KR101843427B1 - cell line for porcine isoglobotrihexosylceramide synthase knock-out and generation method for the same - Google Patents

Abstract

The present invention relates to an isoglobotryhexosyl ceramide synthase (iGb3s) knockout cell line of pigs, and a method for producing the same, and more particularly, to a method for controlling the expression of a gal epitope by controlling the expression of a gal epitope, The present invention relates to a cell line in which pig iGb3s and GT are knocked out at the same time capable of producing reduced pigs, and a method for producing the same.

Description

[0001] The present invention relates to isoglavobutyric acid synthase knockout cell lines,

The present invention relates to a porcine isoglobotrihexosyl ceramide synthase knockout cell line and a method for producing the same. More specifically, the present invention relates to a method for the treatment and prophylaxis of a primary immunodeficiency disease, comprising the steps of administering to a mammal an alpha 1, 3 galactosyltransferase (alpha1 (GT- ^{MCP / -MCP} ) somatic cells transformed with knock-out and knocked-in of CD46 (MCP: membrane cofactor protein), 3-galactosyltransferase (IGb3s) knockout cell line and a method for producing the same.

According to the Korean Network for Organ Sharing, the number of transplant recipients waiting for solid organ transplants such as kidney and liver is slightly different depending on the organ, but about 10% is donated. According to the International Diabetes Federation, the number of diabetes patients by country in 2013 was reported to be 3.32 million (20th) in Korea.

It is reported that the number of potential transplant recipients is increasing due to the diabetic patients who are increasing rapidly as well as the complications. Recently, development of biotechnology, development of stem cell field, and 3D printing technology have made practical use of bio-organs much higher than in the past.

In line with these trends, research to produce heterologous transgenic pigs has been actively pursued in advanced countries such as the United States, Japan, and Europe, and the production of transgenic cloned pigs and the production of pig organs by immune- Transplantation studies are underway.

However, one of the first issues to be addressed in order to realize heterologous transplantation is the development of immune rejection control technology that minimizes the heterologous immune rejection reaction occurring when transplanting a heterologous organ, Production of transgenic cloned pigs.

The immune rejection stage is divided into hyperemia, acute, cellular, and chronic, and pigs in which the GT, which is a triggering immune rejection gene, are transformed with an additional immune response gene have been developed. .

However, when xenotransplantation of biotransplant produced from GT ^{- / -} transgenic pigs is carried out globally, the expression of GT gene was partially confirmed in transplanted heterologous organs, which is still a problem. It is also reported that the GT gene is not controlled completely by some GT genes even in the case of homozygous (GT ^{- / -} ) pigs controlled on both chromosomes.

KR 10-2006-7021553

Therefore, it is necessary to develop a pig that can control the immune rejection, iGb3s, and pigs that can reduce the immune rejection significantly by mating with the newly developed pigs.

Under these technical backgrounds, the present inventors have found that a transformed cell line (iGb3s ^{- / -} ) transformed into a GT- ^{MCP / -MCP} porcine cell with a porcine iGb3s gene or a fragment thereof and a vector containing Cas9- To complete the present invention.

One object of the present invention is to provide a method for the production of pigs which can control the expression of a gal epitope and thereby produce pigs with significantly reduced immunosuppressive response, To provide the hexosyl ceramide synthase (iGb3s) gene.

Another object of the present invention is to provide an iGb3s gene or an iGb3s gene fragment comprising the nucleotide sequence of SEQ ID NO: 1 capable of producing a porcine in which a super immune rejection reaction is significantly reduced by controlling the expression of a gal epitope; And a vector comprising the gene for the iGb3s gene deletion.

It is still another object of the present invention to provide an iGb3s gene knockout cell line transformed with the above vector into cells.

It is still another object of the present invention to provide a method for producing an iGb3s gene knockout cell line, which comprises transforming a cell with the vector.

Of the present invention further object is iGb3s gene or fragment thereof, and Cas9-GFP gene into a vector containing the GT ^{-MCP / -MCP} porcine cell transformation in transformed cell lines ^{(GT -MCP / -MCP + iGb3s -} / - ).

It is another object of the present invention to provide a method for producing the transformed cell line (GT- ^{MCP /} -MCP + iGb3s ^{- / -} ).

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 an aspect of the present invention, there is provided a separated isoglobotrihexylsaccharide ceramide synthase (iGb3s) gene comprising the nucleotide sequence shown in SEQ ID NO: 1.

According to another aspect of the present invention, there is provided a vector comprising the gene.

According to still another aspect of the present invention, there is provided an iGb3s gene fragment or an iGb3s gene fragment comprising the nucleotide sequence of SEQ ID NO: 1; And a vector comprising the gene for the iGb3s gene deletion.

According to another aspect of the present invention, there is provided a cell line transformed with said vector.

According to another aspect of the present invention, there is provided a fertilized oocyte harvested by nuclear transfer using the cell line.

According to another aspect of the present invention, there is provided a method for producing an iGb3s gene knockout cell line, which comprises transforming a cell with the vector.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: transforming a cell with a vector comprising the vector described above and a selection marker; And a step of culturing the transformed cells and then screening for cells that have undergone homologous recombination.

According to another aspect of the present invention, there is provided a method for producing a vector comprising the steps of: introducing the vector described above into a porcine cell; Selecting a cell into which the vector has been introduced and performing somatic cell nuclear replacement; And developing the nuclear-substituted cell. The present invention also provides a method for producing an iGb3s gene knockout pig.

According to one embodiment of the present invention, it is possible to provide a separate isoglucoborotrihexosyl ceramide synthase (iGb3s) gene consisting of the nucleotide sequence shown in SEQ ID NO: 1, so that the primitive immune rejection reaction is remarkably reduced The expression of the gal epitope can be efficiently controlled so that the pig can be produced.

According to an embodiment of the present invention, there is provided a transformed cell line transformed into a pig cell with a vector containing a porcine iGb3s gene or a fragment thereof and a Cas9-GFP gene, and a gal epitope It is possible to develop pigs with significantly reduced expression and to use them for heterologous transplantation studies.

FIGS. 1A and 1B are diagrams showing a guide RNA (gRNA) position candidate group and a selection process thereof for eliminating the primitive immune rejection inducing gene iGb3s using the Cas9 system according to an embodiment of the present invention.
2 is a schematic diagram showing a Cas9-GFP-iGb3s vector construct according to an embodiment of the present invention.
FIGS. 3A and 3B are graphs showing results of selecting Cas9-GFP-iGb3s positive cells. FIG.
FIG. 4 is a diagram showing the results of identifying iGb3s knock-out cell lines by sequencing. FIG.

Certain terms are hereby defined for convenience in order to facilitate a better understanding of the present invention. Unless otherwise defined herein, the scientific and technical terms used herein should have the meaning commonly understood by one of ordinary skill in the art. Also, unless the context clearly indicates otherwise, the singular form of the term also includes plural forms thereof, and plural forms of the term should be understood as including its singular form.

As used herein, the term " knock-out "refers to modifying or eliminating a particular gene in a nucleotide sequence so that it can not be expressed.

As used herein, the term " knock-in "refers to the nucleotide sequence of a foreign species or an exogenous foreign sequence that was not originally present in the organism, to a DNA sequence derived from the genome or the organism of the living organism, It means to insert.

As used herein, " iGb3s " refers to the isoglucoborotrihexosyl ceramide synthase. As used herein, the term " guide RNA " refers to RNA that is specific to target DNA, capable of forming a complex with Cas protein, and bringing Cas protein to target DNA.

As used herein, "GT" refers to alpha 1,3-galactosyltransferase.

"Homology" in the homologous recombination used in the present invention indicates the degree of identity between the first region or the second region and the nucleic acid sequence of the corresponding gene, and is at least 90% identical, preferably at least 95% identical Do.

As used herein, the term " selection marker "is used to select a cell transformed with a gene-targeting knockout vector as a cell. It is used for drug resistance, nutritional requirement, tolerance to cytotoxic agents, May be used, and there are a positive selection marker and a negative selection marker.

The positive selectable marker means a marker that enables positive selection by allowing only the cells expressing the specific marker to survive in the environment in which the selective agent has been treated and the positive selectable marker gene means a gene encoding the positive selectable marker, For example, neomycin phosphotransferase is used to select stable transfected cells in eukaryotic cells, by providing antibiotic resistance that allows eukaryotic cells to survive in the medium to which the antibiotic neomycin is added.

The negative selection marker gene is a marker gene that enables negative selection to selectively remove cells that have undergone random insertion, and selectively kills only cells expressing the gene, thereby preventing transfection by random insertion Role.

"Transformation" as used in the present invention means that DNA is introduced into a host and the DNA becomes replicable as an extrachromosomal factor or by chromosome integration completion. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Transformation of eukaryotes by plasmid or naked DNA is sometimes referred to as "transfection" in order to distinguish it from transformation as a means of tumorigenization of the cells, Is used.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, there is provided a separated isoglucoborhytosylceramide synthase (iGb3s) gene comprising the nucleotide sequence of SEQ ID NO: 1 and a vector comprising the same.

In the present invention, the isoglobotrihexosylceramide synthase (iGb3s) gene is one of the family of glycosyltransferases.

For the purpose of the present invention, the iGb3s gene is an object to be knocked out in cells, and the origin of the iGb3s gene may vary depending on the kind of cells to be knocked out, and is not particularly limited, but mammalian- May be derived from a mini-pig or a pig.

Information on the iGb3s gene can be obtained from known data such as GeneBank of the National Institutes of Health (NCBI Reference Sequence: NM_001009819.2), but is not limited thereto. In one embodiment of the present invention, a base sequence represented by SEQ. ID. NO. 1 capable of enhancing iGb3s gene knockout efficiency using the iGb3s gene information derived from pigs is provided as an iGb3s gene-specific guide RNA. DNA encoding the guide RNA is also included in the scope of the present invention.

The nucleotide sequence shown in SEQ ID NO: 1 includes not only a sequence identical to the sequence of the target gene but also a sequence having homology if the sequence is capable of homologous recombination. Preferably, the sequence of the target gene A sequence showing homology of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more is also included in the scope of the present invention if homologous recombination is possible.

FIG. 1A and FIG. 1B are diagrams showing a guide RNA (gRNA) position candidate group and a selection process thereof for eliminating the primitive immune rejection inducing gene iGb3s using the Cas9 system according to an embodiment of the present invention. As shown in the figure, the swine isobarbital trihexylsaccharide ceramide synthase (iGb3s) gene fragment consisting of the nucleotide sequence shown as # 4 (SEQ ID NO: 1) was found to have the highest indel and was useful for constructing the iGb3s deficient cell line .

Accordingly, the present invention can provide a vector containing the gene fragment capable of producing a pig in which the expression of gal epitope (epitope) is remarkably reduced in the immunological rejection reaction.

In the present invention, the vector means a vector containing a homologous base sequence of a specific gene to be knocked out so that homologous gene recombination occurs at a specific gene position of the genome. Such a vector may be circular or linear.

According to still another aspect of the present invention, there is provided an iGb3s gene fragment or an iGb3s gene fragment comprising the nucleotide sequence of SEQ ID NO: 1; And a vector comprising the gene for the iGb3s gene deletion.

The genes for the iGb3s gene deletion can be various known genes and methods, and there is no particular limitation. For example, ZFN and TALEN techniques can be used. In addition to the results of studies on WNK-depleted cell lines affecting the NaCl co-transporter using this experimental technique, it has been reported that fumarylacetoacetate hydrolase (FAH ), The transgenic animal is made by microinjecting directly into the mouse oocyte by generating a hit vector. This technique has recently been used in a variety of fields such as cell line development for gene editing and animal production.

According to an embodiment of the present invention, the gene for the iGb3s gene deletion may be Cas9, and may be, but is not limited to, hSpCas9.

&Quot; Cas " of the present invention means an essential protein element in the CRISPR / Cas system, and when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) Nuclease, or niacase. In the present invention, the Cas protein may be any Cas protein if it has endonuclease or nicase activity when it forms a complex with the guide RNA. Cas9 protein or variant thereof may be suitable, and hSpCas9 (humanized S. pyogenes Cas9) may be more suitable. In addition, the method of developing a hit vector and a cell line using the CRISPR / Cas9 system has an advantage in that the time and cost for development can be reduced as compared with the previous gene correction method (ZFN or TALEN) And TALEN, respectively, the hit vector operates only when 6 and 15 amino acids are matched, whereas the CRISPR / Cas9 method requires 20 bases to be matched. .

Information on cas genes and proteins can be obtained from, but is not limited to, GenBank from NCBI.

According to one embodiment of the present invention, the vector may comprise various known selection markers. The vector may have the structure of Cas9-GFP-iGb3s vector, though it is not limited thereto.

According to an embodiment of the invention, the vector may be the vector shown in FIG. The vector contains the iGb3s gRNA after the U6 promoter and the hSpCas9 gene and the selectable marker GFP gene for gene deletion. These two genes are transcribed by the efficient CBh promoter in the Cas9 system and NLS (Nuclear localization sequence) is inserted on both sides of the hSpCas9 gene for effective nuclear internalization.

According to another aspect of the present invention, there is provided a cell line transformed with said vector.

The vector production can be carried out using genetic recombination techniques well known in the art. For site-specific DNA cleavage and linkage, enzymes generally known in the art can be used. The transformation also includes any method of introducing the nucleic acid molecule into an organism, cell, tissue or organ, and may be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, A lithium acetate-DMSO method, and the like, but the present invention is not limited thereto.

The cell to be used as the cell line is not limited and may be an animal cell or an animal cell-derived cell, preferably a mammal, more preferably a porcine or a miniature pig-derived somatic cell, Or miniature swine liver, kidney, spleen, bone, bone marrow, thigh, heart, muscle, lung, brain, testes, ovary, islet, intestine, marrow, ear, skin, gallbladder, prostate, bladder, embryo ), An immune system and a hematopoietic cell, and most preferably a fibroblast derived from a pig or a miniature pig, or a cell of an ear tissue, but is not particularly limited thereto.

According to one embodiment of the present invention, the cell line is knocked out by alpha1,3-galactosyltransferase (GT) and CD46 (MCP: membrane cofactor protein) knock-in transformed pig (GT- ^{MCP / -MCP} ) somatic cells.

The α1,3 GT- ^{MCP /} -MCP pig was knocked out by alpha1,3-galactosyltransferase (GT) as a primitive immune rejection gene and human CD46 (MCP : membrane cofactor protein) is a knock-in transgenic pig used as an endogenous organ donor.

In the present invention, the α1,3-galactosyl transferase (α1,3-GT) binds UDP-galactose with a sugar, sphingosine, ceramide, diacylglycerol, hydroxylysine (1, 3) Gal, which does not exist in humans but exists only in pigs. It is believed that this enzyme is involved in the synthesis of Galα It is known. For the purpose of the present invention, the above-mentioned? 1,3-GT is used as an object for removing the activity from the above-described transplantation organs in order to suppress the generation of an immunological rejection reaction by the Galα (1,3) Gal , And is not particularly limited thereto. In general, the alpha 1, 3-galactosyltransferase gene of pigs is composed of 8 introns and 9 exons, and the enzyme activity of alpha 1,3-galactosyltransferase at the protein site corresponding to exon 9 (Yifan Dai, Nature, 20: 251-255, 2002), it is possible to inhibit the function of the alpha 1,3-galactosyltransferase gene more effectively when knocked vectors are prepared to inhibit the function of exon 9 There are advantages to be able to.

In the present invention, the CD46 (MCP: membrane cofactor protein) may be a human CD46 (membrane cofactor protein). CD38 (MCP), a membrane co-regulatory protein, catalyzes the degradation of C3b and C4b during the process of developing into a protein complex (C9) that binds (C1) with an antigen-antibody and destroys the cell membrane. CD55 (DAF) And C5, and inhibits complement activation. CD46 and CD55 inhibit cell death by complement, as well as inhibit the production of opsonin, a substance that stimulates the production of C3a and C5a and the phagocytosis of white blood cells on the cell surface. It has been reported that despite the removal of the alpha1,3-galactosyltransferase gene, a serious rejection may occur during organ transplantation through complement activation induced by other pathways (Tanemura, M. et al. Biochem. Biophys. Res. Commun., 235: 359-364, 1997; Komoda, H. et al., Xenotransplantation, 11: 237-246, 2004). Thus, CD46 is more effective in suppressing cell death due to complement activation by the non-common pathway as well as the general pathway, and thus can more effectively inhibit the rejection reaction in organ transplantation. For the somatic cells of the pig (GT- ^{MCP /} -MCP), the method described in KR 10-1236724DP can be used, and all of these patents are incorporated herein by reference.

According to another aspect of the present invention, there is provided a method for producing an iGb3s gene knockout cell line, which comprises transforming a cell with the vector.

Such transformation includes any method of introducing the nucleic acid molecule into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, A lithium acetate-DMSO method, and the like, but the present invention is not limited thereto.

The cell to be used as the cell line is not limited and may be an animal cell or an animal cell-derived cell, preferably a mammal, more preferably a porcine or a miniature pig-derived somatic cell, Or miniature swine liver, kidney, spleen, bone, marrow, thymus, heart, muscle, lung, brain, testes, ovary, islet, intestine, marrow, ear, skin, gallbladder, prostate, bladder, embryo ), An immune system and a hematopoietic cell, and most preferably a fibroblast derived from a pig or a miniature pig, or a cell of an ear tissue, but is not particularly limited thereto.

According to another aspect of the present invention, there is provided a method for producing a recombinant vector comprising the steps of: transforming a cell with a vector comprising the vector described above and a selection marker; And a step of culturing the transformed cells and then screening for cells that have undergone homologous recombination.

According to one embodiment of the present invention, the porcine cells are knocked out with alpha1,3-galactosyltransferase (GT), and CD46 (MCP: membrane cofactor protein) may be somatic cells of knock-in transformed pig (GT- ^{MCP / -MCP} ).

Such transformation includes any method of introducing the nucleic acid molecule into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art. Such methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, A lithium acetate-DMSO method, and the like, but the present invention is not limited thereto.

The cell to be used as the cell line is not limited and may be an animal cell or an animal cell-derived cell, preferably a mammal, more preferably a porcine or a miniature pig-derived somatic cell, Or miniature swine liver, kidney, spleen, bone, marrow, thymus, heart, muscle, lung, brain, testes, ovary, islet, intestine, marrow, ear, skin, gallbladder, prostate, bladder, embryo ), An immune system and a hematopoietic cell, and most preferably a fibroblast derived from a pig or a miniature pig, or a cell of an ear tissue, but is not particularly limited thereto.

The vector can inactivate or remove the? 1,3-GT gene by homologous recombination. In order to improve the efficiency of homologous recombination, the vector having homology to the polynucleotide sequence of the? 1,3- Nucleotide sequences.

At this time, somatic cells of the pig may be suitable to use cells obtained from the ear of a 10-day-old pig, and culturing of the cells may be carried out by culturing the ear tissue in a culture medium containing glucose and 15% FBS, non- essential amino acid, sodium pyruvate and beta-mercaptoethanol For example, subculture in DMEM medium, but the medium conditions, culture conditions and the like are not particularly limited.

In addition, the method for screening the transformed somatic cells may be carried out using an apparatus or a treatment agent capable of recognizing cells expressing the positive selection marker contained in the vector. For example, a cell into which the vector has been normally introduced using GFP fluorescence can be selected only by cells expressing GFP fluorescence using a cell sorter. In addition, when neomycin is used as a positive selection marker, the transformed somatic cells can be sorted and selected by using a selection medium containing G418 as a treatment agent. At this time, the treatment concentration of G419 contained in the selection medium is 200 ~ 400 / / ml, and the incubation period may be, but not limited to, 11 to 12 days.

According to another aspect of the present invention, there is provided a fertilized oocyte harvested by nuclear transfer using the cell line.

In the present invention, the nuclear transfer means that the nucleus of a cell is transplanted into an oocyte which has already been removed from the nucleus, and an individual born by implanting such a nuclear transfer embryo can transfer the genetic material of the nuclear donor cell to the nucleus- Genetically, it is a completely identical replicating entity. The nuclear transfer embryo according to the present invention is characterized in that GT and iGb3s are simultaneously knocked out.

According to another aspect of the present invention, there is provided a method for producing a vector comprising the steps of: introducing the vector described above into a porcine cell; Selecting a cell into which the vector has been introduced and performing somatic cell nuclear replacement; And developing the nuclear-substituted cell. The present invention also provides a method for producing an iGb3s gene knockout pig.

The pig may be an NIH-miniature replica pig, although not limited thereto. According to an embodiment of the present invention, a transformed NIH miniature pig in which the vector is targeted to the alpha 1,3-galactosyltransferase gene can be provided. The nucleus substitution method used in the production of the cloned animal can be carried out using a method well known in the art, but more preferably, US6,781,030B, US6,603,059B, US6,235,969B, US7,355,094B, US7 , 071,372B, KR862298B, KR500412B, KR807644B, JP4153878B, US6,700,037B, US7,291,764B, US6,258,998B, US6,548,741B, WO03 / 089632A, US7,371,922B, It is more preferable to use the methods described in KR500412B, KR807644, JP4153878B, US6,700,037B, US7,291,764B, US6,258,998B, US6,548,741B, WO03 / 089632A or US7,371,922B. All of these patents are incorporated herein by reference.

The step of performing the nucleus substitution is a step of introducing the nuclei of the selected cells containing the vector into the nucleus-removed oocytes. The nucleus-free oocyte can be prepared from oocytes using physical methods, chemical methods, centrifugation using cytochalasin B, and the like, and a physical method using a glass micropipette is used Can be suitable.

The method of introducing the iGb3s gene-targeted somatic cells into the nucleus-removed oocyte may be cell membrane fusion, intracellular microinjection, etc. The cell membrane fusion method is simple and is advantageous for producing large-scale embryos, My microinjection has the advantage of maximizing the contact between the nucleus and the material in the egg. The fusion of the somatic cell with the nucleus-removed oocyte can be recombined by changing the viscosity of the cell membrane through electrical stimulation, and an electric fusion machine capable of freely adjusting micro current and voltage can be used.

Nuclear transplanted embryos are preferably implanted into surrogate cells after implantation, and are preferably implanted with surrogate mothers. It is desirable to lower the activity of cell signaling substances such as MPF and MAP kinase, which are cell cycle arresting factors. For this purpose, In order to increase the ion, the cell membrane permeability is modified, the calcium influx is rapidly increased from the extracellular medium, or chemical substances such as ionomycin and 6-DMAP are used to directly inhibit the activity of the cell cycle arresting factor Method or the like can be used.

The pigs produced by this method can be used to produce human-tailored organs more effectively by inhibiting the immune response of the transplanted organ.

Hereinafter, the present invention will be described in more detail by way of examples. These examples are provided only for illustrating the present invention in more detail, and the present invention is not limited to the examples.

Example

Selection of Cas9-GFP-iGb3s gRNA site (site)

A guide RNA (guide RNA, gRNA) position candidate group was selected in order to delete the primitive immunity-inducing gene iGb3s using the Cas9 system (FIG. 1A). A total of four candidates were selected, and 20 bases were selected before the PAM (Protospacer Adjacent Motif) sequence of NGG, respectively. T7E1 assay (T7E1 assay) was performed to confirm the efficiency of gRNA. As a result, the highest indel was found in vector No. 4 (FIG. 1b), and this vector was used to construct an iGb3s-deficient cell line.

Construction of the Cas9-GFP-iGb3s vector construct

The basic vector used to delete the iGb3s gene was purchased from Addgene and used [pSpCas9 (BB) -2A-GFP (PX458)], and the structure of the Cas9-GFP-iGb3s vector is as follows. The iGb3s gRNA was inserted after the U6 promoter and the hSpCas9 gene and the selectable marker GFP gene for gene deletion were inserted. These two genes were transcribed by the efficient CBh promoter in the Cas9 system and NLS (Nuclear localization sequence) was inserted on both sides of the hSpCas9 gene for efficient nuclear internalization. In addition, bGH-pA was introduced after GFP to induce an efficient transcription process. In order to more easily select gRNA-inserted vectors, the ampicillin resistance gene was inserted to facilitate vector selection and construction.

Mini-pig somatic cell culture

As a host cell for introducing the prepared vector, somatic cells derived from the ear of α1,3 GT- ^{MCP /} -MCP pig were used. For the culturing of somatic cells, 1 × 10 ⁶ cells were cultured in a 10 cm culture dish, The components of the culture medium used are as follows. DMEM was used as a basic medium for cell culture by adding 20% defined Fetal Bovine Serum, 1% 100X Non-essential amino acid, 1% 100X Sodium pyruvate and 0.1% 1000X b-mercaptoethanol.

Transformation of Cas9-GFP-iGb3s vector

The prepared Cas9-GFP-iGb3s vector was introduced into somatic cells derived from the cultured α1,3 GT- ^{MCP /} -MCP pig ear using Lipofectamine, and transformants were induced. When the cells showed about 80 ~ 90% cell number in a 10 cm culture dish, the transformation was induced. 12 μg of Cas9-GFP-iGb3s vector, 12 μg of Plus regent and 750 μl of Opti-MEM were mixed and reacted at room temperature for 5 minutes. Lipofectamine 2000 and 750 μl of Opti-MEM were mixed and reacted at room temperature for 5 minutes. After incubation at room temperature, the cells were incubated at room temperature for 20 min. The cells were mixed in a 10-cm culture dish containing 10 ml of Opti-MEM and gently mixed. After incubation in a 37 ° C / 5% CO ₂ incubator for 4 hours, the cells were replaced with a somatic cell culture medium and cultured.

Selection of Cas9-GFP-iGb3s positive cells

Cells were transfected with Cas9-GFP-iGb3s vector using lipofectamine, and then cells were observed with a fluorescence microscope (FIG. 3A). As a result, GFP fluorescence could be confirmed in the cells, which means that the vector was normally introduced into the cells. When these cells were compared with a control (α1,3 GT- ^{MCP /} -MCP porcine somatic cell) in which a Cas9-GFP-iGb3s vector was not introduced using a BD FACSAria cell sorter, cells expressing GFP fluorescence GT- ^{MCP /} -MCP + iGb3s ^{- / -} ) (Fig. 3B).

Identification of iGb3s knock-out cell lines by sequencing

The selected cells were cultured and proliferated by FACS sorting, and the gene sequences were analyzed. As a result, # 46 was identified as wild type and 3 base deletion, # 47 as wild type and 1 base insertion, # 42 as 3 base deletion and 10 base deletion. This means that the three cell lines are cell lines lacking iGb3s, which is a priming immunodeficiency triggering gene due to frame shift (Fig. 4).

To confirm the α-Gal epitope expression characteristics, wild-type and transformed cell lines were measured by FACS Aria using BS-I-B4 lectin antibody. α1,3GT- ^{MCP /} -MCP cells (<0.1%) were significantly down-regulated for wild-type fibroblasts (99.4%) (p <0.05). To analyze the function of the inert iGb3s α1,3GT ^{gene, α1,3GT -MCP / -MCP, GT -MCP} / -MCP + iGb3s - / +, and ^{GT -MCP / -MCP + iGb3s - /} - of the α-Gal Epitope expression was measured. The range was gradually lowered to 95.8%, 94.2% and 73.6%, respectively. This ^{α1,3GT-iGb3s} combined and ^{- / -} indicates that inhibit the α-Gal epitope expression in pig cells.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> cell line for porcine isoglobotrihexosylceramide synthase          knock-out and generation method for the same <130> NPF29322 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> guide RNA <400> 1 gccatgcccc gcctgctgct 20

Claims (13)

delete delete An iGb3s gene fragment consisting of the nucleotide sequence shown in SEQ ID NO: 1; And a caspase 9 gene for the iGb3s gene deletion knock-out of alpha1,3-galactosyltransferase (GT) and CD46 (MCP: membrane cofactor protein) A cell line prepared by transforming somatic cells of this knock-in transformed pig (GT- ^{MCP / -MCP} ).
delete 4. The cell line according to claim 3, wherein the vector is a vector comprising a U6 promoter, a gene fragment of SEQ ID NO: 1, a CBh promoter, NLS, hSpCas9, NLS, GFP and bGHpA.
delete delete 5. Nuclear transplanted embryo using the cell line according to claim 3 or 5.
An iGb3s gene fragment consisting of the nucleotide sequence shown in SEQ ID NO: 1; And a caspase 9 gene for the iGb3s gene deletion knock-out of alpha1,3-galactosyltransferase (GT) and CD46 (MCP: membrane cofactor protein) And transforming the somatic cells of the knock-in transformed pig (GT- ^{MCP /} -MCP) into an iGb3s gene knockout cell line.
An iGb3s gene fragment consisting of the nucleotide sequence shown in SEQ ID NO: 1; Cas9 gene for the iGb3s gene deletion; And alpha-1,3-galactosyltransferase (GT) are knocked out and a CD46 (MCP: membrane cofactor protein) is inserted into a vector containing a selection marker, transforming somatic cells of a knock-in transformed pig (GT- ^{MCP / -MCP} ); And
Culturing the transformed cells, and selecting the transformed cells as homologous recombination cells.
delete An iGb3s gene fragment consisting of the nucleotide sequence shown in SEQ ID NO: 1; Cas9 gene for the iGb3s gene deletion; And a selection marker were knocked out by alpha1,3-galactosyltransferase (GT) and inserted into CD46 (MCP: membrane cofactor protein) introducing into a somatic cell of a knock-in transformed pig (GT- ^{MCP / -MCP} );
Selecting a cell into which the vector has been introduced and performing somatic cell nuclear replacement; And
And developing said nucleated cells. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
delete

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