatcttag agtttcca 3268 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 3 gtcgacgggt gctgttcccc aacagaagag attc 34 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 4 atcgatagga ggcagcgggt acaggaacct agag 34 <210> 5 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 5 gatatcgtca ctgggataca gagggtctcc gtctagc 37 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 6 gcggccgctg gaaactctaa gatgactgcc agtgaggatg 40 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 7 catctggggt ctatccaagc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 8 cacggcgact actgcactta 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 9 ttatccagga accgcatctc 20

Claims (13)

A knock-in vector using a whey protein gene having the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2. The method according to claim 1,
The nucleotide sequence of SEQ ID NO: 1 is a knock-in vector using a swine whey protein gene, which is a pWAP 5'-arm derived from exon 1 and a promoter region of whey acid protein (WAP) gene of pig. The method according to claim 1,
The nucleotide sequence of SEQ ID NO: 2 is pWAP 3'-arm derived from exon 3 of the whey acid protein (WAP) gene of pigs, a knock-in vector using a swine whey protein gene. The method according to claim 1,
In order to enable the pWAP 5'-arm, htPA-bGHpA, EF1-neo / GFP-bGHpA, and pWAP 3'-arm of the nucleotide sequence of SEQ ID NO: 2 to function, Contained knock-in vector using the whey protein gene. The method according to claim 1,
PWAP 5'-arm, hG-CSF-bGHpA, EF1-neo / GFP-bGHpA of the nucleotide sequence of SEQ ID NO: 1 and pWAP 3'-arm of the nucleotide sequence of SEQ ID NO: 2 are operable A knock-in vector using the whey protein gene in the sequence, in sequence. A transformed cell line into which the knock-in vector according to any one of claims 1 to 5 has been introduced. An embryo produced by nuclear transfer of the transformed cell line of claim 6. A transgenic cloned animal produced by nuclear transfer of the transformed cell line of claim 6. 9. The transgenic animal according to claim 8, wherein the transgenic cloned animal is a pig. A method for producing an exogenous protein, comprising the step of producing a foreign protein in mammalian cells and a transgenic cloned animal using the knock-in vector according to any one of claims 1 to 5. 11. The method of claim 10,
The exogenous protein may be selected from the group consisting of erythropoietin (EPO), tissue plasminogen activator (tPA), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM- CSF), thrombopoietin , Interleukin, interferon, growth factor, insulin or an insulin-like growth factor. 12. The method of claim 11,
Wherein the exogenous protein is a human tissue plasminogen activator (tPA). 12. The method of claim 11,
Wherein the exogenous protein is a human granulocyte colony stimulating factor (G-CSF).

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