KR100963896B1 - A knock-out vector constructed by using GFP reporter knock-in vector and methods for preparing thereof and knocking out gene in animal cell - Google Patents

Abstract

The present invention relates to a knockout vector prepared using a green fluorescent protein reporter knock-in vector, a method for preparing the same, and a method for knocking out genes in animal cells using the same, more specifically, as a reporter of a knockout vector. Using a green fluorescent protein, the start codon of the gene to be knocked out is replaced with an Open Reading Frame (ORF) sequence containing the start codon of the green fluorescent protein gene, and fused with the promoter sequence of the knockout gene to knock out the vector. To prepare, and to introduce the knockout vector to the animal cells to knock out the function of a specific gene.

According to the present invention, in a knockout vector using a green fluorescent protein as a reporter, ATG, which is a start codon of a knockout gene, is replaced with an ORF including an initiation codon of a green fluorescent protein, and the ORF is a promoter sequence of a knockout gene. By linking to the reporter, the reporter can be expressed more stably and effectively than the existing IRES system.

Green fluorescent protein (GFP) reporter knock-in vector, knock-out vector, knockout gene

Description

Knockout vector constructed by using green fluorescent protein reporter knock-in vector, a method for preparing the same, and a method for knocking out genes in animal cells using the same preparing definitely and knocking out gene in animal cell}

The present invention relates to a knockout vector prepared using a green fluorescent protein reporter knock-in vector, a method for preparing the same, and a method for knocking out genes in animal cells using the same, more specifically, as a reporter of a knockout vector. Using a green fluorescent protein, the start codon of the gene to be knocked out is replaced with an Open Reading Frame (ORF) sequence containing the start codon of the green fluorescent protein gene, and fused with the promoter sequence of the knockout gene to knock out the vector. To prepare, and to introduce the knockout vector to the animal cells to knock out the function of a specific gene.

The most direct and efficient way to study the function of a gene is to examine the phenotype through a knock-out transformant. In order to make an animal free of expression of a specific gene, the strategy is usually designed using a knock-out vector containing a positive selection marker and a negative selection marker. Positive selection markers are located between two gene groups to be used for homologous recombination and negative selection markers are to the left or right of the two gene groups. This allows embryonic stem cells that have undergone homologous recombination to survive by positive selection markers, and randomly inserted vectors that do not rely on homologous recombination to prevent them from living by negative selection markers. It is designed to get cells.

However, there is a problem that it takes time, human resources, and very expensive to produce a gene knockout transgenic animal using such stem cells. It has been around 30 years since the technology was developed, but it has not been greatly improved in terms of its utility. Therefore, it is not widely used in the world. One of the most important problems is that a large number of gene knockout transgenic animals are not even reported because they do not show a clear phenotype. Although no statistical data have been reported, it is estimated that 7-8 out of 10 are not even reported to academia because the phenotype cannot be found clearly.

Meanwhile, genes whose phenotypes are analyzed in transformed organisms are referred to as "reporter genes", which are used for gene deletion of regulatory regions, that is, gene knockout analysis. Prior to performing the deletion assay, a method of analyzing the effect of the deletion on the expression of the cloned gene must be determined, and the effect can only be observed by cloning the gene into the species of origin of the gene. If a plant gene is cloned into bacteria, it is not suitable for the regulation of light in plants. Recently, cloning vectors that can be applied to genes of all individuals have been developed to facilitate gene cloning into the original host. However, in most cases, the host has the same copy as the cloned gene, so the reporter gene is used to distinguish the change in expression of the cloned gene from the normal expression of the copy of the gene in the host. will be. It is a test gene located earlier in the gene to be studied, preferably replacing the original cloned gene. The expression pattern of the reporter gene that has been cloned into the host is controlled by the same regulation as the original gene, so that it is almost similar to that of the original gene.

The first condition for selecting a reporter gene should be a phenotype that the reporter gene does not express in the host, and should be easy to assay when cloned into the host and capable of quantitative analysis of the phenotype. Various reporter genes satisfying these conditions are used for gene regulation studies, among which LacZ (β-Galactosidase) and fluorescence proteins are widely used. In particular, the fluorescent protein is not expressed in the host cell as a reporter, and it is one of the most widely used reporters because it is easy to assay by anyone.

In order to stably express the reporter, a system for expressing a reporter gene using IRES (Internal Ribosome Entry Segment) has been widely used, but a reporter gene is generated from ATG, the start codon of the gene to be knocked out. There is no system to implement. IRES refers to the nucleotide sequence recognized by the ribosome complex as the beginning of the translation process during translation of the transcript, and is mainly used by cloning with a reporter between exons of the knockout gene. Therefore, there are many disadvantages in that reporter expression is affected by the kind of IRES used and the gene knocked out.

Therefore, the present invention has been made to improve the disadvantages of the prior art, it is possible to stably and efficiently express reporter genes without IRES when knocking out a specific gene in the animal using the green fluorescent protein as a reporter. An object of the present invention is to provide a green fluorescent protein reporter knock-in vector and a knock-out vector prepared using the same.

Another object of the present invention to provide a method for knocking out genes in animal cells using the knockout vector.

In order to achieve the above object, the present invention provides a Green Fluorescent protein (GFP) reporter knock-in vector named GFPKI-PS having the nucleotide sequence shown in SEQ ID NO: 1. .

In addition, the present invention is a gene containing a green fluorescent protein reporter gene characterized in that by replacing the start codon ATG of the gene to be knocked out with an ORF (Open Reading Frame) sequence containing the start codon of the green fluorescent protein gene Provide a knock-out vector.

In addition, the present invention

i) mutating the green fluorescent protein (GFP) gene sequence comprising ATG, the start codon, to prepare the ORF sequence of the GFP gene set forth in SEQ ID NO: 2;

ii) preparing a polynucleotide consisting of a promoter base of a gene to be knocked out and a base linked to it, a deleted base at the 5 'end of GFP deleted in preparation of the GFP ORF sequence in step i); And

iii) linking the promoter sequence and the polynucleotide prepared in step ii) to the 5 'end of the ORF sequence of GFP prepared in step i);

Provided is a method of preparing a knockout vector comprising a green fluorescent protein reporter gene.

The present invention also provides isolated host cells transformed, transfected or infected with the knockout vector.

In addition, the present invention provides a method for knocking out genes on mammalian genomes except humans by homologous recombination using the knockout vectors.

The present invention also provides a transgenic animal other than humans, in which the function of transforming growth factor β-inducible early gene 1 (TIEG1) obtained by the knockout method of the gene is knocked out.

The present invention uses a green fluorescent protein as a reporter when knocking out a specific gene in the manufacture of a knockout animal, but replaces ATG, the start codon of the knockout gene, with an ORF including the start codon of the green fluorescent protein. By linking the ORF with the promoter sequence of the knockout gene, there is an excellent effect that the reporter is expressed more stably and effectively than the existing IRES system.

In addition, when the green fluorescent protein reporter knock-in vector of the present invention is used for knockout animal development, accurate expression of the knockout gene expression pattern is possible, and thus, very useful information can be provided for the functional study of the gene.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present inventors produced a green fluorescent protein expression vector obtained by adding an Eco RV cleavage site to the green fluorescent protein ORF (Open Reading Frame), and finally, a green fluorescent protein level by using gene-specific primer design and PCR (Polymerase chain reaction). A porter knock-in vector was produced. When the green fluorescent protein reporter knock-in vector is used for the development of a knockout animal, it is possible to accurately investigate the expression pattern of the knockout gene, thereby providing very useful information for the study of gene function.

Accordingly, the present invention provides a green fluorescent protein reporter knock-in vector (hereinafter also referred to as a "GFP reporter knock-in vector"), a GFPKI-PS vector, having the nucleotide sequence set forth in SEQ ID NO: 1.

According to an embodiment of the present invention, a PCR product obtained by performing a mutant strand synthesis reaction using PCR using a phrGFPII-C vector (Stratagene, Cat. Number: 240144) as a template and primer pairs of SEQ ID NOs: 3 and 4 Is transformed into Top10 competent cells to obtain a clone containing the Eco RV cleavage site in the ORF of GFP and named it "GFPKI-RV vector". Next, a GFPKI-RV vector was used as a template, and a PCR product obtained by performing mutant strand synthesis reaction using PCR using primer pairs of SEQ ID NOs: 5 and 6 was transformed into Top 10 competent cells, thereby cutting Sal I cleavage site. Obtain a clone containing and name it "GFPKI-S vector". Finally, the GFPKI-S vector was used as a template, and a PCR product obtained by performing mutant strand synthesis using PCR using primer pairs of SEQ ID NOs: 7 and 8 was transformed into Top10 competent cells to prepare a Pac I cleavage site. The clones were obtained and finally produced a "GFPKI-PS vector", which is a GFP reporter knock-in vector (FIG. 1).

In the present invention, the gene "knock-out" means modifying or removing a specific gene from the base sequence so that it cannot be expressed, and the gene "knock-in" expresses a specific foreign gene. It is meant to be introduced into the genome of the host so that it can be.

In the present invention, "ORF (Open Reading Frame)" refers to a coding sequence (CDS) and refers to a portion that is a template (template) in protein synthesis.

In the present invention, the "reporter gene" is a gene whose phenotype is analyzed in a transformed organism, and is used for gene deletion of a regulatory region, that is, gene knockout analysis. The present invention uses an appropriate GFP gene as a reporter gene as a reporter for confirming the knockout pattern of the gene because it is not originally expressed in a host cell, which is an animal cell, and is easy to assay, followed by ATG, which is an initiation codon of ORF of GFP. It has a nucleotide sequence represented by SEQ ID NO: 2 to which an EcoR V cleavage site was added.

In the present invention, " EcoR V cleavage site" refers to a DNA site that can be cut by Eco RV restriction enzyme, and refers to a GAT / ATC site.

 The GFP reporter knock-in vector of the present invention is designed so that an ORF including an initiation codon of GFP can be easily replaced from ATG, a start-out codon of a knock-out gene. By effectively eliminating the expression of and allowing the expression of the knockout gene to be replaced by the expression of the GFP gene, the GFP gene can be cloned more stably, and the expression of the reporter can be more stably and efficiently performed.

In addition, the present invention comprises a green fluorescent protein reporter gene characterized in that ATG, which is the start codon of the gene to be knocked out, is replaced with an ORF (Open Reading Frame) sequence including the start codon of the green fluorescent protein gene. Provide a knock-out vector.

According to one embodiment of the present invention, the knockout vector is designed such that ATG, the start codon of the gene to be knocked out, is replaced with an ORF including ATG, the start codon of the GFP gene, followed by ATG, the start codon of the GFP. By constructing a GFP reporter knock-in vector added with an EcoR V cleavage site, it is possible to easily replace the ORF of GFP from ATG, the start codon of the gene, for all genes regardless of the nucleotide sequence of the gene to be knocked out. The way is to make it possible.

In addition, the knockout vector of the present invention is designed to be linked to the promoter sequence of the gene to be knocked out by the ORF of GFP, and the promoter is a 5'-primer designed to have restriction enzyme sites at the 5 'end (SEQ ID NO: 12 ) And PCR amplification using a 3'-primer (SEQ ID NO: 13) designed to include the 5 'terminal polynucleotide (SEQ ID NO: 10) of the GFP deleted in preparation of the GFP reporter knock-in vector, as described in SEQ ID NO: Use it. In addition, since the promoter sequence of the gene to be knocked out and the ORF sequence of the green fluorescent protein reporter gene are directly connected instead of the existing IRES, the effect on the expression of the reporter gene according to the type of IRES and the knockout gene is influenced. By removing it, the reporter gene was cloned more stably, and the expression of the reporter gene was made to be stable and efficient.

Gene to be knocked out of the present invention is transforming growth factor β-inducible early gene 1 (TIEG1), but is not limited thereto.

The knockout vector may further connect the nucleotide sequence of the 3 'end of the knockout gene to the 3' end of the ORF of GFP. In other words, the ORF of the GFP refers to the exon region other than the exon (exon 1) of the knockout gene deleted by fusion to the promoter sequence of the knockout gene.

According to one embodiment of the present invention, the 3 'terminal sequence includes a portion of exon 3 and exon 4 of TIEG1.

As used herein, the term "vector" refers to a gene construct capable of expressing a target protein or target RNA in a suitable host cell and comprising essential regulatory elements operably linked to express the gene insert. The term "operably linked" means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the target protein or RNA are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the nucleic acid sequence encoding. Operative linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. In addition, the expression vector includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, includes a replication origin. According to the present invention, the knockout vector may include MCI neo (MCI promoter; neo: poly A-less neomycin resistant gene) and PGK-DTA (PGK: phosphoglycerate kinase promoter; DTA: diphtheria toxin-A chain gene) sequences as selection markers. Include.

According to the object of the present invention, if the host is an animal cell, a suitable signal sequence may include, but is not limited to, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, and the like.

Knockout vectors of the invention can be transformed, transfected or infected into host cells to express reporter genes. According to the present invention, the host cell is an R1 mouse embryonic stem cell, or any cell capable of expressing a reporter gene by introducing a knockout vector is not limited thereto.

In addition, the present invention

i) mutating the green fluorescent protein (GFP) gene sequence comprising ATG, the start codon, to prepare the ORF sequence of the GFP gene set forth in SEQ ID NO: 2;

ii) preparing a polynucleotide consisting of a promoter base of a gene to be knocked out and a base linked to it, a deleted base at the 5 'end of GFP deleted in preparation of the GFP ORF sequence in step i); And

iii) linking the promoter sequence and the polynucleotide prepared in step ii) to the 5 'end of the ORF sequence of GFP prepared in step i);

Provided is a method of preparing a knockout vector comprising a green fluorescent protein reporter gene.

According to one embodiment of the invention, a primer is designed to amplify a portion (promoter sequence, SEQ ID NO: 11) that is about 4 kb upstream 5'-from the start codon ATG of the TIEG1 gene (SEQ ID NOs: 12 and 13), PCR amplification was used to prepare a promoter sequence (SEQ ID NO: 14) of about 3.9 kb and cloned into the 5 'end of the GFP gene included in the GFP knockin vector.

The 5'-primer (SEQ ID NO: 12) for amplifying the promoter sequence includes a restriction enzyme site designed for recombination with a GFP reporter knockin vector, for example, a nucleotide sequence corresponding to Hind III (AAG CTT) but It is not limited to this. In addition, the 3'-primer (SEQ ID NO: 13) is designed to include a polynucleotide deleted at the 5 'end of the GFP, according to the present invention, comprising the base sequence of CTG CTT GCT CAC CAT (SEQ ID NO: 10) It features.

The 5'-arm of the cloned recombinant knock-in vector was cloned into the Osdupde1 vector, the base vector used for knockout, and part of the exon 3 and approximately 3.4 kb of recombinant knock-in vector containing exon 4 of the TIEG1 gene. The '-arm is cloned directly behind the GFP gene of the Osdupde1 vector cloned with the 5'-arm to produce a knockout vector.

The present invention also provides a method for knocking out genes on mammalian genomes except humans by homologous recombination using the knockout vectors.

According to one embodiment of the present invention, the TIEG1 knockout vector of the present invention is introduced into R1 mouse embryonic stem cells to induce homologous recombination. This homologous recombination replaces the green fluorescent protein whole ORF, starting with TITG1 gene start codon ATG, starting with GTG start codon ATG.

It is desirable to confirm the accuracy of the knockout vector by measuring the fluorescence level according to the expression of the green fluorescent protein. If the knockout clone using the embryonic stem cells is expressed in the embryonic stem cells, the green fluorescent protein It is possible to check easily and accurately by the expression of.

The present invention also provides a transgenic animal other than humans, in which the function of transforming growth factor β-inducible early gene 1 (TIEG1) obtained by the knockout method of the gene is knocked out.

Preferably, when constructing a knockout transgenic animal, it is preferable to select a knockout gene and allogeneic animals as knockout target animals. For example, if the TIEG1 gene is a mouse, the mouse is selected as the knockout animal.

The following examples are only for illustrating the present invention, and it is apparent to those skilled in the art that these specific techniques are only preferred embodiments, and thus the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Example 1 Preparation of GFP Reporter Knock-in Vector

The GFPKI-PS vector (GFP reporter knock-in vector) was inserted by inserting the Eco RV, Sal I, Pac I cleavage site into the phrGFPII-C vector through a mutation from the phrGFPII-C vector (Stratagene, Cat. number: 240144). Produced.

A: (200518 Stratagene, Cat Number. ) More specifically, the QuikChange ^® Site-Directed Mutagenesis Kit to phrGFPⅡ-C vectors to addition of Eco RV cleavage site produced with primers (SEQ ID NOS: 3 and 4), and mutations (mutagenesis) It was used and carried out according to the instruction manual (see Table 1). The PCR product was transformed into Top10 competent cells to obtain clones in which the Eco RV cleavage site was successfully obtained, and finally confirmed by sequencing, named "GFPKI-RV vector".

Next, mutations were performed as described above using primers (SEQ ID NOs: 5 and 6) to add Sal I cleavage sites to the GFPKI-RV vector (Table 1). The PCR product was transformed into Top10 competent cells to obtain a clone in which the Sal I cleavage site was successfully formed, and finally confirmed by sequencing, named "GFPKI-S vector".

Finally. In GFPKI-S vector Mutations were performed as above using primers (SEQ ID NOS: 7 and 8) to add Pac I cleavage sites (Table 1). The PCR product was transformed into Top10 competent cells to obtain a clone that successfully produced the Pac I cleavage site, and finally confirmed by sequencing, 4.8kb containing Eco RV, Sal I and Pac I cleavage site. The GFP reporter knock-in vector, GFPKI-PS vector, was completed (FIG. 1). The base sequence of the vector is shown in SEQ ID NO: 1, the ORF of the mutated GFP is shown in SEQ ID NO: 2.

As shown in Figure 1, by preparing a new GFP reporter knock-in vector with the addition of the Eco RV cleavage site to the ORF of GFP, from the start codon ATG to the ORF of the GFP for all knockout genes regardless of the nucleotide sequence of the knockout gene An alternative method is possible. The method is described through the following examples.

Addition of Eco RV cleavage site in ORF of GFP primer SEQ ID NO: 3 (GFPKI-RV-5 '):
5'-GCCGCACCATGGTGAGCAAG gAtATC CTGAAGAAC ACCGG-3 ' SEQ ID NO: 4 (GFPKI-RV-3 '):
5'-CCGGTGTTCTTCAG GATaTc CTTGCTCACCATGGT GCGGC-3 ' Underlined sequences are recognized by Eco RV restriction enzyme, and the sequences are modified by lowercase letters. Mutant Strand Synthesis Reaction for PCR 5 μl 10 × reaction buffer PCR conditions Cycle 1: 95 ° C., 30 seconds
Cycle 17: 95 ° C., 30 seconds / 55 ° C., 1 minute / 68 ° C., 5 minutes
Cycle 1: 4 ° C., 2 minutes
(1 μl Dpn I restriction enzyme is added)
Cycle 1: 37 ° C., 1 hour 1 μl phrGFPII-C vector (50 ng) 1 μl GFPKI-RV-5 'primer (10pmole) 1 μl GFPKI-RV-3 'primer (10pmole) 1 μl dNTP mix 41 μl ddH ₂ O 1 μl PfuTurbo DNA polymerase (2.5 U / μl) Sal I cutting site added primer (SEQ ID NO: 5) GFPKI-SI-5 '): 5'-GCATGCTGGGGC GtCGaC GCGCGTAAATTGTAAGC-3' (SEQ ID NO: 6) GFPKI-SI-3 '): 5'-GCTTACAATTTACGCGC GtCGaC GCCCCAGCATGC-3' Underlined sequences recognized by Sal I restriction enzymes, of which the lowercase sequences are modified Mutant Strand Synthesis Reaction for PCR 5 μl 10 × reaction buffer PCR
Condition Cycle 1: 95 ° C., 30 seconds
Cycle 17: 95 ° C., 30 seconds / 55 ° C., 1 minute / 68 ° C., 5 minutes
Cycle 1: 4 ° C., 2 minutes
(1 μl Dpn I restriction enzyme is added)
Cycle 1: 37 ° C., 1 hour 1 μl GFPKI-RV vector (50 ng) 1 μl GFPKI-SI-5 'primer (10pmole) 1 μl GFPKI-SI-3 'primer (10pmole) 1 μl dNTP mix 41 μl ddH ₂ O 1 μl PfuTurbo DNA polymerase (2.5 U / μl) Pac I cutting site added primer (SEQ ID NO 7) GFPKI-PacI-5 '):
5'-CGCGCGTAAATTGTAAGCG TTAATtaa TTGTTAAAATTCGC-3 ' (SEQ ID NO: 8) GFPKI-PacI-3 '):
5'-GCGAATTTTAACAA ttaATTAA CGCTTACAATTTACGCGCG-3 ' Underlined sequences are recognized by Pac I restriction enzymes, and the sequences are modified by lowercase letters. Mutant Strand Synthesis Reaction for PCR 5 μl 10 × reaction buffer PCR
Condition Cycle 1: 95 ° C., 30 seconds
Cycle 17: 95 ° C., 30 seconds / 55 ° C., 1 minute / 68 ° C., 5 minutes
Cycle 1: 4 ° C., 2 minutes
(1 μl Dpn I restriction enzyme is added)
Cycle 1: 37 ° C., 1 hour 1 μl GFPKI-S vector (50 ng) 1 μl GFPKI-PacI-5 'primer (10pmole) 1 μl GFPKI-PacI-3 'primer (10pmole) 1 μl dNTP mix 41 μl ddH ₂ O 1 μl PfuTurbo DNA polymerase (2.5 U / μl)

Example 2 Fabrication of Knockout Vector Using GFP Reporter Knock-in Vector

The GFP reporter knock-in vector prepared in Example 1 includes the 5 'terminal sequence of the GFP ORF, which was deleted during preparation of the GFP reporter knock-in vector of Example 1 from the promoter sequence of the knockout gene and the ATG of the GFP gene linked thereto. The knockout vector was cloned in association with the gene fragment.

First, primers were designed to be connected to the 5 'end of the GFP gene included in the GFP reporter knock-in vector to convert the start codon of the knockout gene into an ORF including the start codon of GFP (FIG. 2).

The boxed nucleotide sequence (AAG CTT) portion of the 5 'primer is designed to select and use a useful restriction enzyme among the restriction enzymes that can be used for the cloning portion of the GFP reporter knock-in vector. Hind III, Kpn I, Bam HI, Sma I and the like can be used and are not limited to these specific restriction enzymes. Three restriction sequences (CCG) were added at the 5 'end to designate restriction enzymes (SEQ ID NO: 9). Corresponds to the nucleotide sequence on the 5 'side of the 5' fragment of the 3 'knockout gene (promoter sequence).

The yellow nucleotide sequence (CTG CTT GCT CAC CAT, SEQ ID NO: 10) portion of the 3'-primer represents the base sequence of 15bp from the ATG of the 5 'end of the GFP ORF, which was deleted when preparing the GFP reporter knock-in vector. Complement of this made a complete GFP ORF and confirmed its expression as a reporter. The 3 'end of the 15bp sequence is ATG, from which a primer was designed using the 5' direction sequence of ATG, the start codon of the knockout gene.

As a result, the ORF of GFP was substituted from the ATG of the knockout gene and expressed as a reporter regardless of the nucleotide sequence of the knockout gene.

A knockout vector was designed to knock out the transforming growth factor β-inducible early gene 1 (TIEG1) gene of the mouse and to determine its expression pattern by using the GFP reporter knock-in.

TIEG1 start codon from ATG to the 5'-portion of about 4kb upstream of the gene (3963bp fragment, SEQ ID NO: 11) the designed primer of Bam HI restriction enzyme place a primer designed to have 5 ' (SEQ ID NO: 12: TIEG1 / KO / 5'-1: 3'-primer designed to contain 5'-CCCGGATCCTTCTCCCACTATCCTAGAAGTTCC-3 'and the deleted polynucleotide on the 5' end of GFP (SEQ ID NO: 13: TIEG1 / KO / 3'-1: 5'-CTGCTTGCTCACCATGGTTGGCTGCCTGGCCG 0.5 μl of template (10 ng of plasmid DNA), primer pair (10 pmole) using -3 ' A total of 25 μl of a reaction solution was prepared from 1 μl / 1 μl, 10 × buffer 2.5 μl, dNTP (10 mM) 1 μl, Taq Polymerase 0.5 μl (2.5U) and dH ₂ O 18.5 μl, 94 ° C., 2 minutes; 30 cycles of 94 ° C, 60 seconds / 60 ° C, 60 seconds / 72 ° C, 2 minutes; PCR amplification was carried out at 72 ° C. for 10 minutes.

The PCR product (SEQ ID NO: 14) was treated with BamH I restriction enzyme, and the 5 'end of the GFP gene of the GFP reporter knock-in vector prepared in Example 1 was cut with BamHI / EcoR V and cloned.

The 5'-arm of the cloned recombinant vector was cloned between the Hind III and Pac I sites of MCS1 of Osdupde I (donated by Dr. Heiner Westphal of the National Institutes of Health, USA), the base vector used for knockout. In addition, the 3'-arm of the recombinant vector of about 3.4 kb (3425 bp fragment, SEQ ID NO: 15) containing a part of exon 3 and exon 4 of the TIEG1 gene was cut with Kpn I / EcoR I to clone the 5'-arm. It was cloned into the Pml I site of MCS2 of the OsdupdeI vector, which was located behind the GFP gene (FIG. 3).

The TIEG1 knockout vector thus prepared was introduced into R1 mouse embryonic stem cells (donated by Heiner Westphal) to induce homologous recombination, and Southern blots were performed using 5'- and 3'-probes.

As shown in FIG. 4, clone numbers (87, 88, 90) of R1 mouse embryonic stem cells were confirmed to have homologous recombination with knockout vectors, and clone numbers (89, 91) were negative. It was confirmed that 25 clones out of 182 clones were replaced by ORTG of green fluorescent protein by ATG, which is the start codon of TIEG1.

The knocked out genomic DNA vector was transfected into NS20Y cells, which are neuroblastoma cells (donated by Dr. Maral Mouradian of the National Institutes of Health, USA) according to a conventional method to measure fluorescence.

As shown in FIG. 5, when the vector containing the GFP reporter knock-in vector is transfected, fluorescence does not appear at all, whereas when the GFP reporter knock-in vector is transfected, the GFP reporter knock-in vector is transfected by the TIEG1 promoter. It was confirmed that fluorescence appeared.

Example 3 Genotyping of TIEG1 by PCR

PCR was performed to confirm the genotype of TIEG1. Primers (SEQ ID NOs: 16, 17, and 18) were used, and the primers of SEQ ID NOs: 16 and 17 can identify TIEG1 mutant clones using the primers of SEQ ID NOs: 16 and 18.

TIEG1 5'-1 (SEQ ID NO: 16): 5'-TGTACACGCTCCACTGACAG -3 '

TIEG1 3'-1 (SEQ ID NO: 17): 5'-CCTTGTCTCCTGGATACC -3 '

GFPKI-PS 3'-1 (SEQ ID NO: 18): 5'-CGAACAGGATGTTGCCCT TGC-3 '

1.TIEG1 5'-1 and TIEG1 3'-1 (591 bp for wild-type)

Template 0.5 μl (genomic DNA prep), 1 μl / 1 μl primer pair (10 pmole), 2.5 μl 10 × buffer (w / o MgCl ₂ ), 1.5 μl MgCl ₂ (50 mM), 1 μl dNTP (10 mM), Taq A total of 25 µl of the reaction mixture was prepared from 0.5 µl of Polymerase (2.5 U) and 17 µl of dH ₂ O, 94 ° C., 2 minutes; 30 cycles of 94 ° C, 30 seconds / 58 ° C, 30 seconds / 72 ° C, 30 seconds; PCR was performed at 72 ° C. for 3 minutes.

2.TIEG1 5'-1 and GFPKI-PS 3'-1 (424 bp for mutant)

Template 0.5 μl (genomic DNA prep), 1 μl / 1 μl primer pair (10 pmole), 2.5 μl 10 × buffer (w / o MgCl ₂ ), 1 μl MgCl ₂ (50 mM), 1 μl dNTP (10 mM), Taq A total of 25 µl of the reaction mixture was prepared from 0.5 µl of Polymerase ( _2.5 U) and 17.5 µl of dH ₂ O, and 94 ° C. for 2 minutes; 30 cycles of 94 ° C, 30 seconds / 62 ° C, 30 seconds / 72 ° C, 30 seconds; PCR was performed at 72 ° C. for 3 minutes.

As a result of PCR, wild type of TIEG1 was 591bp and mutant was 424bp.

1 shows a cleavage map of a GFP reporter knock-in vector into which a green fluorescent protein (GFP) reporter including an Eco RV cleavage site is inserted.

Figure 2 is a recombinant vector comprising 15 base sequences deleted in the preparation of the GFP start codon and GFP reporter knock-in vector from ATG, the start codon of the knockout gene to prepare a knockout vector using the GFP reporter knock-in vector The primer design structure for making the 5 'terminal gene fragment is shown.

Figure 3 shows a vector production schematic showing the whole process of cloning the knockout vector for knocking out the TIEG1 gene using the GFP reporter knock-in vector.

Figure 4 shows the Southern blot results showing that homologous recombination of the knockout vector of the present invention in embryonic stem cells.

Figure 5 shows the results of measuring the fluorescence by transfecting the knockout vector of the present invention to NS20Y cells.

Figure 6 shows the genotyping (Genotyping) of TIEG1 by PCR.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A knock-out vector constructed by using GFP reporter knock-in          vector and methods for preparing pretty and knocking out gene in          animal <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 4849 <212> DNA <213> Artificial Sequence <220> <223> GFPKI-PS vector <220> <221> variation 222 (790) .. (795) <223> EcoRV site <220> <221> variation (222) (1780) .. (1785) <223> Sal I site <220> <221> variation (1808) .. (1813) <223> PacI site <220> <221> gene (222) (778) .. (1494) <223> Open Reading Frame of Green Fluorescent Protein <400> 1 aggcgcgccg cgatgtacgg gccagattta cgcgttgaca ttgattattg actagttatt 60 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 120 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 180 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 240 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 300 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 360 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 420 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 480 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 540 caaaatgtcg taacaactcc gccccattga agcaaatggg cggtaggcgt gtacggtggg 600 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg 660 aaattaatac gactcactat agggagaccc aagcttctgg aggcccgggc tttcagggta 720 ccgaagaagg atccaaggag gaattctgca gatatccatc acactggcgg ccgcaccatg 780 gtgagcaagg atatcctgaa gaacaccggc ctgcaggaga tcatgagctt caaggtgaac 840 ctggagggcg tggtgaacaa ccacgtgttc accatggagg gctgcggcaa gggcaacatc 900 ctgttcggca accagctggt gcagatccgc gtgaccaagg gcgcccccct gcccttcgcc 960 ttcgacatcc tgagccccgc cttccagtac ggcaaccgca ccttcaccaa gtaccccgag 1020 gacatcagcg acttcttcat ccagagcttc cccgccggct tcgtgtacga gcgcaccctg 1080 cgctacgagg acggcggcct ggtggagatc cgcagcgaca tcaacctgat cgaggggatg 1140 ttcgtgtacc gcgtggagta caagggccgc aacttcccca acgacggccc cgtgatgaag 1200 aagaccatca ccggcctgca gcccagcttc gaggtggtgt acatgaacga cggcgtgctg 1260 gtgggccagg tgatcctggt gtaccgcctg aacagcggca agttctacag ctgccacatg 1320 cgcaccctga tgaagagcaa gggcgtggtg aaggacttcc ccgagtacca cttcatccag 1380 caccgcctgg agaagaccta cgtggaggac ggcggcttcg tagagcagca cgagaccgcc 1440 atcgcccagc tgaccagcct gggcaagccc ctgggcagcc tgcacgagtg ggtgtaagct 1500 cgagcatgca tctagagggc cctattccct ttagtgaggg ttaattgcta gagctcgctg 1560 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 1620 ttccttgacc ctggaaggtg ccactctcac tgtcctttcc taataaaatg aggaaattgc 1680 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 1740 gggggaggat tgggaagaca atagcaggca tgctggggcg tcgacgcgcg taaattgtaa 1800 gcgttaatta attgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc 1860 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga 1920 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag 1980 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt 2040 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta 2100 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag 2160 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg 2220 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa 2280 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 2340 cctgataaat gcttcaataa tattgaaaaa ggaagaatcc tgaggcggaa agaaccagct 2400 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat 2460 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc 2520 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac 2580 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 2640 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 2700 cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc atgattgaac 2760 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact 2820 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc 2880 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caagacgagg 2940 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg 3000 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt 3060 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc 3120 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag 3180 cacgtactcg gatggaagcc ggtcttctcg atcaggatga tctggacgaa gaacatcagg 3240 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc 3300 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt 3360 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg 3420 ctacccgtga tattgctgaa gaacttggcg gcgaatgggc tgaccgcttc ctcgtgcttt 3480 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct 3540 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg 3600 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga 3660 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag 3720 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc 3780 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg 3840 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg 3900 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc 3960 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga 4020 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 4080 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 4140 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 4200 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 4260 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt 4320 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 4380 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 4440 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 4500 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 4560 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 4620 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 4680 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 4740 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 4800 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcc 4849 <210> 2 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> Open Reading Frame of Green Fluorescent Protein <400> 2 atggtgagca aggatatcct gaagaacacc ggcctgcagg agatcatgag cttcaaggtg 60 aacctggagg gcgtggtgaa caaccacgtg ttcaccatgg agggctgcgg caagggcaac 120 atcctgttcg gcaaccagct ggtgcagatc cgcgtgacca agggcgcccc cctgcccttc 180 gccttcgaca tcctgagccc cgccttccag tacggcaacc gcaccttcac caagtacccc 240 gaggacatca gcgacttctt catccagagc ttccccgccg gcttcgtgta cgagcgcacc 300 ctgcgctacg aggacggcgg cctggtggag atccgcagcg acatcaacct gatcgagggg 360 atgttcgtgt accgcgtgga gtacaagggc cgcaacttcc ccaacgacgg ccccgtgatg 420 aagaagacca tcaccggcct gcagcccagc ttcgaggtgg tgtacatgaa cgacggcgtg 480 ctggtgggcc aggtgatcct ggtgtaccgc ctgaacagcg gcaagttcta cagctgccac 540 atgcgcaccc tgatgaagag caagggcgtg gtgaaggact tccccgagta ccacttcatc 600 cagcaccgcc tggagaagac ctacgtggag gacggcggct tcgtagagca gcacgagacc 660 gccatcgccc agctgaccag cctgggcaag cccctgggca gcctgcacga gtgggtg 717 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> GFPKI-RV-5 ' <400> 3 gccgcaccat ggtgagcaag gatatcctga agaacaccgg 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> GFPKI-RV-3 ' <400> 4 ccggtgttct tcaggatatc cttgctcacc atggtgcggc 40 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> GFPKI-SI-5 ' <400> 5 gcatgctggg gcgtcgacgc gcgtaaattg taagc 35 <210> 6 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> GFPKI-SI-3 ' <400> 6 gcttacaatt tacgcgcgtc gacgccccag catgc 35 <210> 7 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> GFPKI-PacI-5 ' <400> 7 cgcgcgtaaa ttgtaagcgt taattaattg ttaaaattcg c 41 <210> 8 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> GFPKI-PacI-3 ' <400> 8 gcgaatttta acaattaatt aacgcttaca atttacgcgc g 41 <210> 9 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> 5'primer: designed by containing restriction site <400> 9 ccgaagctt 9 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> 3'primer: designed by containing polynucleotides deleted from ORF          of GFP <400> 10 ctgcttgctc accat 15 <210> 11 <211> 3963 <212> DNA <213> Mus sp. <220> <221> promoter (222) (1) .. (3963) <223> promoter of TIEG1 (Transforming Growth Factor beta-inducible early          gene 1) <400> 11 cttctcccac tatcctagaa gttccttcaa cacagggaat gtttttatga tggttctcca 60 gtgttcacta atgcctgaca tgtggtacag ccaaaaaggg atgaacaagg atgagacttg 120 gtgactgtac agagcaaaga gaagctcgct ggtaggtaca atgtgttcta agatcagtat 180 ctgaactggg gagtctctat cttcaatatt ctgactgaag ggaaaccaac accattagca 240 ataagggtca gcatctgagg tatcacttcc tacagccaaa ttaagtctcc tctcttattc 300 atgcccaaga tgactacatc tatgaagggc cttgtaacag cacaatctaa gagaagggtt 360 tggaaggact cgtacttcca ctcacatatt ggcccacatt gtctgcaatc ccatggggtc 420 ttcagaaaca atctagtatt ttatttaatt cattgggaaa aatgtatcaa gtacctatta 480 aaagaacaaa ttgttaataa gaaagatact atggaactgg gcacaacagc tcatgtcggg 540 tgatcacagc acttcaggag ctgaggcagg aggcttgctg agagttccag gccagcttga 600 gtgtatatat gaagggacat tatgaaatga ccactgtctt ataggagttt agatctgagt 660 ggggtgtggg caggattcaa tactataaaa ggttttgcaa taacatatat atatatatat 720 atatatatat atatatatat atatatctcc ttacttataa cattgctttg gaggagaggc 780 aggcaaataa ttcagcctgg agctatcaag gaaggcttca ccaacatggc aaggaaacac 840 tgaaaccaga gagaaaaggg gccttcagaa aaaaaaaaaa aagcatctct tccaagagag 900 tctagcctct ctttgcacca agtcttgtgt cattccatag cctgtaaatt cttcagtagt 960 cagtctccac ctgaaactat gtagcccagg ctggtctcat ttatgacaat cttgtcttag 1020 cctcccatgt ggcacaccct tgtgatccca gtagttggca gataaaggca gaagattcaa 1080 gagctcgaga ccagtctact ggccggccta gaatacgcag atgctcaata acattggttg 1140 aatgcatcag tgaacagagg cctgaaggac agtgcttatt gaatgggggt gtggcatgga 1200 gatgtatgca ctgtatgatg gctctgggca agggaagaac ttactaaaag gctgaaaagt 1260 tgaatgagta gctggagacc cctagctcat aaaggggtag ccttttatct cccgataaca 1320 atagacaaag gtaatttatt taagtaggga ttgttgtgat cttaagttta agcatggcat 1380 ctgggctctg gaaaacaagt tgtgatgtgg gtggatgtcc tgggattaca gacagaaata 1440 gcttcgtgca agctagaggt gaaacactga gccaagtgcc tgcctggtgc ccattagccc 1500 agtacttggg aggctgtggc aggagggttg tctaggtagc cagggctaca cagaatttgt 1560 cagccagcca gggctgcata gtgagaccct gttactgaag aacaatgggg ggaggggggt 1620 gaagacaggc agacagagta ccttgtaaaa caaacagtaa gtaggtgttt ctgaggcaaa 1680 gctgtctaga gctggggact aactagatct cacttaagtt tttgagggag ggaggcaact 1740 gtggctagac tggcaatgat caccccttaa gaccacaaag aaagaggagt tttagatttg 1800 gcgatattag gtctgaagtg cctggggaag gctttctctg aatctccagg tcaccactac 1860 ccagtttctc ctaccccttt ctctcctctt tcctgctgtg atcactccaa gtcccaaacg 1920 ctccttacaa ctcagcctgc agagtgggtg tgggggcacc acgccagaca cagggttcag 1980 gcttggtggg tctggctgtg acctctcccg gaggcggctc cctgacagga aggcaggcag 2040 gcaggcctgc cactgggcac acacaagagc ttcctgcgct gcagcaggtg cccaggccag 2100 ctgctccagc ccttcagact cagatcaaca ggatgggcca gcccaagcgg aaggaagcct 2160 gcagggaagg acactcctgc agacagaagc agggcaccgg gtggagagag gcaggaagag 2220 ctgcctgggt tgctgagttg gcgctcctcc agcggattca gagcaggcag tgacaggagg 2280 ccattcagtt atctttcccc cactttccac ccccacccca gggcttctct ggcccacgtt 2340 attatcatta catttgttgc tggctggctt taggtatacc agaaggaacc atcagataga 2400 tgttggtaga gctggaggcc tctaaaacaa attccctcct ttacctagtc tgtggtgact 2460 gagaaagggc acctgcttta gcccttttag cccagcaggc tgaaaaagcc cacaggaggg 2520 cagtgacttt tgtctcatga tctgcctgtc ccagggagaa aataaaaatc tctagtttgc 2580 tttaagcccc gttatcactc cctccggctc caacagctgc cgtgaacccg cacaaacggg 2640 cgagggggcg gggcaatagg gcgccaacga ggagccaaga gctctcagac taggaggcta 2700 cagcctcagc cgggctgctg cacccacaag ctggcttccc taagaagctc agcttgccct 2760 caatccctgg agcggtggag ggggcaggga caggaactcg atgtttgacc gggtcaggaa 2820 tcccacctgc aagcatgctt tcctcacccc atcccctcaa gcggtttctt ggtagggcgt 2880 cggcgaagac tgtggtcccg cgggtggggg aagtggagtc caccttggaa atagctcgaa 2940 cattgtattt taaacttcaa ctacatcaaa ctcaaactcg attctgccac cgagcgtcgg 3000 gtctggggct ccctgccacg ccactagctc tttggtaccg gcttccctta gtccccactc 3060 caaccgccca gaaggacggg agggacgcgc agaggcgggc cctgctgcta gtgcctgtgc 3120 tgcagatggg aggacgagcc agcgcctgcc gccttgggcg tgggcggtga gtgcgagcag 3180 gtgtctgttc cgtgacaggg ctcccgccgc caccatctcg ccctggttct ccccagtcct 3240 ccggcttaga gtgacaaaac ccggcaaatg ctcagaaatg ccgctcagtg gttcatccat 3300 cccttgcttc cacgcaagac ccacgagaac cacgctgcca aagaacgtct ggtggctcgg 3360 agccgcgctc gcagcctccg cctccgcctc ctgctcgggg tcccgcccac tccctggcgg 3420 gcccagacgc tgtgggcgcg aaggggactg ggcggagcgg gcttccctcc ccacccccca 3480 cgtggggccg gctctccgca aagcctagtc gccgccaccg cgcctcccag ctatgccccg 3540 ccgccggcgc cgcccccggg catgggcgga ggactgaagg ctagggttgg gcgaggggcg 3600 tcacggagtc aggtggtccc cggagttccc cggggctgga ccgagggaac acggtgcctc 3660 gggttgtgta cacgctccac tgacagagcc tcttgcagcc gggcagccgc tgatcacgcg 3720 tggctctgcc agctcattgg ctgaggcctc acacaccttt gccgctgatt ggtcgacact 3780 aggccccgcc ctctaccccg cccgcggcgc ggggcaggca gagcggctac ctgcacgggg 3840 aggggggcag acagcgcgga gcgcggtggc gtcgacgtct agtgtctcag tgctcccgtc 3900 tgtggctaac taagcagcca gcagccaggc agctcgcgac ctgcggccag gcagccaacc 3960 atg 3963 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> TIEG1 / EN / 5'-1 <400> 12 cccggatcct tctcccacta tcctagaagt tcc 33 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> TIEG1 / EN / 3'-1 <400> 13 ctgcttgctc accatggttg gctgcctggc cg 32 <210> 14 <211> 3983 <212> DNA <213> Mus sp. <220> <221> promoter (222) (1) .. (3983) <223> promoter region of TIEG1 containing PCR primer sequence <400> 14 cccggatcct tctcccacta tcctagaagt tccttcaaca cagggaatgt ttttatgatg 60 gttctccagt gttcactaat gcctgacatg tggtacagcc aaaaagggat gaacaaggat 120 gagacttggt gactgtacag agcaaagaga agctcgctgg taggtacaat gtgttctaag 180 atcagtatct gaactgggga gtctctatct tcaatattct gactgaaggg aaaccaacac 240 cattagcaat aagggtcagc atctgaggta tcacttccta cagccaaatt aagtctcctc 300 tcttattcat gcccaagatg actacatcta tgaagggcct tgtaacagca caatctaaga 360 gaagggtttg gaaggactcg tacttccact cacatattgg cccacattgt ctgcaatccc 420 atggggtctt cagaaacaat ctagtatttt atttaattca ttgggaaaaa tgtatcaagt 480 acctattaaa agaacaaatt gttaataaga aagatactat ggaactgggc acaacagctc 540 atgtcgggtg atcacagcac ttcaggagct gaggcaggag gcttgctgag agttccaggc 600 cagcttgagt gtatatatga agggacatta tgaaatgacc actgtcttat aggagtttag 660 atctgagtgg ggtgtgggca ggattcaata ctataaaagg ttttgcaata acatatatat 720 atatatatat atatatatat atatatatat atatctcctt acttataaca ttgctttgga 780 ggagaggcag gcaaataatt cagcctggag ctatcaagga aggcttcacc aacatggcaa 840 ggaaacactg aaaccagaga gaaaaggggc cttcagaaaa aaaaaaaaaa gcatctcttc 900 caagagagtc tagcctctct ttgcaccaag tcttgtgtca ttccatagcc tgtaaattct 960 tcagtagtca gtctccacct gaaactatgt agcccaggct ggtctcattt atgacaatct 1020 tgtcttagcc tcccatgtgg cacacccttg tgatcccagt agttggcaga taaaggcaga 1080 agattcaaga gctcgagacc agtctactgg ccggcctaga atacgcagat gctcaataac 1140 attggttgaa tgcatcagtg aacagaggcc tgaaggacag tgcttattga atgggggtgt 1200 ggcatggaga tgtatgcact gtatgatggc tctgggcaag ggaagaactt actaaaaggc 1260 tgaaaagttg aatgagtagc tggagacccc tagctcataa aggggtagcc ttttatctcc 1320 cgataacaat agacaaaggt aatttattta agtagggatt gttgtgatct taagtttaag 1380 catggcatct gggctctgga aaacaagttg tgatgtgggt ggatgtcctg ggattacaga 1440 cagaaatagc ttcgtgcaag ctagaggtga aacactgagc caagtgcctg cctggtgccc 1500 attagcccag tacttgggag gctgtggcag gagggttgtc taggtagcca gggctacaca 1560 gaatttgtca gccagccagg gctgcatagt gagaccctgt tactgaagaa caatgggggg 1620 aggggggtga agacaggcag acagagtacc ttgtaaaaca aacagtaagt aggtgtttct 1680 gaggcaaagc tgtctagagc tggggactaa ctagatctca cttaagtttt tgagggaggg 1740 aggcaactgt ggctagactg gcaatgatca ccccttaaga ccacaaagaa agaggagttt 1800 tagatttggc gatattaggt ctgaagtgcc tggggaaggc tttctctgaa tctccaggtc 1860 accactaccc agtttctcct acccctttct ctcctctttc ctgctgtgat cactccaagt 1920 cccaaacgct ccttacaact cagcctgcag agtgggtgtg ggggcaccac gccagacaca 1980 gggttcaggc ttggtgggtc tggctgtgac ctctcccgga ggcggctccc tgacaggaag 2040 gcaggcaggc aggcctgcca ctgggcacac acaagagctt cctgcgctgc agcaggtgcc 2100 caggccagct gctccagccc ttcagactca gatcaacagg atgggccagc ccaagcggaa 2160 ggaagcctgc agggaaggac actcctgcag acagaagcag ggcaccgggt ggagagaggc 2220 aggaagagct gcctgggttg ctgagttggc gctcctccag cggattcaga gcaggcagtg 2280 acaggaggcc attcagttat ctttccccca ctttccaccc ccaccccagg gcttctctgg 2340 cccacgttat tatcattaca tttgttgctg gctggcttta ggtataccag aaggaaccat 2400 cagatagatg ttggtagagc tggaggcctc taaaacaaat tccctccttt acctagtctg 2460 tggtgactga gaaagggcac ctgctttagc ccttttagcc cagcaggctg aaaaagccca 2520 caggagggca gtgacttttg tctcatgatc tgcctgtccc agggagaaaa taaaaatctc 2580 tagtttgctt taagccccgt tatcactccc tccggctcca acagctgccg tgaacccgca 2640 caaacgggcg agggggcggg gcaatagggc gccaacgagg agccaagagc tctcagacta 2700 ggaggctaca gcctcagccg ggctgctgca cccacaagct ggcttcccta agaagctcag 2760 cttgccctca atccctggag cggtggaggg ggcagggaca ggaactcgat gtttgaccgg 2820 gtcaggaatc ccacctgcaa gcatgctttc ctcaccccat cccctcaagc ggtttcttgg 2880 tagggcgtcg gcgaagactg tggtcccgcg ggtgggggaa gtggagtcca ccttggaaat 2940 agctcgaaca ttgtatttta aacttcaact acatcaaact caaactcgat tctgccaccg 3000 agcgtcgggt ctggggctcc ctgccacgcc actagctctt tggtaccggc ttcccttagt 3060 ccccactcca accgcccaga aggacgggag ggacgcgcag aggcgggccc tgctgctagt 3120 gcctgtgctg cagatgggag gacgagccag cgcctgccgc cttgggcgtg ggcggtgagt 3180 gcgagcaggt gtctgttccg tgacagggct cccgccgcca ccatctcgcc ctggttctcc 3240 ccagtcctcc ggcttagagt gacaaaaccc ggcaaatgct cagaaatgcc gctcagtggt 3300 tcatccatcc cttgcttcca cgcaagaccc acgagaacca cgctgccaaa gaacgtctgg 3360 tggctcggag ccgcgctcgc agcctccgcc tccgcctcct gctcggggtc ccgcccactc 3420 cctggcgggc ccagacgctg tgggcgcgaa ggggactggg cggagcgggc ttccctcccc 3480 accccccacg tggggccggc tctccgcaaa gcctagtcgc cgccaccgcg cctcccagct 3540 atgccccgcc gccggcgccg cccccgggca tgggcggagg actgaaggct agggttgggc 3600 gaggggcgtc acggagtcag gtggtccccg gagttccccg gggctggacc gagggaacac 3660 ggtgcctcgg gttgtgtaca cgctccactg acagagcctc ttgcagccgg gcagccgctg 3720 atcacgcgtg gctctgccag ctcattggct gaggcctcac acacctttgc cgctgattgg 3780 tcgacactag gccccgccct ctaccccgcc cgcggcgcgg ggcaggcaga gcggctacct 3840 gcacggggag gggggcagac agcgcggagc gcggtggcgt cgacgtctag tgtctcagtg 3900 ctcccgtctg tggctaacta agcagccagc agccaggcag ctcgcgacct gcggccaggc 3960 agccaaccat ggtgagcaag cag 3983 <210> 15 <211> 3425 <212> DNA <213> Mus sp. <220> <221> exon (222) (1) .. (3425) <223> Exon 3-4 of TIEG1 <400> 15 ggtaccccag cccgttgtgc agagcccaag gcctccagtg gtgagcccca gtggcaccag 60 actgtctccc attgcccctg ctcctggatt ctctccttca gcagcaaggg tcactcctca 120 gattgactcg tccagagtaa gaagtcacat ctgtagccac ccagggtgtg gcaagactta 180 ctttaaaagt tcccatctga aggcccacgt gaggacacac acaggtactg accgagtggg 240 ctctgtcttt gaagtgggtg tgtatgtgga gttagactgt ggacacaaat gcaagggagg 300 gtgaggtaga tgactcattg tctttaaaat tagtcagttt gtccctaggc ctggtgccac 360 acccttgtaa tcccagcact tgggaattgg aggcagagga agcagcacat ctggtaatat 420 atacatgata tatatgtatt tattacatta tttattcagg cggggtttct ttgtgcagcc 480 ctggctaccc tggaactcag tctgtagacc aggctgatct tgaactcaga tttctttctg 540 cctctatctc cagagtgctg ggattaaagg tgtgaaccat ggcgccaagc tttgataatt 600 tatgaactga ccagctttca attttaaact ctgtgcgtca atgtatagta accatctggc 660 tttctttgct tcaggggaaa aacctttcag ctgcagctgg aaaggctgtg aaaggaggtt 720 tgctcgctcc gatgaactgt ccagacaccg gcggacacac acaggtgaga agaagtttgc 780 ctgtcccatg tgtgaccgtc ggtttatgag gagcgaccat ttaaccaagc atgcccgacg 840 ccacctatca gccaagaagc tgccaaactg gcaaatggaa gttagcaagt taaatgacat 900 tgctctgcct ccgacccctg cttccgcaca gtgacggcca gaagatggag acgcagaata 960 aactttggtc agagtcagga gccagtgatg gtgtcaagtg cttctgcaag gctgtggccc 1020 tccaaaaggg cctaaagtag aagccctggc ctgggggagg ccccgcctgg gtgaaatgac 1080 aagaagtgct tcagccacag gcaggtcaca gaggacaggg ctcagttctt accacagaga 1140 gagaggagaa cccttttatt cctcccttat tttagtctgg aaagtttcgg ctgaggtgag 1200 cgcagcacag gttttgaatc acatacacat tggggacttt gtttttgcca tttatacttg 1260 agaccagctt tgcagtgtga ttctttcaaa ggattggttt caagaatata gaggctggaa 1320 attacggtac agaaatggag ctagaaaatg agtttgtgtt acacagagat gtcatcttct 1380 cctagagtta tcttgtttct tattcctagt ctttccagtc aaatccgtgg atgtagctaa 1440 gtatatctaa aactcatttt tccactattg ttggtatttg aagttgaaca gctgtacatt 1500 gctgtggggg agccaaagga ttggaaccct cattaattta attgcttgga aatgcagcta 1560 aaattcttct ttggcatttt gttttgaaag tttaggcatt ttactctact ttagatttta 1620 gtttgcttgc agttttttgt gtagatttga aaattgtata ccaatgtgtt ttctgtaggc 1680 ttaaaataca ctgcactttg tttagaaaaa aatctggaga tgaaaatatg tattataaag 1740 aagagatgtc aagaatttga gataactcct tgagaaagtt ggctttatgt catcagcaaa 1800 ggacacttaa cgtcaagcat acactgtggt ttttttgttt ttttgttttt ttttttcaaa 1860 ttagaaagtt taatgaccgt tacagatgga cagtgtcttt ttatttatag gagtttttca 1920 ggatgtcaga gtagataggt aggaaaattg ttattagaac attcgcttct accttgaaaa 1980 ggatgttaat gtggtcatgt tcttagcacc acagtgtctg ggcatctggg aaactccgag 2040 acttttttaa agtgtcatga tgtgatcaca cctgcagttt ggggcatcga atccagggcc 2100 ttgcatgtct tctgtaagag ctctcatcgc tgacctgtat cccccgcaag agcaatgact 2160 tttgctaaca gtatttcttt tctgttgtaa agtggacaga tgatacactt ggtcgcaaag 2220 gtaaattatt caaaatccac agtgaaaacc tcaccacact ttcccattta aactatttcc 2280 atatctcaga ggtttctgac atgcaaactt gaacccttga aagaagagtt ttcttaaaaa 2340 ttataaaaaa tcacgagtta caatttgcac aatatttttt gttgaacttt ataccttgtt 2400 tacaataaag acttttcttt ggtatacaca gtgagtctgt tgtatgtgtt aaaacccatt 2460 ctccatctca ccttcatact gacctcattc agatcagaac ctgaggccag attgctttaa 2520 gatgatgtaa ctgctatgac caacaagaga aggctgacct gtcatctgga gggacttgtt 2580 caagttgtac tcactcagac caggaagaga tactctagat ataacagata aactggtcct 2640 taagcagtgt gagaggccag gccacctctg cagagacact tgctagtcac ttaggagcgt 2700 tgtaagtagg gtatgacttt tttcttttaa taaggtcact attttagttg gcccaaaagt 2760 ctcactgctc ttgagaaaat ctgcaaaggc ccctgcttga acacctggct cactttttga 2820 gacaaagtct tattgtagcc caggctagcc tggaactcaa atagcccagg gggtcgcaat 2880 tcttctgtgc ctctggaatg ctaggattca ccacgattcc ttaccccaac ctcttgattt 2940 gttattttta ccatccttgt atgaatcttg tttgaggcaa attattgtgg tattctaatt 3000 cttttgttgt atatttttgg ggattctata ggaaagacta gtttagtctc cttagttatt 3060 cctgtcgata tggatttgtg gagacatata tcattctctt aagttataag ggttttgtta 3120 ctcagctgtc ccagtgtgat ccttggtttc ttgttttgtt ttgttttgtt ttgttggttt 3180 ttcaggacag ggtttctctc ttttttgttt gtttgtttgt ttgtttgtga gacagggttt 3240 ctctgtgtag ccctggctgt cctggaactc actctgtaga ccaggctggc ctcgaactca 3300 gaaatctgct tgcctctgcc tcccaagtgc tgggattaaa ggcgtgcgcc accaccaccc 3360 agctcagtgt ggtccttgtg aactcctgac tggttaacca ttttgattga caaagatggg 3420 aattc 3425 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TIEG1 5'-1 <400> 16 tgtacacgct ccactgacag 20 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TIEG1 3'-1 ( <400> 17 ccttgtctcc tggatacc 18 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GFPKI-PS 3'-1 <400> 18 cgaacaggat gttgcccttg c 21  

Claims (17)

SEQ ID NO: 1 having the base sequence described in SEQ ID NO: 1, wherein the sequence comprises the ORF sequence of the Green Fluorescent Protein (GFP) gene described in SEQ ID NO: 2, the green fluorescent protein reporter knock-in vector ( knock-in vector). The promoter sequence of the gene to be knocked out; And An ORF sequence of a green fluorescent protein gene having the nucleotide sequence set forth in SEQ ID NO: 2, The promoter sequence is linked to the 5 'end of the ORF sequence, and the knockout vector comprising a green fluorescent protein reporter gene, characterized in that by replacing the ORF sequence to replace the start codon of the gene to be knocked out (knock-out vector). The method of claim 2, A knockout vector comprising a green fluorescent protein reporter gene, wherein the ORF sequence of the green fluorescent protein gene is derived from the green fluorescent protein reporter knockin vector of claim 1. delete The method of claim 2, A knockout vector, characterized in that the promoter sequence is set forth in SEQ ID NO: 11. The method of claim 2, A knockout vector, wherein the gene to be knocked out is TIEG1 (Transforming Growth Factor β-inducible early gene 1). i) preparing a GFP reporter knock-in vector having the nucleotide sequence set forth in SEQ ID NO: 1 comprising the ORF sequence of the GFP gene set forth in SEQ ID NO: 2 prepared by mutating a green fluorescent protein (GFP) gene sequence; ii) preparing a polynucleotide consisting of a promoter sequence of a gene to be knocked out and a base linked to the 5 'terminal of the GFP deleted in preparation of the GFP ORF sequence in step i); And iii) included in the GFP reporter knock-in vector prepared in step i) Linking the promoter sequence and the polynucleotide prepared in step ii) to the 5 'end of the ORF sequence of GFP, Method for producing a knockout vector comprising a green fluorescent protein reporter gene. The method of claim 7, wherein A promoter sequence is a method for producing a knockout vector comprising a green fluorescent protein reporter gene, characterized in that the nucleotide sequence of SEQ ID NO: 11. The method of claim 7, wherein A method for producing a knockout vector comprising a green fluorescent protein reporter gene, wherein the polynucleotide sequence is a nucleotide sequence set forth in SEQ ID NO: 10. The method of claim 7, wherein A method for producing a knockout vector comprising a green fluorescent protein reporter gene, wherein the gene to be knocked out is TIEG1 (Transforming Growth Factor β-inducible early gene 1). The method of claim 7, wherein To the 3 'end of the ORF of GFP, further comprising linking the nucleotide sequence of the remaining exon of the 3' end of the knockout gene except for the exon deleted due to the insertion of the ORF of GFP. Method for producing a knockout vector comprising. The method of claim 11, The base sequence of the 3 'terminal of the knockout gene is a method for producing a knockout vector comprising a green fluorescent protein reporter gene, characterized in that described in SEQ ID NO: 15. An isolated host cell transformed, transfected or infected with the knockout vector according to claim 2. The method of claim 13, Host cells that are R1 mouse embryonic stem cells. A method of knocking out genes on a mammalian genome except for humans by homologous recombination using the knockout vector according to claim 2. The method of claim 15, A method for knocking out genes on mammalian genomes other than humans, characterized in that the knockout is determined by measuring the fluorescence level depending on whether the green fluorescent protein is expressed. Transgenic animals other than humans knocked out the function of transforming growth factor β-inducible early gene 1 (TIEG1) that can be obtained according to the method of claim 15.

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