KR101893702B1 - Methods for Analyzing a Genotoxicity Using Non-fluorescent Fluorescence Proteins - Google Patents

Abstract

The present invention provides a method for screening a non-fluorescent fluorescent protein comprising: (a) transforming a fish with a nucleotide sequence encoding a non-fluorescent fluorescent protein having a mutation; (b) treating the transformed fish with the test substance; And (c) measuring fluorescence generated by mutation of the non-fluorescent fluorescent protein into a fluorescent protein by generating a back mutation in the introduced nucleotide sequence in the fish treated with the test substance, And a method for analyzing genotoxicity of a substance. According to the present invention, the MutaFish system, which is a line of fluorescent protein variants of the present invention, preferably a wild-type EGFP mutant transformed with a Brachydanio rerio line, is capable of producing fluorescence (fluorescence) induced by reversion of a fluorescent protein variant It provides an excellent animal system that can produce genetic toxicity of the test substance by producing fry. Thus, the MutaFish system of the present invention can very simply and quickly analyze the genotoxic effects of test substances in eukaryotes, which was a limitation of prior art (e.g., Ames test).

Description

Methods for Analyzing a Genotoxicity Using Non-fluorescent Fluorescence Proteins}

The present invention relates to a method for analyzing genotoxicity using a non-fluorescent fluorescent protein.

The introduction of chemicals into the surrounding environment poses a significant risk to the environment and human health. Accordingly, recent legislation in Europe and other industrial countries requires risk assessment data for the registration of chemicals, pesticides, biocides and pharmaceuticals (Commission of the European Communities, 1967, 1991, 1992, 1993a, b, 1994; CVMP/VICH, 2000; EMEA/CHMP, 2006; VICH, 2004). Among the various genotoxicity assays developed to evaluate the toxicity of chemicals, the Ames assay is the most frequently used (Ames, Durston et al. , 1973; Maron and Ames, 1983). This method is based on detecting the reversal of a point mutation in his gene of a Salmonella typhimurium defective mutant by quantifying growth in histidine-deficient cultures. However, the Ames assay has a major drawback: Salmonella is unable to perform the intracellular mechanisms in vertebrates required to convert procarcinogens into reactive metabolites. Thus, MutaMouse (Gossen, de Leeuw et al. , 1989), BigBlue mouse (Kohler, Provost et al. , 1991), gpt-Δ transgenic mouse system (Nohmi, Katoh et al. , 1996), and rpsL transgenics. Various transgenic animal systems such as zebrafish (Amanuma, Takeda et al. , 2000) have been developed. However, in all the systems described above, all of the transgenic reporter genes had to be evaluated by extracting DNA from animal organs of prokaryotic origin and then introducing them into appropriate test bacteria (E.coli).

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have tried to develop a novel genotoxicity analysis method for test substances. As a result, the present inventors developed a genotoxicity analysis method (eg, MutaFish model) using cells transformed with a fluorescent protein variant, preferably an enhanced green fluorescent protein (EGFP) mutant (EGFP ^{mut) or a zebrafish.} And, by using this, the genotoxicity of the test substance can be visualized by fluorescence of the EGFP revertant mutant, and by confirming that the genotoxicity can be tested more sensitively and easily compared to the previous model, the present invention was completed. Became.

It is an object of the present invention to provide a method for analyzing genotoxicity.

Another object of the present invention is to provide a composition for genotoxicity analysis.

Another object of the present invention is to provide a wild-type EGFP mutant (EGFP ^mut).

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

According to one aspect of the present invention, the present invention provides a method for analyzing genotoxicity of a test substance comprising the following steps: (a) a fish with a nucleotide sequence encoding a non-fluorescent fluorescent protein having mutations. Transforming; (b) treating the transformed fish with a test substance; And (c) measuring fluorescence generated when a back mutation occurs in the introduced nucleotide sequence in the fish treated with the test substance and the non-fluorescent fluorescent protein is mutated into a fluorescent protein.

According to another aspect of the present invention, the present invention includes a fish transformed with a nucleotide sequence encoding a non-fluorescent fluorescent protein having a mutation, and the transformed fish is treated with a test substance to perform a reverse mutation ( It provides a composition for genotoxicity analysis, characterized in that observing the occurrence of back mutation).

According to another aspect of the present invention, the present invention provides a nucleotide sequence of SEQ ID NO: 1 or 2, or a complementary nucleotide sequence thereof, encoding an ^{EGFP mutant (EGFP mut).}

The present inventors have tried to develop a novel genotoxicity analysis method for test substances. As a result, the present inventors developed a genotoxicity analysis method (eg, MutaFish model) using cells transformed with a fluorescent protein variant, preferably an enhanced green fluorescent protein (EGFP) mutant (EGFP ^{mut) or a zebrafish.} By using this, it was confirmed that the genotoxicity of the test substance can be visualized by fluorescence of the EGFP revertant mutant, and the genotoxicity can be tested more sensitively and easily compared to the previous model.

Short-term test methods have been developed to measure the degree to which a chemical substance reacts with DNA or the degree to which DNA is modified, and the positive results obtained using these methods can sufficiently prove the carcinogenicity of the chemical substance. It is not possible, but it does present possibilities and has often been used in the selection of chemicals for biometric quantification in animals. Such short-term testing methods include mutagenicity testing (eg, Ames test), chromosome effect testing (eg, measuring changes in the number or structure of chromosomes), and mammalian cell transformation tests (eg, measuring changes in cells). .

Of these short-term testing methods, the Ames test is the most common method used to assess whether a chemical is a carcinogen or not. According to the Ames test, a measure of the degree to which a chemical causes back mutations or revertants of a specific genetic factor in a strain of Salmonella typhimurium. Chemicals that show positive results in the Ames test show carcinogenicity in animal tests in more than 90% of cases. The Ames test has the following advantages: (a) low cost; (b) ease; And (c) swift. However, the Ames test has the following fatal flaws. Since Salmonella used in the Ames test is prokaryotic, it cannot fully reflect the intracellular mechanisms of eukaryotes (eg, humans). Salmonella, for example, is unable to perform the intracellular mechanisms of vertebrates required to convert procarcinogens into reactive metabolites. Therefore, the present inventors have developed a novel genotoxicity analysis method for a test substance using a mutant non-fluorescent fluorescent protein in order to overcome the above-described disadvantages.

The present invention provides a method for genotoxicity analysis of a test substance using cells transformed with wild-type EGFP mutant (EGFP ^mut ) (eg, Salmonella typhimurium; CH4001 and CH4003 strains) or fish (eg, MutaFish).

According to the present invention, the present invention provides a method for analyzing genotoxicity comprising the following steps:

(a) transforming a cell or fish with a nucleotide sequence encoding a non-fluorescent fluorescent protein with mutation;

(b) treating the transformed cells or fish with a test substance; And

(c) measuring fluorescence generated when a back mutation occurs in the introduced nucleotide sequence in the cells or fish treated with the test substance and the non-fluorescent fluorescent protein is mutated into a fluorescent protein.

As described above, the simplest method of examining whether a test substance functions as a mutagenic factor is a method of performing a reversion experiment using a vegetative mutant (eg, Ames test). The simplest reversion experiment is to cultivate a certain number of mutant bacteria in a medium containing a mutant and count the number of revertants that occur there. If the test substance is a mutagenic factor, the number of revertants is increased compared to the medium in which the test substance has not been treated.

The present inventors have developed a method for more efficiently analyzing the genotoxicity of a test substance using a transformant obtained by transforming a non-fluorescent fluorescent protein into cells or fish, and the method of the present invention comprises the non-fluorescent fluorescent protein. It is possible to analyze genotoxicity more easily and quickly by visualization through fluorescence generated by mutation into this fluorescent protein. Therefore, the method of the present invention is a very excellent method capable of overcoming the limitations of the prior art (Ames test) that cannot be applied to eukaryotes due to the use of prokaryotic cells.

As used herein, the term “mutation” or “mutation” refers to a mutation occurring in a nucleotide sequence encoding a non-fluorescent fluorescent protein that can be used in the present invention, and preferably a transformed cell Or it refers to a mutation (back mutation or revertant) in which the non-fluorescent fluorescent protein is returned to a fluorescent protein in fish.

In general, DNA mutations are caused by various factors (e.g., ultraviolet rays, viruses, transposons, mutagens, etc.), and can be caused by organisms themselves (e.g., hypermutation that occurs during cellular processes). have. In addition, mutations can lead to changes in the DNA sequence in various forms. For example, nonsense mutations, frame shift mutations, missense mutations, and the like. In the missense mutation, a protein expressed according to a codon formed by a nucleotide mutated by a point mutation in which one nucleotide is replaced or substituted with another nucleotide in the DNA sequence is determined. The frame shift mutation is a mutation caused by the insertion or deletion of several nucleotides, and the protein expressed is determined according to the number of nucleotides inserted or deleted. Nonsense mutations are point mutations in which one nucleotide is changed to cause a mutation to a codon encoding another amino acid, and the expressed mutant protein sometimes loses its original activity.

According to a preferred embodiment of the present invention, the mutant non-fluorescent fluorescent protein of the present invention is (i) a frame shift mutation or (ii) a missense mutation at the level of the nucleotide sequence of the wild type fluorescent protein. It is mutated by a substitution mutation that causes According to the present invention, the mutation occurring in the nucleotide sequence encoding the non-fluorescent fluorescent protein of the present invention includes the above-described mutation. That is, the mutations induced in the present invention cause various types of mutations on the nucleotide sequence encoding the non-fluorescent fluorescent protein by the test substance, and the non-fluorescent fluorescent protein (variant) is returned to the fluorescent protein, causing fluorescence. . When the non-fluorescent fluorescent protein (variant) is returned to the fluorescent protein by the treatment of the test substance and shows fluorescence, it is determined that the test substance exhibits genotoxicity in transformed cells or fish, which means that the test substance is a carcinogen ( carcinogen) candidate.

According to a preferred embodiment of the present invention, the reverse mutation of the present invention mutates a non-fluorescent fluorescent protein having a frame shift mutation into a fluorescent protein, and mutates a non-fluorescent fluorescent protein having a substitution mutation into a fluorescent protein. More preferably, the reverse variant of the present invention is a non-fluorescent fluorescent protein encoded by the nucleotide sequence of the first or second sequence of the sequence listing encoding the ^{EGFP mutant (EGFP mut), or a complementary nucleotide sequence thereof, as a fluorescent protein.} Mutate. The term “substitution mutation” as used herein refers to mutating a non-fluorescent fluorescent protein into a fluorescent protein by mutating the nucleotide sequence encoding the non-fluorescent fluorescent protein of the present invention, which is a point mutation. , Insertion or deletion, and the like, and more preferably refers to a mutation caused by a missense mutation.

According to a preferred embodiment of the present invention, the fluorescent protein that can be used in the analysis method of the present invention is GFP (green fluorescent protein), RFP (red fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow fluorescent protein), BFP (blue fluorescent protein), EGFP (enhanced green fluorescent protein), ECFP (enhanced cyan fluorescent protein), EYFP (enhanced yellow fluorescent protein), ERFP (enhanced red fluorescent protein), mCherry, mTomato, or EBFP (enhanced blue fluorescent protein) Including, but is not limited to this. Most preferably, the fluorescent protein of the present invention is EGFP.

According to a preferred embodiment of the present invention, the EGFP mutant (EGFP ^mut ) of the present invention does not exhibit fluorescence.

According to a preferred embodiment of the present invention, fish to which the method of the present invention can be applied include, but are not limited to, zebrafish.

According to a preferred embodiment of the present invention, the method of the present invention further comprises the step of culturing and hatching embryos of a transgenic fish treated with a test substance after the above-described step (b) to obtain a lavae.

According to a preferred embodiment of the present invention, fluorescence analysis in step (c) of the present invention may be performed using various methods known in the art, and most preferably, it is performed using flow cytometry. .

According to a more preferred embodiment of the present invention, the present invention comprises the steps of: (a) treating the above-described transformed cells or fish with a test substance; And (b) a method for screening mutagens comprising the step of analyzing the fluorescence of the EGFP mutant, wherein the test substance is a nucleotide sequence of SEQ ID NO: 1 or 2 or a complementary nucleotide sequence thereof. When the fluorescence of the wild-type EGFP mutant protein is induced, it is considered to be a mutagenic substance.

Since the method of the present invention includes the above-described EGFP variant of the present invention as an active ingredient, descriptions of overlapping content between the two are omitted in order to avoid excessive complexity of the present specification due to redundant description.

The term “test substance” used in referring to the screening method of the present invention refers to a non-fluorescent fluorescence consisting of a nucleotide sequence encoding a variety of non-fluorescent fluorescent proteins or a nucleotide sequence complementary thereto, or an amino acid sequence encoding the nucleotide sequence described above. It refers to an unknown substance that induces a mutation in a protein to generate a revertant mutant, and more preferably, the nucleotide sequence of the first or second sequence of the sequence listing of Fig. 2 or a complementary nucleotide sequence thereof, or the third of the sequence listing. It refers to a substance that generates a fluorescent protein by causing mutation ^{in a wild-type EGFP mutant (EGFP mut} ) protein consisting of a sequence or an amino acid sequence of the fourth sequence.

Subsequently, a mutation of a non-fluorescent fluorescent protein to a fluorescent protein in the cells or fish treated with the test substance is detected by fluorescence. More preferably, the above-described wild-type EGFP mutant (EGFP ^mut ) protein detects a mutation exhibiting fluorescence in non-fluorescence by fluorescence.

The test substance can be obtained from a library of synthetic or natural compounds or from natural materials. Methods for obtaining libraries of synthetic or natural compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA), and a library of natural compounds is available from Pan Laboratories (USA). ) And commercially available from MycoSearch (USA).

Test substances can be obtained by various combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, and deconvolution. It can be obtained by the required synthetic library method, the “1-bead 1-compound” library method, and the synthetic library method using affinity chromatography selection. Synthesis methods for molecular libraries are described in DeWitt et al., Proc. Natl . Acad . Sci . USA . 90, 6909, 1993; Erb et al. Proc . Natl . Acad . Sci. USA . 91, 11422, 1994; Zuckermann et al., J. Med . Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew . Chem . Int . Ed. Engl. 33, 2059, 1994; Carell et al., Angew . Chem . Int . Ed. Engl . 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, etc.

On the other hand, the present invention provides a recombinant vector comprising the above-described EGFP mutant or a cell infected with a transcript thereof and a transformed cell or transformant by introduction of a gene.

Since the transformed cell or transformant of the present invention contains the EGFP mutant of the present invention described above as an active ingredient, the overlapping content between the two is omitted in order to avoid excessive complexity of the present specification due to redundant description. do.

The recombinant vector of the present invention comprises a nucleotide sequence of the first or second sequence of the sequence listing or a nucleotide sequence that is complementary thereto. The recombinant vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the recombinant vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

Preferably, the recombinant vector of the present invention comprises (i) a nucleotide sequence encoding the expression target material of the present invention described above; (Ii) a promoter operably linked to the nucleotide sequence of (i) and acting in an animal cell to form an RNA molecule, and more preferably (i) the first sequence or the first sequence of the present invention described above A nucleotide sequence encoding a nucleotide sequence of two sequences or a nucleotide sequence that is complementary thereto; (Ii) a promoter operably linked to the nucleotide sequence of (i) and acting in animal cells to form RNA molecules; And (iii) a recombinant expression vector comprising a 3'-non-translational site that acts in an animal cell to cause polyadenylation of the 3'-end of the RNA molecule.

Preferably, the above-described expression target material is a non-fluorescent fluorescent protein in which a fluorescent protein is mutated, and the fluorescent protein includes GFP, RFP, CFP, YFP, BFP, EGFP, ECFP, EYFP, ERFP and EBFP. It is not limited. Most preferably, it is EGFP.

In the present specification, the term "promoter" refers to a DNA sequence that controls the expression of a coding sequence or functional RNA. In the prepared expression vector of the present invention, the expression target material-encoding nucleotide sequence is operably linked to the promoter. As used herein, the term “operatively linked” refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter sequence, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, and In this way, the control sequence regulates the transcription and/or translation of the other nucleic acid sequence.

When the vector of the present invention uses prokaryotic cells as a host, a strong promoter capable of proceeding transcription (eg, tac promoter, lac Promoter, lac UV5 promoter, lpp promoter, p _L ^λ Promoter, p _R ^λ Promoter, rac 5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation and a transcription/translation termination sequence. Even more preferably, the host cell used in the present invention is Salmonella, most preferably Salmonella typhimurium LT2 strain. At this time, as the promoter used in the present invention, lac A promoter is preferred. In addition, when E. coli is used as a host cell , E. coli The promoter and operator site of the tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . , 158: 1018-1024 (1984)) and the left-handed promoter of phage λ (p _L ^λ Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)) can be used as a regulatory site. Meanwhile, vectors that can be used in the present invention include plasmids that are often used in the art (e.g., pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.), phage (e.g., λgt4λB, λ-Charon, λΔz1). And M13, etc.) or a virus (eg, SV40, etc.).

The method of transporting the vector of the present invention into a host cell is, when the host cell is a prokaryotic cell, the CaCl ₂ method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9:2110-2114 (1973)) ), one method (Cohen, SN et al., Proc . Natl . Acac . Sci . USA , 9:2110-2114 (1973); And Hanahan, D., J. Mol . Biol . , 166:557-580 (1983)) and an electroporation method (Dower, WJ et al., Nucleic. Acids Res. , 16:6127-6145 (1988)).

On the other hand, when the recombinant vector of the present invention is applied in a mammal such as fish, a promoter that can be used is preferably operated in an animal cell, more preferably in a mammalian cell to control the transcription of the expression target material of the present invention. As such, it includes a promoter derived from a mammalian virus and a promoter derived from the genome of a mammalian cell, such as a cytomegalo virus (CMV) promoter, a late adenovirus promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, and HSV. in the tk promoter, RSV promoter, EF1 alpha promoter, the metal in thionine promoter, beta-actin promoter, human IL-2 promoter of the gene, the promoter of the human IFN gene, the human IL-4 of the gene promoter, the promoter of the human lymphotoxin gene And the promoter of the human GM-CSF gene, but is not limited thereto. Most preferably, it is the CMV promoter or the EF1 alpha promoter. Preferably, the expression construct used in the present invention comprises a poly anenylation sequence (eg, a bovine growth hormone terminator and a poly adenylation sequence derived from SV40).

More specifically, the recombinant vector for transformation of the present invention has a structure of “promoter-expressing target material encoding nucleotide sequence-polyadenylation sequence”, and more preferably “CMV promoter-non-fluorescent fluorescent protein-SV40-derived” It is composed of “poly adenylated sequence”. In addition, the recombinant vector for transformation is a multi-position gateway-based construct kit (Fisher, et al. , 2006; Kwan, et al. , 2007) to include transposon elements on both sides of the construct. It is manufactured using. The constructed recombinant vector is microinjected into fertilization eggs obtained from zebrafish lines, more preferably into single-cell stage fertilized eggs (Xu, Q. (1999). Microinjection into zebrafish embryos. In “Molecular Methods in Developmental” Biology” (M. Guille, Ed.), Vol. 127, pp. 125-132. Humana Press, Inc., Totowa, NJ.). The above-described fertilized eggs use fertilized eggs not exceeding 20 minutes after fertilization, and include a homozygote for the transformed expression target material by inducing maturity in embryos (F1 embryos) generated after microinjection and mating with each other. Obtain an F2 embryo. Thereafter, F3 embryos are obtained through in-cross between F2 embryos and zebrafish.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference.

The production of transformed animal cells using the recombinant expression vector of the present invention can be carried out by a gene transfer method commonly known in the art. For example, electroporation, liposome-mediated transfer method (Wong, et al., 1980), retrovirus-mediated transfer method (Chen, HY, et al., (1990), J. Reprod. Fert. 41: 173-182; Kopchick, JJ et al., (1991) Methods for the introduction of recombinant DNA into chicken embryos.In Transgenic Animals, ed.NL First & FP Haseltine, pp.275-293, Boston; Butterworth-Heinemann; Lee , M.-R. and Shuman, R. (1990) Proc. 4th World Congr. Genet. Appl. Livestock Prod. 16, 107-110) and microinjection (Zebrafish Course, Mullins, August, 2005; Xu, Q. (1999). Microinjection into zebrafish embryos. In “Molecular Methods in Developmental Biology” (M. Guille, Ed.), Vol. 127, pp. 125-132. Humana Press, Inc., Totowa, NJ.). More preferably, it is carried out by microinjection.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a method for analyzing genotoxicity using a fluorescent protein variant, preferably a wild-type EGFP variant (EGFP ^mut).

(b) According to the present invention, the fluorescent protein variant of the present invention, preferably the wild-type EGFP variant transformed zebrafish ( Danio rerio ) line, MutaFish system provides a very excellent animal system that can measure the genotoxicity of test substances by producing fluorescent fry in which the reversion of fluorescent protein variants is induced by the treatment of the test substance.

(c) The MutaFish system of the present invention can very simply and quickly analyze the genotoxicity of a test substance in eukaryotes, which was a limitation of the prior art (eg, Ames test).

1 shows a vector map ^{of pEGFP mut ::CAT.} The cat gene was cloned at the stuI site of pEGFP (Clonetech).
Figure 2 shows the DNA (A) and amino acid sequence (B) of wild-type EGFP in which the DNA sequence of the ^{mutant EGFP mut is indicated.} In ^{the case of EGFP mut1} , it is a mutation substituted with L137P (CTG to CCC), in ^{the case of EGFP mut2} , it is a mutation in which another Gly is added after the 51st Gly (T G ⁵¹ G KLPVPWPT), and in the case of ^{EGFP mut3, 51} The 51st Gly with a -1 frame shift mutation with two Glys added after the 1st Gly Then it stops at the 10th amino acid, the 62nd amino acid. The sequence of EGFP ^mut3 is as follows: NH ₂ -MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGG SCPCPGPPS -OH.
3 shows a vector map of pCMV-Mut-EGFP::pCMLC2-mCherry. The EGFP ^mut was cloned between the two att positions in the tol2 kit.
4 is a result of observing Salmonella LT2 carrying an EGFP variant plasmid under UV light. A, pEGFP::cat; B, pEGFP ^mut1 ::cat; C, pEGFP ^mut3 ::cat; D, pEGFP ^mut2 ::cat.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental materials and test methods

Salmonella strains

Ames test S. typhimurium strains TA98 and TA were purchased and cultured according to the manufacturer's instructions (Xenometrix AG, Switzerland). ^{EGFP mut} on chromosome CH4001 and CH4003 strains (Table 1) carrying DNA were prepared as described in the experimental results.

Strain and plasmid used in this study. Strain Items to be stated Reference LT2
TA100
TA98
CH4001
CH4003 Wild-type Salmonella Typhimurium
Ames test strain
Ames test strain
TA100 carrying EGFP ^{mut1 on chromosome}
TA100 carrying EGFP ^{mut3 on chromosome}
Barnes et al. (1982)
Isono & Yourno (1974)
This study
This study Plasmid pEGFP
pEGFP::CAT
pEGFP ^mut1 ::CAT
pEGFP ^mut2 ::CAT
pEGFP ^mut3 ::CAT
pCMV-Mut-EGFP::
pCMLC2-mCherry Clontech
Transporting CAT to the stuI location of pEGFP
Leu ¹³⁷ (CTC) replaced by Pro (CCC)
Carrying GGG after Gly ^{51 (GGG)}
Carrying GG after Gly ^{51 (GGG)}
Carrying related EGFP ^mut in Tol2 kit
Yang, Cheng et al. (1996)
This study
This study
This study
This study
This study

EGFP ^mut Manufacture of

Non-fluorescent mutated EGFP was obtained through site directed mutagenesis using a mutant-inducing primer. The following four sets of primers were used: lac A forward primer (primer 1) containing the BamHI position of pEGFP (Clontech Laboratories, Palo Alto, CA) (Yang, Cheng et al. , 1996) upstream of the promoter and a reverse primer containing the EcoRI position downstream of the stop codon ( Primer 4) (Table 2). A second set of primers (primer 2-x and primer 3-x) containing the desired mutations were designed to produce the necessary changes (Joseph Sambrook, 2001). To construct EGFP ^mut1 , the first PCR (95°C, 30 seconds; 55°C, 30 seconds; 72°C, 1 minute) was performed using primers 1 and 3d, and the second PCR ( 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 1 minute). To construct EGFP ^mut2 , the first PCR (95°C, 30 seconds; 55°C, 30 seconds; 72°C, 1 minute) was performed using primers 1 and 3a, and the second PCR ( 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 1 minute). To construct EGFP ^mut3 , the first PCR (95° C., 30 seconds; 55° C., 30 seconds; 72° C., 1 minute) was performed using primers 1 and 3b, and the second PCR ( 95°C, 30 seconds; 55°C, 30 seconds; 72°C, 1 minute). The PCR products of the two reactions were mixed, optimized, and annealed, and then used as a template for final PCR using primers 1 and 4. The final PCR product was digested with EcoR1 and BamH1 and religated to the pEGFP empty vector. The plasmids described above contained a non-fluorescent EGFP gene under the control of the lac promoter. ^{EGFP mut with} Salmonella chromosome To locate the gene, the chloramphenicol acetyltransferase (CAT) gene was added to the StuI of the plasmid. ^{The pEGFP mut} ::CAT plasmid was made by inserting at the position (Fig. 1). The sequence modification of EGFP ^mut is described in FIG. 2.

Primers used in the preparation of pEGFP ^mut. primer Sequence (5’-3’) Items to be stated One mEGFP-F CCTGCAGGTCGACTCTAGAG Forward primer 4 mEGFP-R CCGGCGCTCAGTTGGAATTC Reverse primer 2a G ⁵¹ GGG-F ATCTGCACCACCGG G GGGAAGCTGCCCGTGCCC G51+GGG(Gly-Gly)
(Template for frame shift mutation) 3a G ^51G GG-R GGGCACGGGCAGCTTCCCCCCGGTGGTGCAGAT 2b G ⁵¹ GGG+GF CATCTGCACCACC GGG GGG G AAGCTGCCCGTGCCC G51+GGG+G 3b G ⁵¹ GGG+GR GGGCACGGGCAGCTTCCCCCCCGGTGGTGCAGATG G51+GGG+G 2f L137P-F CTCGTGACCACCCTGGCCTACGGCGTGCAGTGC Base pair replacement 3f L137P-R GCACTGCACGCCGTAGGCCAGGGTGGTCACGAG Base pair replacement

EGFP ^mut Insertion of DNA into the Salmonella chromosome

EGFP ^mut DNA is approximately about 5 min away from the end (ter) region of chromosome It was inserted into the chromosome of the Salmonella typhimurium LT2 strain using the putA site (Elliott, 1992). LacP- EGFP ^mut ::CAT DNA was amplified using the following primers containing the sequence of putA position of Salmonella enterica serotype typhimurium : 5'primer , 5'-GAAATCGCCTGTTAATGGTACCAATAGCCTTGACGCAATAGAGTAATGAC TCACTCACTCATTAGGCACCCCAGGCTTT-3' and 3'primers , 5'- CGTCATTGTCAGTCTCTTACAGAAAGATTACACGATTATTTCATCGGCAGGAGAC GCGTAGCACCAGGCGTTTAAG-3'. The above-mentioned primers are a pEGFP ^mut :: CAT plasmid as template lacP -nonEGFP :: cat embellish pieces of 50-nt or 55-nt putA (underlined in the sequence), and a 25-nt sequence or the 22-nt sequence Each was made up. The 1.4-kbp PCR product was purified and transformed into bacteria having a red helper plasmid (pKD46) by electroporation (Datsenko and Wanner, 2000). Linear DNA included the chloramphenicol gene as a marker for P22 transduction with TA100 and TA98, Salmonella typhimurium strains of the Ames test, and as a result, CH4001 and CH4003 were prepared, respectively (Davis, et al. , 1980).

Ames test

A plate-incorporation assay was performed as previously described (OECD, 1997). Briefly, bacterial cells from the stock solution were incubated at 37° C. for 12 hours in liquid medium. 0.1 mL of the bacterial suspension was added to 2 mL of Top Agar (Difco) medium containing 0.6% agar, 0.6% NaCl, 20.96 μg l-histidine and d-biotin (Sigma). In addition, test solution (or solvent control, or formulation positive control) and S9-mix (S9-mix; 0.5 mL/plate; 10 v/v% S9; Sigma), or potassium phosphate buffer (0.5 mL/plate; 0.2 M , pH 7.4) (Sigma) was added to the top agar and mixed, and then the Vogel-Bonner minimal medium agar plate was plated (in the case of the treatment experimental group, 3 independent replicate groups per administration and the solvent control group, independent per administration). 6 replicates). Colonies were incubated at 37° C. for 48 hours and then counted. The largest volume of the test solution added to the plate was limited to 100 μl in the first experiment, and increased to 200 μl in the reconfirmation experiment.

S9-mixture

Composition: 1 mL of S9-mixture contains 0.1 mL of S9, 0.01 mL of 0.4 M MgCl ₂ (Sigma), 0.01 mL of 1.65 M KCl (Sigma), 0.5 mL of 0.2 M phosphate buffer (pH 7.4; Sigma), 0.04 mL of 0.1 M NADP (Sigma), 0.005 mL of 1 M glucose-6-phosphate (Sigma), and 0.335 mL of distilled water were included. All co-factors were filtered through a 0.45-µm sterile membrane (Millipore) before use.

Preparation of mutagen

ENU (isopack N-3385; Sigma Chemical) in 105.5 mL of 0.008% Instant Ocean salts (Senju Pharmaceutical, Hyogo, Japan) buffered with 5 mM 2-(N-morpholino)ethanesulfonic acid (pH 6.0) (Sigma). Co., St. Louis, MO, USA) 1 g was dissolved to prepare a stock solution of 81 mM ENU and stored at -80°C. A stock solution of 2 mg/mL ICR-191 (Polyscience Inc., Warrington, PA, USA) was prepared by dissolving 20 mg of ICR-191 in 10 mL of sterile distilled water.

EGFP ^mut Construction of zebrafish expression vector carrying

pEGFP ^mut :: EGFP ^{mut in CAT plasmid} The gene was amplified by PCR using a forward primer (attB1 ) and a reverse primer ( attB2). The primer sequence is as follows: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCACCATGGTGAGCAAGGGCGAG-3'(forward);5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTGGCTGATTATGATCTAGAG-3' (reverse). The amplified PCR product was gateway cloned into the Tol2 plasmid, and the EGFP ^mut gene was expressed by the CMV promoter (Tol2 kit: a multi-position gateway-based construction kit for the Tol2 transposon transformation construct (Fisher, et al. , 2006). ; Kwan, et al. , 2007)). The Tol2 plasmid carries the mCherry gene regulated by the heart-specific CMLC2 promoter, which can be used as an indicator of germline transduction. The final construct pCMV-Mut-EGFP::pCMLC2-mCherry (Fig. 3) was confirmed through restriction enzyme digestion and DNA sequencing.

Creation of transgenic zebrafish

^{EGFP mut} constructed as described above Plasmids were microinjected into single-cell stage fertilized eggs made at the AB* zebrafish line (Oregon University, Corvallis, OR, USA) with transporase mRNA. The microinjected embryos induced sexual maturity and were mate with wild type AB* zebrafish. Thereafter, it was screened for the presence or absence of mCherry fluorescence in the heart of the resulting embryos. To confirm the genomic integration (insertion) of the mutated EGFP, RT-PCR, Western blotting and Southern blotting were performed on screened embryos (F1). Maturity was induced again in the F1 embryo and mated with each other to generate the F2 embryo, which is homozygous for ^{the EGFP mut.} F3 embryos generated through mating between F2 zebrafish again induced maturity.

Mutagen treatment

Fertilized eggs were obtained by mating transgenic F3 female and non-transgenic male fish. The present inventors classified 24-hour embryos still surrounded by corion into three groups (266 embryos/group), and then 0, 2 or 5 mM ENU 10 contained in 0.1% Instant Ocean salts (Senja Pharmaceutical, Hyogo, Japan). mL was treated and reacted at 26° C. for 30 minutes. Thereafter, the mutant embryos were rinsed and maintained in an incubator at 28.5° C. for 3 days. Almost all embryos hatched during the incubation period. The combined surviving laba were counted for morphological abnormal laba and EGFP ^{+ laba.}

Statistical analysis

All analyzes were performed using SPSS version 11.0 for Windows statistical package. Statistical analysis of the experimental values obtained in the Ames test was performed by a parametric method, namely, Dunnett's t -test (Dunnett, 1955; Dunnett, 1964) and linear regression analysis (Kirkland, 1994) observing the response according to the dose. . For the SCE test, after one-way variance analysis (ANOVA) was performed, Student's t -test and post-hoc Dunnett's t -test were performed. TF analysis was performed using a χ ^{2 -test.} In all tests conducted, a p -value of ≤0.05 was considered to be statistically significant.

Experiment result

Preparation of non-fluorescent EGFP

In an attempt to create a visual genotoxicity test system using EGFP that is genetically similar to the Ames test, the present inventors noted two mutations adopted in the Ames test, hisG46 (TA100) and hisD6610 (TA97). hisG46 The mutation has a base pair in which Leu(CTC) is replaced by Pro(CCC), hisD6610 The mutation results in 6 cytosines (-CCCCCC-) by a C1 frame shift mutation. These mutations are sensitive to several mutagenes that revert the -1 frame shift mutation of hisD3052 (TA98), which affects the reading frame of the repetitive CGCGCGCG- sequence (Isono and Yourno, 1974; Levin, Yamasaki et al. , 1982). . The present inventors used a site-directed mutagenesis method to generate a non-fluorescent protein by inducing a similar mutation in EGFP. First of all, we replaced ^{Leu 137} with Pro similar to the hisG46 mutation. This replacement changed the mutant EGFP to a non-fluorescent protein (Figure 4). This was named ^{EGFP mut1} and was expected to be able to detect base pair replacement by mutagenes like hisG46 mutant. Then, the present inventors have added another Gly (GGG) after ^{Gly 51} (GGG) located at the site of α-helix (reviewed in Tsien, 1998) connected to a β-barrel that forms an empty cylinder as the core structure of GFP. A mutant EGFP was made. This is followed by Gly ⁵¹ (GGG), where additional glycine has little effect on the EGFP molecule, producing a fluorescent protein. 4 is a result showing that the EGFP protein (EGFP ^{mut2) having an additional Gly exhibits fluorescence.} Meanwhile, a hotspot DNA sequence with 6 guanosine for frame shift mutation was constructed. Therefore, -1G frame shift mutation was induced from ^{EGFP mut2} with five guanosines ^{, thereby producing EGFP mut3} . In this case, the EGFP protein translation is Gly ⁵¹ It can then be stopped at the 10th amino acid, the 62nd amino acid. This protein was found to have non-fluorescent properties (Figure 4). ^{EGFP mut3} carrying five guanosines was expected to be similar to hisD6610 , a strain that can be used to detect frame shift mutations.

Genotoxicity test with Salmonella typhimurium test strains carrying the EGFP ^mut

^{Next, the EGFP mut} gene was transferred to the associated Salmonella typhimurium chromosome to establish a mutation test strain similar to the Ames test. A linear DNA transformation method (Datsenko and Wanner, 2000) using a λ red system was performed so that the EGFP ^mut gene enters the putAP position on the wild-type Salmonella (LT2) chromosome (Elliott, 1992). ^{EGFP mut1} using P22, a general transduced phage And EGFP ^mut3 genes were inserted into TA100 and TA98, respectively, to produce CH4001 and CH4003.

The present inventors tested the mutation frequency of CH4001 and CH4003 together with conventional Ames test strains TA100 and TA98 in the presence of mutagenes (ENU and ICR 191). ENU is known to induce base-pair replacement, while ICR 191 is known to induce frame shift mutations (OECD, 1997). ^{His +} revertants were selected under the minimal medium plate, ^{and about 10 6} colonies were screened ^{for EGFP +} revertants in NA plates that could be individually visualized. In the absence of mutagen, His ⁺ and EGFP ⁺ return mutants both occurred in the TA and CH strains with ^{a frequency of 1.1 to 1.8 x 10 -6} (Table 3). In the presence of ENU (0.1 mg/plate), the frequency of ^{His +} revertant in TA100 and CH4001, test strains for base replacement, increased to ^{5.6 x 10 -6} and 5.2 x 10 ^{-6, respectively.} This frequency of return is within the range reported in previous reports (Lang, 1984). Very interestingly, the present inventors similarly increased the frequency ^{of the EGFP +} revertant mutant in ^{the presence of ENU of 7.1 x 10 -6.} TA98 and CH1003, a test strain for frame shift mutations, were reacted with ICR 191 (1 mg/plate). Similar to previous reports (Hera and Pueyo, 1988), His ⁺ revertant increased with a mutation frequency of ^{420.8 x 10 -6.} In addition, the His ⁺ revertant mutant in TA98 increased with a mutation frequency of 327.6 x 10 ^-6. In the presence of ICR 191, the EGFP ⁺ revertant was similarly increased with a mutation frequency of 456.3 x 10 ^-6. Taken together, the above-described results indicated that CH4001 and CH4003 were functional analogs with TA100 and TA98 ^{in the His + reversion mutation.} Thus, the EGFP ⁺ remutation system of CH strains can replace ^{the His +} remutation system of the Ames test.

Number of revertants that occurred in the Salmonella tester strain ^* . TA100 EGFP ^mut1 TA100 TA98 EGFP ^mut2 TA98 Phenotype His ⁺ His ⁺ EGFP ⁺ His ⁺ His ⁺ EGFP ⁺ process ^** Non-treatment 1.6 1.8 1.2 1.1 1.2 0.5 ENU 5.6 5.2 7.1 3.2 3.5 3.2 ICR 191 - - - 420.8 377.6 456.3

^{* A} total of 10 ⁶ bacteria were plated on a minimal medium plate and an NA plate, respectively, to screen for His ⁺ reversion mutation and EGFP ^{+ reversion mutation.} EGFP ^mut1 carries the L137P amino acid replacement and EGFP ^mut2 carries the G ⁵¹ G ⁵² mutant construct.

^* ENU and ICR 191 were treated at 0.1 mg and 1 mg, respectively, per plate.

Cytotoxicity test using transgenic zebrafish carrying genes

The present inventors ^{injected Tol2 plasmid encoding EGFP mut} and transposase mRNA into single-cell stage zebrafish fertilized eggs. As described in the experimental protocol, F1 progeny by germline transmission were selected by PCR and Southern blot analysis. One high transgene-positive line was identified. When the F3 progeny of this line was obtained by mating F2 zebrafish and non-transgenic zebrafish, F2 zebrafish delivered a transgene to about 50% of the F3 progeny zebrafish, which means that the transgene stably is a zebrafish chromosome. Indicates that it is integrated into one of the As a result, the present inventors MutaFish ^{1 carrying EGFP mut1} And MutaFish ^{3 carrying EGFP mut3} was established.

Using these transgenic zebrafishIn vivoIn order to investigate whether a mutation can be detected in, transgenic zebrafish and non-transgenic zebrafish were mated and treated with mutagen for 30 minutes in 24-hour embryos obtained: MutaFish treated with 5 mM ENU.^One And MutaFish treated with 10 mM ICR 191³. After 3 days, EGFP⁺The number of lavae was measured (Table 4). The background of the frequency of mutants occurring in the absence of mutagenes is approximately 2-3 x 10^{- 5}Was. This is the mouse system (Gondo, Shioyama,et al., 1996) andrplSTransgenic zebrafish (Amanuma, Takeda,et al., 2000), and very low to detect mutations in the genomic DNA of treated embryos. MutaFish processed with ENU^OneSilver 6.3 x 10^-4EGFP with a frequency of⁺The number of laba was increased, which was associated with an increase in morphological abnormalities (20.9%). this isrplSSimilar to the results for transgenic zebrafish carrying (Amanuma, Takeda,et al., 2000). MutaFish treated with ICR 191³Silver 2.32 x 10^-3EGFP with a frequency of⁺The number of laba was increased, which was associated with an increase in morphological abnormalities (24.2%). Taken together, the above data indicated that the MutaFish system could be used to investigate the genotoxicity of mutagenic compounds including alkylating agents and basic adducts.

Incidence of transgene mutations in mutagen-treated embryos ^* . EGFP ^mut1 MutaFish EGFP ^mut3 MutaFish process ^** Survival rate (%) Morphological abnormality (%) Mutation frequency (x10 ^-5 ) Survival rate (%) Morphological abnormality (%) Mutation frequency (x10 ^-5 ) Non-treatment 100 0.075 2 100 0.081 3 ENU 97.3 20.9 63 - - - ICR 191 - - - 79.2 24.2 232

^{* A} total of 10 ⁶ fish were irradiated for EGFP expression. The EGFP ^mut1 MutaFish carries the L137P amino acid replacement and the EGFP ^mut3 MutaFish carries the frame shift mutation in the ^{G 51} G ^{52 mutant construct.}

^* ENU and ICR 191 were treated with 5 mM and 10 mM, respectively.

Further discussion

The MutaFish system using the EGFP remutagenic system provides a selective animal test for genotoxicity testing. The greatest advantage of the MutaFish system is the visualization of genotoxicity: ^{the quantification of EGFP +} Lava under UV light reflects the genotoxicity of the chemical and does not require any further manipulation of the Lava. Regulatory systems for drug registration need to be tested for genotoxicity (Billinton, Hastwell, et al. , 2008); CPM/ICH 1995), these tests are designed to identify compounds that increase the risk of carcinogenicity and/or hereditary mutation by damaging the genome or otherwise modifying the genome. Although a variety of selective approaches to predicting the environmental hazards of chemicals can be used, the present inventors have demonstrated in this study that MutaFish is the most convenient animal assay system. Zebrafish are unique in terms of available research content, techniques and approaches. ^{However, EGFP mut} applied to zebrafish in the present invention The visual technique of the present invention using the system is medaka ( Oryzias; latipes ) and fathead minnow ( Pimephales) promelas ) can also be applied to other experimental model fish species. In addition, lava of these species may be proposed as an alternative to acute toxicity testing for fry/adult fish (Braunbeck, Boettcher, et al. , 2005; Scholz, Fischer, et al. , 2008).

In order to improve the sensitivity of the MutaFish system, fish with defects in DNA repair enzymes in the future must be considered. Many invoices In mouse models, the following strains have been developed and used to more effectively detect many toxic chemicals: XPA ^-/- , XPC ^-/- , Msh2 ^+/- , Msh2 ^-/- and p53 ^+/- mutant mice. , Apc (ApcΔ716, Apc 1638N, Apc min) mutant mice, A33 Δ_β-cat knock-in mice (Dashwood, 2003). Several of these models provided a review of the spectrum of mutations induced in vivo in target and non-target organs by chemicals during tumorigenesis. In addition, other genes that are included in the DNA repair system but have not yet been identified can be tested on MutaFish to further increase detection sensitivity.

In this study, although we have used the advantage of fluorescent GFP, the technique of the present invention includes a series of GFP variants (Shaner, Steinbach, et al. , 2005) and Firefly luciferase and Renilla luciferase. It can be extended to other fluorescent proteins, such as light-generating proteins. In addition, it may be possible that another mutation of GFP that can eliminate fluorescence not described in this study has a higher reversion rate in exposure to chemicals than in the present invention.

Over the past few years, zebrafish have become a means of increasing importance to biologists as a model to study development, because zebrafish embryos are transparent and rapidly develop, and zebrafish genes are very similar to humans ( 75%) and are vertebrates (Bradbury, 2004; Scholz, Fischer, et al. , 2008). Given the fact that the developing embryos are very sensitive to toxic damage, the zebrafish system is an attractive in vivo when it comes to visualizing the genotoxicity of chemicals. It is a model. Recently, under European Union legislation for the protection of animals used for experimental and other scientific purposes, the use of vertebrates at the embryonic stage has not been regulated (Commission of the European Communities, 1986). Therefore, experiments using embryos are considered as an alternative to animal experiments (Fleming, 2007). Therefore, the MutaFish system is an excellent model for environmental risk assessment of chemicals in that it can perform small-scale, high-throughput analysis using a modified cell flow analysis method.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

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<110> Chonnam National University <120> Methods for Analyzing a Genotoxicity Using Non-Fluorescent Fluorescence Proteins <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> EGFP mutant 1 <400> 1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat ccccgggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 720 <210> 2 <211> 722 <212> DNA <213> Artificial Sequence <220> <223> EGFP mutant 3 <400> 2 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accgggggaa gctgcccgtg ccctggccca 180 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 240 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 300 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 360 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atccccgggc 420 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 480 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 540 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 600 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 660 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt 720 aa 722 <210> 3 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> EGFP mutant 1 <400> 3 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Pro Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 4 <211> 62 <212> PRT <213> Artificial Sequence <220> <223> EGFP mutant 3 <400> 4 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Gly Ser Cys Pro Cys Pro Gly Pro Pro Ser 50 55 60 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mEGFP-F primer <400> 5 cctgcaggtc gactctagag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mEGFP-R primer <400> 6 ccggcgctca gttggaattc 20 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> G51GGG-F primer <400> 7 atctgcacca ccggggggaa gctgcccgtg ccc 33 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> G51GGG-R primer <400> 8 gggcacgggc agcttccccc cggtggtgca gat 33 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> G51GGG+G-F primer <400> 9 catctgcacc accggggggg aagctgcccg tgccc 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> G51GGG+G-R primer <400> 10 gggcacgggc agcttccccc ccggtggtgc agatg 35 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> L137P-F primer <400> 11 ctcgtgacca ccctggccta cggcgtgcag tgc 33 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> L137P-R primer <400> 12 gcactgcacg ccgtaggcca gggtggtcac gag 33 <210> 13 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> lacP-EGFPmut::CAT forward primer <400> 13 gaaatcgcct gttaatggta ccaatagcct tgacgcaata gagtaatgac tcactcactc 60 attaggcacc ccaggcttt 79 <210> 14 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> lacP-EGFPmut::CAT reverse primer <400> 14 cgtcattgtc agtctcttac agaaagatta cacgattatt tcatcggcag gagacgcgta 60 gcaccaggcg tttaag 76 <210> 15 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> attB1 primer <400> 15 ggggacaagt ttgtacaaaa aagcaggctc caccatggtg agcaagggcg ag 52 <210> 16 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> attB2 primer <400> 16 ggggaccact ttgtacaaga aagctgggtt ggctgattat gatctagag 49

Claims (6)

Wherein the embryo comprises an embryo of a transformed fish with a nucleotide sequence encoding a non-fluorescent fluorescent protein with a mutation, wherein when a test substance is treated in the embryo to generate a back mutation, Wherein the non-fluorescent fluorescent protein is incubated with a larvae mutated into a fluorescent protein. The method of claim 1, wherein the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), blue fluorescent protein protein, enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), enhanced red fluorescent protein (ERFP), or enhanced blue fluorescent protein (EBFP). 3. The composition of claim 2, wherein the fluorescent protein is EGFP. The method according to claim 1, wherein the mutated non-fluorescent fluorescent protein has a substitution mutation that causes (i) a frame shift mutation or (ii) a missense mutation at a nucleotide sequence level of a wild type fluorescent protein wherein the composition is mutated by a substitution mutation. 2. The composition of claim 1, wherein the mutated non-fluorescent fluorescent protein is encoded by a nucleotide sequence of the first or second sequence of the sequence listing. 2. The composition of claim 1, wherein the fish is a zebrafish.

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