JP2001157588A - Gene trap using green fluorescent protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for observing and determining the localization and dynamic behavior of a protein in an intact cell. SOLUTION: A linearized trap vector, which contains an intron and splice donor sequence, a DNA sequence encoding green fluorescent protein(GFP) from which N-terminal methionine and valine ware deleted, an inner ribosome-binding site, a DNA sequence encoding a selective marker, and a polyadenylated signal sequence, is transfected into a cell, and GFP is expressed as a fused protein with an intrinsic protein. Observation of the green fluorescence allows to observe and determine the localization and dynamic behavior of the intrinsic protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel gene trapping method using green fluorescent protein (GFP) as a reporter. More specifically, the present invention relates to an animal cell that expresses a protein localized in a cell by fusing it with a green fluorescent protein, a method for producing such a cell, and a trap vector for producing such a cell.

[0002]

2. Description of the Related Art With the progress of a genome analysis plan for several model organisms including humans, the structure of an enormous number of unknown genes has been revealed. In this plan
At the beginning of the 21st century, the entire nucleotide sequence of the human chromosome will be determined. Therefore, there is no doubt that the research of medical biology in the next century will proceed with the primary structure of all proteins present in the human body as common knowledge of all scientists. However, elucidation of the functions of all proteins remains an endless goal, and in the current medical biology research that is about to enter the post-genome analysis era, the biological functions of proteins have been It is not an exaggeration to say that systematically elucidating research is the task most demanded for its realization.

[0003] Then, how do we specifically clarify the biological functions of proteins? One apparently productive strategy is to make a thorough comparison of the primary structure of the whole protein with known functional proteins to reveal their relatives and to gain an analogy with known functions Direction. But,
This makes it impossible to understand proteins whose functions are not known at all or have no homology at all with known proteins.

[0004] When a protein of unknown function is obtained, the protein is expressed in any organ or tissue in the living body, and is present in any region inside the cell (or outside the cell) in order to know its function. The amount of information required to consider the function is dramatically increased by clarifying whether or not the function is available. In other words, the first experiment to be performed on a gene product whose sequence has been identified is to search for the organ / tissue specificity of the protein, and to analyze the intracellular (external) localization.

[0005] Here, the former search for organ / tissue specificity uses a DNA base sequence which is a product of the genome project,
Information can be obtained directly by a hybridization technique using a probe. On the other hand, the intracellular localization of a protein cannot be analyzed using a DNA probe, and it is almost impossible with a current knowledge to clarify it a priori from primary structural information.

At present, the most trusted experimental technique is an indirect immunohistochemical method using an antibody specific to a gene product. However, this method has two disadvantages.
That is, first of all, a great deal of effort and expertise is required to produce high quality specific antibodies that can withstand this application. Second, even if antibodies are obtained, it is very difficult to observe living cells (generally, except when targeting a cell surface antigen).

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for observing the intracellular localization of a protein without using a probe specific to the protein, and while keeping the cell alive. .

[0008]

First, in the present invention, (1) an intron and a splice acceptor sequence; and (2) a methionine and a valine at the N-terminus are sequentially deleted from the 5'-terminus toward the 3'-terminus. A trap vector containing a DNA sequence encoding a green fluorescent protein (GFP); (3) an internal ribosome binding site (IRES); (4) a DNA sequence encoding a selectable marker; and (5) a polyadenylation signal sequence. I do.

The present invention further provides (1) transformation by introducing the above trap vector into mammalian cells, (2) culturing the cells in a solid medium, and (3) obtaining colonies from each colony. Selecting a cell expressing the green fluorescent protein for the cell to be obtained, and (4) identifying a protein endogenous to the cell fused to the green fluorescent protein, including expressing the protein endogenous to the cell fused to the green fluorescent protein The present invention also relates to a method for producing animal cells.

[0010] The present invention also relates to an animal cell expressing an endogenous protein fused to a green fluorescent protein, which can be produced by the above method. If the cells are observed with a fluorescence microscope, the intracellular localization and dynamic behavior of the protein fused to the green fluorescent protein can be easily observed and determined.

Therefore, the present invention further provides a method for observing the localization and dynamic behavior of an endogenous protein fused to a green fluorescent protein in a cell by observing the fluorescence emitted by the green fluorescent protein of the cell. Related.

The outline of the present invention will be described with reference to FIG. Intron and splice acceptor sequence (SA); DNA sequence encoding green fluorescent protein (GFP) deleted of N-terminal methionine and valine; internal ribosome binding site (IRES); D encoding selectable marker
And a linear trap vector (pG) having an NA sequence (bsr); and a polyadenylation signal sequence (pA).
When the cells are transfected with FTV, FIG. 1A), the trap vector is integrated at random into the chromosome to form a fusion gene (FIG. 1B. The shaded frame encodes a protein in the chromosome). Exons, white boxes indicate untranslated exon regions). The fusion gene is transcribed (FIG. 1C), spliced and translated, and a green fluorescent protein and a marker protein fused to the protein are expressed (FIG. 1D). The cells expressing the GFP fusion protein can be easily selected by the green fluorescence, and their intracellular localization can be easily determined (E in FIG. 1).

The trap vector of the present invention has the following three features. (1) Introns and splices are added to the 5 '
It has an acceptor site. Many structural genes on the mammalian genome contain introns that divide the exons that contain the protein-encoding region, and when a DNA fragment is incorporated into the structural gene, it can be inserted into the intron region It is considered highly likely. The vector was provided with an intron and a splice acceptor site on the 5 'side so that the fusion protein could be expressed while incorporated into the intron. This results in the expression of a transcript in which the exon portion 5 ′ upstream of the insertion site and GFP are fused. Structural genes that do not contain introns are also known, but in order for the fusion protein to be correctly expressed when inserted into such a gene, the intron sequence on the vector is also placed in the same reading frame as GFP. Is designed to have a structure that does not include a stop codon.

(2) A mutant GFP obtained by deleting two amino-terminal amino acids from the GFP gene is used as a reporter gene. From the results of preliminary experiments performed using GFP (dM-GFP) in which the wild-type full-length or start codon had been deleted during the development of this vector, the efficiency of these gene traps was extremely low, and the signal of GFP was low. It revealed that the unfused protein was expressed in most of the cells seen. Therefore, for the purpose of reducing the background of the non-fusion protein, a deletion mutant dMV-GFP (green fluorescent protein from which N-terminal methionine and valine were deleted) was used. As a result, a cell line that efficiently expresses the GFP fusion protein can be obtained. The use of this deletion mutant was essential for practical use of this method.

(3) Use of internal ribosome binding site Generally, in the translation of eukaryotic mRNA, the ribosome binds to the mRNA.
Begin translation by scanning from the 5 'end until the start codon is found. In contrast, in picornavirus mRNA such as encephalomyocarditis virus (ECMV), ribosomes bind to an internal site called IRES and initiate translation.
By connecting this IRES sequence to an unrelated gene, a polycistronic mRNA can be produced. In this bicistronic mRNA, translation of a fusion gene of the target gene and GFP from the 5 'end and translation of a marker gene from ECMV IRES can be performed by a single mRNA.
Marker genes (eg, antibiotic resistance marker genes) are translated only from vectors inserted in the forward direction into the transcription unit of the target gene, so that the percentage of gene-trapped strains among antibiotic-resistant cell lines is increased It becomes possible to raise it.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Construction of Trap Vector The gene trap vector of the present invention can be constructed as follows. That is, each DNA fragment constituting the trap vector is prepared, inserted into an appropriate plasmid, ligated, and then cut out as a continuous DNA fragment using a restriction enzyme. (1) Intron and splice acceptor site The general structure of the intron is 5'-GU (A / G) AGU ..... (Py) 6X (C / U) AG-3 'is known, the GU (A / G) AGU part on the 5' side is a splice donor site, and the (Py) 6X (C / U) AG part on the 3 'side is a splice Functions as an acceptor site. The intron excluding the splice donor site functions as a functional intron when integrated into the intron region on the chromosome in the correct orientation.

[0017] In general, the cell type specificity and the animal type specificity of the signal have not been reported between mammals, and any sequence having splicing acceptor activity may be used. However, it is necessary to use a gene that does not contain a coding region of another gene or a sequence of a splice acceptor other than a splice donor and a splice acceptor at the 3 ′ end. For example, the rabbit β-globin gene (Bru
ce Alberts, Dennis Bray, Julian Lewis, Martin Raf
f, Keith Roberts, and James D. Watsomn, Molecular
Biology of the Cell (3rd ed.), Garland Publishin
g, Inc., New York & London (1994) p372 Figure 8-5
1) Remove the splice donor site from the vector containing intron II (for example, Stratagene expression vector pSG5) and replace the sequence containing the splice acceptor site by PCR.
An XhoI site is added at the 5 ′ end and a SalI site is added at the 3 ′ end for use.

(2) GFP gene In the trap vector of the present invention, green fluorescent protein (GFP) (G of Aguoria victoria)
Based on the cDNA of FP, an artificial sequence in which a codon frequently used in mammalian cells is used and a mutation for enhancing the fluorescence intensity is introduced (methionine and valine 2
A DNA sequence encoding a mutant GFP (SEQ ID NO: 1) with three amino acids deleted is used. For example, GFP cDNA
Was compared to the pCMX-SAH / Y145F plasmid (Ogawa, H., Inouye, S., Tsuji, FI, Yasuda,
K., and Umesono, K. (1995), "Localization, traffick
ing and temperature-dependence of the Aquorea gree
n fluorescent protein in cultured vertebrate cell
Natl. Acad. Sci. USA, 93: 4845-4850.)
Thus, a mutant GFP in which two amino acids of methionine and valine at the amino terminus are deleted is prepared by PCR. At the 5 'end
An EcoRI site is added to the SalI site and the 3 ′ end for use.

(3) Internal ribosome binding site In the trap vector of the present invention, an internal ribosome binding site (IRES) (Mountford, PS, and Smith, A .;
G., "Internal ribosome entry sites and dicistronic
RNAs in mammalian transgenesis ", Trends in Genetic
s 11: 179-184 (1995); Ramage, AD, Clark, AJ, Sm.
ith, AG, Mountford, PS, and Burt, DW, "Improv
ed EBV-based shuttle vector system: dicistronic mR
NA couples the synthesis of the Epstein-Barr nucle
ar antigen-1 protein to neomycin resistance ", Gene
197: 83-89 (1997)).

Currently known internal ribosome binding sites derived from animal viruses have been used in various vertebrate species, and their biological activities have been confirmed. The activity of invertebrates has been confirmed at least in insects such as Drosophila. Other animal species have been poorly reported to date. No effects on plants have been reported to date. It has been reported that there is no activity in yeast cells. Therefore, it is clear that this trap vector cannot be applied to yeast cells. Currently, various IRES sequences are available on nucleic acid databases, and the sequences can be fully synthesized and prepared. It can also be obtained by contacting the reporter. At present, an expression plasmid containing an IRES sequence is commercially available from Clontech or the like, and can be purchased. For example, an internal ribosome binding site derived from encephalomyocarditis virus was obtained from plasmid 6P-T3-Neolres LacZ-BS-MO.
It can be obtained as EcoRI and NcoI fragments.

(4) Marker gene Any marker can be used as long as it is a resistance gene of an antibiotic to which the host cell is sensitive. However, in this method, the expression level of the resistance gene product depends on the transcription level of the trapped gene determined by the promoter activity of the trapped gene. For this reason, it is considered that the use of a resistance gene that allows the host cell to acquire resistance even when the expression level in the cell is extremely small will enable trapping of even a gene with a small transcription amount. . Therefore, the use of a resistance gene having such properties is preferred. For example, blasticidin S resistance gene
From the plasmid pSVbsr manufactured by vitrogen, Eco was added to the 5 'end by PCR.
An XhoI site can be added to the RI site and 3 ′ end for use.

(5) Polyadenylation signal A mammalian cell polyadenylation signal can be used as the boria adenylation signal sequence. In general, cell type specificity and animal type specificity of a signal have not been reported, and any sequence having a polyadenylation signal activity may be used. For example, the polyadenylation signal is derived from the K14 human type I keratin gene by EcoRI at the 5 'end by PCR.
And an EcoT22I site and a PstI site at the 3 ′ end.

(6) Construction of Plasmid Holding Trap Vector Next, (1) intron (including splice acceptor site but not splice donor site) and (2) cDNA of mutant GFP obtained as described above The DNA fragment of the fragment, (3) IRES, (4) drug resistance gene, and (5) polyadenylation signal sequence are ligated in this order as an inserted DNA fragment on a plasmid vector to construct a plasmid vector holding a trap vector. . As the plasmid vector, a general-purpose vector used in ordinary recombinant DNA experiments can be used, but a plasmid with a high yield is desirable in order to prepare a large amount of DNA fragments after completion. In other words, it is desirable to use one that has a high copy number in E. coli.

(7) Acquisition of trap vector Next, the trap vector is cut out from the plasmid vector as a continuous DNA fragment using an appropriate restriction enzyme. That is, the restriction | limiting which cut | disconnects both ends (that is, (1) the 5 'end of an intron, and (5) the 3' end of a polyadenylation signal) without cutting the inside of a trap vector with the plasmid containing a completed gene trap vector is carried out. The enzyme is digested to release the trap vector from the plasmid backbone. At this time, the molecular weights of the trap vector and the DNA of the plasmid backbone should be different so that they can be separated by the agarose gel electrophoresis to be performed next. The DNA mixture after the digestion reaction is separated by agarose gel electrophoresis and subsequently purified to obtain a linearized trap vector. For example, a plasmid containing the completed gene trap vector is cut with three kinds of restriction enzymes XhoI, PstI and AseI, separated by agarose gel electrophoresis, and a 3 kb DNA fragment is adsorbed on an anion exchange membrane. The linearized trap vector is obtained by purification using

Cell Transfection Next, the trap vector thus obtained is introduced into animal cells. As the animal cells, any animal cells can be used as long as the used IRES has activity and can be selected with the drug resistance marker to be used. For example, PC12ASK1 cells derived from rat pheochromocytoma can be used. It is necessary to transfect the trap vector under the condition that only one site is integrated on the chromosome.
Any method may be used as long as it is an introduction method that allows efficient integration into one random place on the transfected chromosome per cell. Electroporation is an example of a DNA introduction method suitable for such a purpose. For example, after incubating the cells in a phosphate buffer (PBS (-)) containing no calcium and magnesium at 37 ° C. for 10 minutes, 10 ml
Single cells are obtained by passing 10 times through a 21-gauge injection needle with a plastic syringe. The cells are suspended in PBS (-) at a concentration of 5 × 10 ⁶ cells / ml, and 0.8 ml of the cell suspension and 20 μg of the linearized vector DNA are mixed, and DNA is introduced by electroporation.

Next, desired cells are selected from the transfected cells. The selection of cells determines the drug to be used according to the drug resistance gene used. Preliminary experiments determine the drug concentration at which the host cells without the introduction of the trap vector are completely killed. The trap vector-introduced cells are cultured at a higher drug concentration to form colonies. Next, the resistant colonies are observed as they are with viable cells using a fluorescence microscope, and cells emitting a GFP fluorescent signal are isolated and used for the following analysis. For example, when plasmid cidin S is used as a marker, the obtained blasticidin S
Transfer resistant colonies one by one to a 6-well culture dish and culture them.
Observe with a fluorescence microscope equipped with a water immersion objective. GFP
The cell line showing green fluorescence is selected and culture is continued, and used for the following analysis.

Analysis of Cell Line The cell line is grown in a 10 cm culture dish and subjected to cryopreservation and protein extraction. Western blot analysis using an anti-GFP antibody identifies cells expressing a fusion protein with a higher molecular weight than unfused GFP (about 27 kD)). Poly (A) + RNA is purified from those cells, and the GFP fusion gene is identified by the 5'RACE method. Hereinafter, the present invention will be described with reference to examples. However, the examples are for explanation, and the present invention is not limited to the examples.

[0028]

Example 1 Preparation of Trap Vector A trap vector was constructed by introducing appropriate restriction enzyme recognition sites into each DNA fragment constituting the trap vector and ligating them sequentially. Restriction enzyme recognition sites required for each DNA fragment were introduced by PCR. That is, an oligonucleotide primer containing an appropriate restriction enzyme recognition site is synthesized, PCR is performed with the primer, restriction enzyme digestion is performed, and the resultant is inserted into a plasmid vector. The operation of the PCR method was performed as follows.

PCR method: The PCR reaction mixture used was as follows. Sterile distilled water 32 μl 10x KOD buffer 5 μl 2 mM dNTP mix 5 μl 25 mM MgCl ₂ 2 μl 10 μM 5 ′ primer 2 μl 10 μM 3 ′ primer 2 μl 10 ng / μl Template plasmid DNA 1 μl KOD DNA polymerase (2.5 U / μl) 1 μl Total 50 μl Note) Composition of 10xKOD buffer 1.2M Tris-HCl (pH 8.
0); 100 mM KCl; 60 mM (NH ₄ ) ₂ SO ₄ ; 1% Triton X-100;
0.1 mg / ml bovine serum albumin

The PCR reaction was performed as follows. The reaction tube and the tube containing each component are previously cooled on ice. A mixed solution is prepared by adding the mixture in the above order. After equilibrating the thermal cycler to 98 degrees, transfer the reaction tube to the thermal cycler and start the reaction. The thermal cycler is performed with the following program using Thermal Cycler MP of TaKaRa. One cycle of 98 ° C for 1 minute; 25 cycles of 98 ° C for 15 seconds, 65 ° C for 2 seconds, and 74 ° C for 30 seconds; finally one cycle of 74 ° C for 4 minutes 30 seconds and then 4 ° C overnight. . Eight candidates were prepared for all plasmids to be prepared below, and first, four bases were sequenced (sequencing). Sequencing was performed by capillary electrophoresis using an ABI 310Prism Genetic Analyzer. If no candidate with the correct structure was found in the first four, the remaining four were sequenced to find a candidate for the correct structure.

(1) Preparation of GFP cDNA Fragment 5′-side primer (145-5′H3Nco) Using (SEQ ID NO: 2) as a 3′-side primer (145-3′Eco), (SEQ ID NO: 3) and a plasmid containing GFP (pCMX-SAH /
PC using KOD DNA polymerase with Y145F) as the template
A DNA fragment of about 800 bp was obtained by the R method. After digesting this fragment with EcoRI and HindIII, the pBluescriptII KS (+) vector (St
ratAgene) (hereinafter abbreviated as pBSII) into EcoRI and HindIII sites.

(2) Preparation of marker (bsr) fragment The blasticidin S resistance gene was used as a marker for detection. As 5 'primer (bsr5'EcoNco), Using (SEQ ID NO: 4) as a 3′-side primer (bsr3′Pst) Using (SEQ ID NO: 5), plasmid pSV2bsr containing bsr
(Kaken Pharmaceutical Co., Ltd.) was used as a template to obtain a DNA fragment by a PCR method using KOD DNA polymerase. This DNA fragment was digested with EcoRI and PstI, and inserted into the same site of pBSII.

(3) Preparation of Fragment of K14 Polyadenylation Signal Sequence The polyadenylation signal was obtained from the K14 human type I keratin gene (Marchuk, D., McCrohon, S., and Fuchs, E., "Comp.
lete sequence of a gene encoding a humantype I ker
atin: Sequences homologous to enhancer elements in
the regulatory region of the gene ", Proc. Natl. A
cad. Sci. USA, 82: 1609-1613 (1985)). As 5 'primer (5'K14 EcoR / T22I) Using (SEQ ID NO: 6) as a 3′-side primer (3′K14Pst I) Using (SEQ ID NO: 7), a plasmid pG3K14 Cassette containing a K14 polyadenylation signal was used as a template. KODDNA
Amplified DNA fragment obtained by PCR using polymerase
After digestion with EcoRI and PstI, it was inserted into the same site of pBSII.
This plasmid is called pBS-K14pAEP.

(4) Preparation of Intron-Splice Acceptor Fragment The intron-splice acceptor sequence used was derived from the rabbit-β-brobin gene. 5 'primer (bg-int5'Xho Ver.2) (SEQ ID NO: 8), 3 ′ primer (3 types) (bg-int
3'NcoEco A, B, C) Were used.

A combination of three primers (5 '+ 3'
A, 5 ′ + 3′B, 5 ′ + 3′C), and using the plasmid pSG5 containing intron (manufactured by Stratagene) as a template, the amplified DNA fragment obtained by the PCR method using KOD DNA polymerase was converted into XhoI and EcoR.
After digestion with I, it was subcloned into the same site of pBSII.

(5) Preparation of Internal Ribosome Binding Site Fragment The internal ribosome binding site derived from encephalomyocarditis virus was
EcoRI and NcoI from -T3-Neolres LacZ-BS-MO plasmid
Obtained as fragments. The plasmid is Peter S. Mountford
Granted by Dr.

Next, the thus obtained DNA fragments were sequentially connected as follows. (1) Connection of intron and GFP three types of plasmids containing introns (pBS-bg int A, B,
After linearizing C) by XhoI digestion, a 610 bp fragment was purified by agarose gel electrophoresis by NcoI partial digestion. These three types of intron fragments were transformed with GFP-containing p
Three types of plasmids, pBS-in, which are inserted into the XhoI and NcoI sites of the BS-GFP145 plasmid and have an intron-GFP cassette
tGFP A, B, C were obtained.

(2) Connection between the IRES part and the blasticidin S resistance gene The 6P-T3-NeolresLacZ-BS-MO plasmid was ligated with EcoRI and Nco
A 600 bp IRES fragment obtained by digestion with I was purified by agarose gel electrophoresis. This DNA fragment was transformed into EcoR of plasmid pBS-NcoBSR with blasticidin S resistance gene.
A plasmid pBS-IRES bsr having an IRES-bsr cassette inserted into the I and NcoI sites was obtained.

(3) Connection of two cassettes From pBS-intGFP-A, B and C, three intron-GFP cassettes were obtained by digestion with XhoI and EcoRI. These cassettes were purified as a 1.4 Kbp DNA fragment by agarose gel electrophoresis. This fragment was used as the pBS-IRESbsr E
It was inserted into the coRI and XhoI sites. As a result, three types of plasmids, pBS-intGFP-IRES bsr A, B, and C, were obtained.

(4) Insertion of polyadenylation signal Plasmid pBS-K14 containing polyadenylation signal sequence
The insert (490 bp) from pAEP was digested with EcoT22I and PstI and purified by agarose gel electrophoresis. EcoT
22I has the same structure of the cut surface as the structure of the cut surface of PstI. Therefore, the PstI digested fragment and the EcoT22I digested fragment can be ligated. This DNA fragment was inserted into the three plasmid PstI sites of (3). The resulting plasmid EcoRI was digested with PstI. The plasmid with the correct structure is 1.5k
This generates fragment IRES-bsr-polyA of b. If the fragment is inserted in the PstI site in the opposite direction, a 1.0 kb fragment of IRES-bsr is generated. The one into which the polyadenylation signal was inserted in the correct direction was used as a trap vector having wild-type GFP.

(5) Preparation of IRES-bsr-polyA cassette In order to prepare a trap vector having mutant GFP,
An S-bsr-polyA cassette was prepared. In the same manner as in (4), a polyadenylation signal fragment was prepared, and the plasmid pBS-
It was inserted into the PstI site of IRES bsr. Similarly EcoRI & PstI
Digestion identified candidates for the structure inserted in the forward direction. The completed plasmid is called pBS-IRESbsrpA.

(6) Preparation of dMV-GFP As a 5 ′ primer (145-5′Xho), Using (SEQ ID NO: 12) as a 3 'primer (145-3' Eco) Using (SEQ ID NO: 13), the N-terminal methionine of GFP and
We decided to avoid valine (GTG) and leucine * (CTG), which may have unusual initiation codons, by changing them to leucine (CTC). GFP plasmid (pCMX-SAH / Y145F) by PCR using KOD DNA polymerase
Was digested with EcoRI and XhoI, and inserted into pBSII at the same site.

(7) Re-preparation of intron fragment for binding to dMV-GFP Since the intron of β-globin also has an NcoI cleavage site inside, the intron fragment prepared in (4)
To obtain a DNA fragment cut with the added NcoI to fuse with the initiation codon portion of GFP at the I site, NcoI
Partial digestion with I was required. Since this requires a complicated purification operation, dMV-GFP was used to simplify the operation.
A new intron was also reworked for plate making. 5 'primer (bg-int5'Xho Ver.2) (SEQ ID NO: 14) using the 3′-side primer (bg-int3′Eco
Sal A, B, C) Was used. Combination of three primers (5 '+ 3'A,
Using 5 ′ + 3′B, 5 ′ + 3′C), the amplified DNA fragment obtained by the PCR method using KOD DNA polymerase with the intron-containing plasmid (pSG5) as a template, after digestion with EcoRI and XhoI, It was inserted into the same site of pBSII. The completed plasmid is called pBS-X intSal-A, B, C.

(8) Preparation of Intron-dMV-GFP Cassette An intron fragment was cut out from pBS-X intSal-A, B and C by digestion with XhoI and SalI, and purified by agarose gel electrophoresis. This fragment was converted to Xh of pBS-dMV-GFP plasmid.
Inserted at the oI site. Correctly constructed plasmids are XhoI,
It was confirmed that a 1.4 kb fragment was generated by EcoRI digestion. Insertion of the intron fragment in the reverse direction results in a 0.8 kb fragment. The completed plasmid is called pBS-intdMVGFP-
Call them A, B, C.

(9) The IRES-bsr-polyA cassette of (5) (1.6 k
b) was obtained by digesting pBS-IRESbsrpA with EcoRI and PstI, and then purified by agarose gel electrophoresis. After digesting the three plasmids (8) with EcoRI and PstI, the IRES bsrpA cassette was inserted to complete the plasmid. The completed gene trap vector is XhoI
And, it is obtained as a 3 kb DNA fragment by digestion with PstI. However, the skeleton pBSII
Since these are also the same size, the trap vector portion cannot be purified by agarose gel electrophoresis as it is. In order to solve this problem, they found that Asel was suitable as a restriction enzyme that cuts only the pBSII portion of the backbone and does not cut the trap vector portion. All three plasmids including the gene trap vector completed in this manner were cut with three restriction enzymes XhoI, PstI and Asel, separated by agarose gel electrophoresis, and
The DNA fragment of kb was purified using an adsorption method to an anion exchange membrane. SEQ ID NO: 1
FIG.

Example 2 Introduction of Trap Vector into Cells and Selection of Cells Expressing Green Fluorescent Protein (Establishment of Cell Line) The trap vector obtained in Example 1 was introduced into rat cell PC12ASK1 as follows. . The purified linearized trap vector is dissolved in purified water to a concentration of 1 mg / ml. Two days before transfection, detach the host cells with PBS (-) and remove large clumps of cells by pipetting in the medium. Furthermore, the cells are reciprocated 10 times in a 10-ml syringe equipped with a 21-gauge needle to make the cells almost completely a single cell. Spread it at a concentration of 4 x 10 ⁶ cells / 90 mm diameter dish.

On the day of transfection, the following operations are performed: the medium is removed by suction. 10 ml PBS
Wash with (-) (room temperature). Add 10 ml PBS (-) (room temperature)
Incubate for 10 minutes in a CO ₂ incubator. Cells are removed from the dish by pipetting. Transfer to a 50 ml conical plastic tube. The cells are pelleted by centrifugation at 700 rpm for 2 minutes. The supernatant is removed by aspiration. Add 10 ml of medium to the precipitate. 10 with 21 gauge needle
Pass 5 times through the ml syringe. Count the number of cells. At this time, confirm that cells are 95% or more and single cells, and that live cells are 90% or more. Centrifuge again at 700 rpm, aspirate the supernatant and discard. 5 x cell density
Suspend the cells in PBS (-) to a concentration of 10 ⁶ cells / ml. Keep on ice. Continue to cool in an Eppendorf tube (1.5 ml, all autoclaved) on 0.8 ml of ice for 10 minutes. During this time, 20 ml for the number of transfection
Dispense the medium into a 50 ml conical plastic tube and keep it on ice. At the same time, the cuvette for electroporation is wrapped in wrapping paper and kept in ice to keep it cool.

The setting of Gene Pulser II (BIORAD) was performed under the following conditions. Use 0.4cm gap cuvette; Capacitan
Use ce extender; no parallel resistor connected; 0.4cm gap c
Use uvette; applied voltage 0.25 kV; capacitance 500 μF.

0.8 ml of the mixture of cells and DNA is added to the cuvette, and then an electric pulse is applied once. Quickly transfer the mixture in the cuvette into a 20 ml medium on ice and keep on ice for 30 minutes. During this time, 10 ml of medium is dispensed into five 150 mm dishes (per transfection). Next, add 30 ml of medium and dispense 10 ml into 5 dishes. Place in a CO ₂ incubator and culture at 10% CO ₂ at 37 ° C. Two
After 4 hours, add blasticidin-S to a final concentration of 2.0 μg / ml. Actually, 10 μg / m in medium
Make a 5-fold concentrate containing 1 blasticidin S, and add 5 ml of this solution to each dish. Leave in the CO ₂ incubator for at least 14 days.

The obtained blasticidin S resistant colonies were transferred one by one to a 6-well culture dish, cultured, and observed with a fluorescence microscope equipped with a water immersion objective lens. A cell line showing green fluorescence of GFP was selected and cultured, and used for the following analysis. Of the 24 cell lines that emit green fluorescence (hereinafter referred to as dMV cells), 8 green fluorescence is observed in the whole cell, 11 is observed in the cytoplasm, and 2 is observed in the nucleus. Two things are observed in mitochondria,
One was observed in the nuclear envelope. dMV-8 cells were identified as a cell line that emits fluorescence uniformly throughout. GFP fused to the N-terminal 47 amino acids of a kind of human reticulon rat homolog is expressed. dMV-7 was identified as a cell with fluorescence in the cell nucleus. GFP fused to the N-terminal 196 amino acids of rat ZO-1
Is expressed. dMV-37 was identified as a cell whose fluorescence was observed in the cytoplasm. GFP fused to the N-terminal 51 amino acid region of AnnexinII is expressed. dMV-38 was identified as a cell line that showed fluorescence in mitochondria. GFP fused to the N-terminal 37 amino acid region of ribosomal protein L19 is expressed. 222-17 was identified as a cell line that showed fluorescence in the inner nuclear membrane. GFP fused to the N-terminal 615 amino acid region of Nucleoporin 153 is expressed.

FIG. 2 shows cell lines dMV-8, dMV-7,
It is a microscope photograph of dMV-37, dMV-38, and 222-17. In FIG. 2, a fluorescent image of each cell, a fluorescent image of a DAP1-stained nucleus, and a phase contrast image are shown in order from the left. In cell line dMV-8, GFP is present throughout the cell,
In the case of dMV-7, GFP is present in the nucleus, in the case of dMV-37, GFP is present in the cytoplasm, in the case of dMV-38, GFP is present in the mitochondria, and in the case of 222-17, GFP is present in the nuclear membrane. Cell lines dMV-7, dMV-8, dMV
-37 and dMV-38 are Rat pheochromocytoma PC
12ASK1dMV-7, Ratpheochromocytoma PC12ASK1dMV-8, Ra
t pheochromocytoma PC12ASK1dMV-37 and Rat pheochrom
ocytoma PC12ASK1dMV-38, accession numbers FERM P-17645, FERM P-17646, FERM P-17647 and F, respectively
Deposited as ERM P-17648 with the Research Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

Example 3 Analysis of Cell Line The cell line showing GFP green fluorescence obtained in Example 2 was
Propagated in 10 cm culture dishes, cryopreserved and protein extracted. The extracted protein was analyzed by Western blotting using an anti-GFP antibody. Rabbit anti-GFP
Antibodies are available from Clontech or MBL;
Used at a concentration of 0 dilution. By western blotting analysis, cells expressing a fusion protein having a higher molecular weight than unfused GFP (about 27 kD) were identified. FIG. 3 shows the results of Western blot analysis. The numbers in the figure indicate the serial numbers of the cell lines, and the right end indicates the case of non-fused GFP.

As described in detail below, poly (A) + RNA was purified from those cells, and GFP was purified by the 5'RACE method.
The fusion gene was identified, and the sequence of the cDNA of the protein fused to the green fluorescent protein was determined and identified. Culture the cell line showing GFP fluorescence in a 10 cm dish,
i Using BioMAC μMACS mRNA Isolation Kit,
Poly (A) + RNA was directly purified from 1 × 10 ⁷ cells per cell line. 2-10 μg of Poly (A) + RNA was obtained, and the 5′RACE method was performed using 500 ng of Poly (A) + RNA. The 5'RACE method is based on the 5'RACE System of LIFE TECHNOLOGIES.
m, using a version 2.0 kit. All operations were performed according to the manufacturer's instructions. The sequences of the GFP-specific primers used to synthesize and amplify the cDNA are as follows. OgGFP3: 5'-GAA CTT CAG GGT CAG CTT GCC-3 '(SEQ ID NO: 19) OgGFP5: 5'-TGA ACT TGT GGC CGT TCA CGT CGC CGT CCA
GCT C-3 '(SEQ ID NO: 20) OgGFP7: 5'-GAC CAG GAT GGG CAC CAC CCC GGT GAA CAG
CTC-3 '(SEQ ID NO: 21)

(1) First, cDNA is synthesized by reverse transcriptase using OgGFP3. (2) Next, poly dC is synthesized at the 5 'end of the cDNA using terminal deoxynucleotidyl transferase. (3) After purifying this cDNA, OgGFP5 and 5 'RACE Abridged Anchor Primer 5'-GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG attached to the kit
PCR was performed using -3 ′ (SEQ ID NO: 22). (4) Dilute the PCR product and add OgGFP7 primer and A
bridged Universal Amplification Primer (AUAP)
Nested-PCR was performed using 5′-GGC CAC GCG TCG ACT AGT AC-3 ′ (SEQ ID NO: 23) to obtain the desired product. (5) This PCR product was purified by agarose gel electrophoresis, and the T-vector (pT7Blue-T vecto
r) and introduced into Escherichia coli strain DH5a.
And the nucleotide sequence was determined. Table 1 shows the proteins fused to GFP identified as described above, together with the cells expressing the proteins and the intracellular localization of the fusion proteins.

[0055]

[Table 1] Cell line Fusion protein Intracellular localization of protein fused to GFP dMV-8 Whole cell human reticulon rat homolog dMV-7 nucleus human ZO-1 rat homolog dMV-37 cytoplasm Annexin II dMV-38 mitochondrial ribosomal protein L19 222 -17 Nuclear membrane nucleoporin 153

Example 4 Preparation of GFP Expression Plasmid and Confirmation of Cell Localization Based on the nucleotide sequence of the GFP fusion protein expression plasmid analyzed by the 5′RACE method, a PCR sense primer containing the initiation codon portion of the fusion protein was used. RT with the antisense primer corresponding to the carboxy terminus of GFP
-The PCR product obtained by performing PCR is transferred to the expression vector pCIneo (Prome
(manufactured by ga Co., Ltd.).
Next, the expression plasmid for the GFP fusion protein was introduced into parent PC12ASK1 cells by lipofection using Lipofectamine Plus (Lifetech Oriental). Between 24 hours and 48 hours after transfection, the cells were observed under a fluorescent microscope to confirm cell localization.

[0057]

Although no practical gene trap method using GFP as a reporter has been reported so far, this method has been realized for the first time by this method. The feature of this method is the reporter gene
The fact that GFP can be observed in living cells, which has not been realized by other conventional gene trapping methods. A characteristic feature is that the fusion protein of the trapped gene fragment and GFP can be directly observed, so that the intracellular localization of the trapped gene product can be identified with sufficient accuracy. This was not possible with the conventional method using the β-galactosidase gene as a reporter. According to the method of the present invention, GF
Cell lines exhibiting various subcellular localizations of the P fusion protein are obtained. Since it is relatively easy to analyze which gene is actually fused, a cell line that can be applied to various cell function studies can be created. On the other hand, by using the present GFP gene trap method, it is possible to clarify the intracellular localization of each gene product without preparing a probe specific to the gene product. You can track your location. That is, it can provide an experimental technique complementary to the indirect immunohistochemistry method.
A cDNA fragment having a signal for distributing the fusion protein to various regions in a cell, and an expression plasmid expressing the GFP fusion protein can be obtained. By applying this method, it is possible to develop a technique for labeling specific organelles with living cells.

The present vector is considered to be applicable to a wide variety of animal cells, and the vector DNA itself can be used as a research tool, can be commercialized, and has industrial applicability. In other words, it is an experimental method that does not require special techniques or experimental equipment in the experimental technique and can be performed with a relatively general set of animal cell culture devices, an electroporation device, and a fluorescence microscope. It has the potential to be used. The gene trap experiment using this vector is also a chromosome modification technique, and if applied to embryonic stem cells (ES cells), gene-disrupted animals can be produced. A collection of ES cells that identified the disrupted gene after gene disruption at random was considered a valuable research resource for analyzing the function of the gene at the individual animal level or for producing disease model animals. Has already been commercialized (US Lexicon Genetics Incorporated Om
ni Bank Program).

Since cells produced by this method can observe specific organelles alive, they provide a valuable experimental system for screening drugs that target organelles and inhibit or promote their formation. can do. Therefore, the cells of the present invention also have industrial applicability.

[0060]

[Sequence List] Sequence Listing <110> Osaka Bioscience Institute <120> Gene trap using green fluorescence protein <130> 167063 <160> 23

<210> 1 <211> 260 <212> PRT <213> Aguorea victoria <400> Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu 5 10 15 Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly 20 25 30 Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr 35 40 45 Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Ala 50 55 60 Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His 65 70 75 80 Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr 85 90 95 Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys 100 105 110 Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp 115 120 125 Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe 130 135 140 Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile 145 150 160 165 Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln 170 175 180 Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val 185 190 200 Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys 205 210 215 Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr 220 225 230 Ala Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys Ser Gly Gly 235 240 245 250 Ser Thr Ile Ser Gly Ser Trp Pro Ala Ser 255 260

<210> 2 <211> 27 <212> DNA <213> Artificial Sequence <400> actttggcag ataagcttct aggtaac 27

<210> 3 <211> 27 <212> DNA <213> Artificial Sequence <400> tttgaattcc tagctagctg gccagga 27

<210> 4 <211> 36 <212> DNA <213> Artificial Sequence <400> tttgaattcc ccatggtgaa aacatttaac atttct 36

<210> 5 <211> 39 <212> DNA <213> Artificial Sequence <400> tttctgcagc atatggtaaa acttttaatt tcgggtata 39

<210> 6 <211> 36 <212> DNA <213> Artificial Sequence <400> tttgaattca tgcatcctag gaggcccccc gtgtgg 36

<210> 7 <211> 30 <212> DNA <213> Artificial Sequence <400> tttctgcaga ttctccaagg aatccctgat 30

<210> 8 <211> 27 <212> DNA <213> Artificial Sequence <400> tttctcgagc tttctttttc gctattg 27

<210> 9 <211> 31 <212> DNA <213> Artificial Sequence <400> gaattcccat ggaccaaaat gatgagacag c 31

<210> 10 <211> 35 <212> DNA <213> Artificial Sequence <400> gatgaattcc catggaacca aaatgatgag acagc 35

<210> 11 <211> 36 <212> DNA <213> Artificial Sequence <400> gatgaattc ccatggaaacc aaaatgatga gacagc 36

<210> 12 <211> 48 <212> DNA <213> Artificial Sequence <400> aagaagctcg agagcaaggg cgaggagctc ttcaccgggg tggtgccc 48

<210> 13 <211> 27 <212> DNA <213> Artificial Sequence <400> tttgaattcc tagctagctg gccagga 27

<210> 14 <211> 30 <212> DNA <213> Artificial Sequence <400> tttctcgagc tttcttittt ttcgctattg 30

<210> 15 <211> 34 <212> DNA <213> Artificial Sequence <400> gatgaattcg tcgacaccaa aatgatgaga cagc 34

<210> 16 <211> 35 <212> DNA <213> Artificial Sequence <400> gatgaattcg tcgacaacca aaatgatgag acagc 35

<210> 17 <211> 36 <212> DNA <213> Artificial Sequence <400> gatgaattcg tcgacaaacc aaaatgatga gacagc 36

[0078] <210> 18 <211> 2917 <212> DNA <213> Artificial Sequence <400> gtgagtttgg ggacccttga ttgttctttc tttttcgcta ttgtaaaatt catgttatat 60 ggagggggca aagttttcag ggtgttgttt agaatgggaa gatgtccctt gtatcaccat 120 ggaccctcat gataattttg tttctttcac tttctactct gttgacaacc attgtctcct 180 cttattttct tttcattttc tgtaactttt tcgttaaact ttagcttgca tttgtaacga 240 atttttaaat tcacttttgt ttatttgtca gattgtaagt actttctcta atcacttttt 300 tttcaaggca atcagggtat attatattgt acttcagcac agttttagag aacaattgtt 360 ataattaaat gataaggtag aatatttctg catataaatt ctggctggcg tggaaatatt 420 cttattggta gaaacaacta caccctggtc atcatcctgc ctttctcttt atggttacaa 480 tgatatacac tgtttgagat gaggataaaa tactctgagt ccaaaccggg cccctctgct 540 aaccatgttc atgccttctt ctctttccta cagctcctgg gcaacgtgct gcaacgtgct 600 ggttgttgtg ctgtctcatc attttccatg gtgagcaagg gcgaggagct gttcaccggg 660 gtggtgccca tcctggtcga gctggacggc gacgtgaacg gccacaagtt cagcgtgtcc 720 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 780 ggcaagctgc ccgtgccctg gcccacacta gtgaccacct tcgcttacgg cgtgcagtgc 840 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 900 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 960 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 1020 aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacgta 1080 tacatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 1140 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 1200 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 1260 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 1320 cacggcatgg acgagctgta caagtcaggc gggtcgacga tatcaggatc ctggccagct 1380 agctaggaat tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg 1440 gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc 1500 aatgtgaggg ggcccccccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg 1560 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttct 1620 ctggaagctt cttgaaga ca aacaacgtct gtagcgaccc tttgcaggca gcgggaaccc 1680 cccacctggc gacaggggtt gccctctgcg gccaaaagcc acgtgtataa gatacacctg 1740 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat 1800 gggctctcct caagcgtatt caacaagggg ctgaaggatt ttgcccagaa agggtaaccc 1860 cattgtatgg gatctgatct ggggcctcgg tgcacatgct tttacatgtg tttagtcgag 1920 gttaaaaaac gtctaggccc cccccgaacc acggggacgt ggttttcctt tgaaaaacac 1980 gatgataagc ttRgccacaa ccatgaaaac atttaacatt tctcaacaag atctagaatt 2040 agtagaagta gcgacagaga agattacaat gctttatgag gataataaac atcatgtggg 2100 agcggcaatt cgtacgaaaa caggagaaat catttcggca gtacatattg aagcgtatat 2160 aggacgagta actgtttgtg cagaagccat tgcgattggt agtgcagttt cgaatggaca 2220 aaaggatttt gacacgattg tagctgttag acacccttat tctgacgaag tagatagaag 2280 tattcgagtg gtaagtcctt gtggtatgtg tagggagttg atttcagact atgcaccaga 2340 ttgttttgtg ttaatagaaa tgaatggcaa gttagtcaaa actacgattg aagaactcat 2400 tccactcaaa tatacccgaa attaacctag gaggcccccc gtgtggacac agatcccact 2460 ggaagatccc ctctcctgcc caa gcacttc acagctggac cctgcttcac cctcaccccc 2520 tcctggcaat caatacagct tcattatctg agttgcataa ttctcgcctc tctctggtca 2580 ttgttaggag tgggggtggg gagaaagtgg gagagcatct ctttggagct tgtcatgcac 2640 ctggctatgg cccctgggac tgggagaaaa gtcctggggg tgggttgggc tcaggtccca 2700 ggatatcttt cgccatctca gaagacacag atagatgtgt gtaccaggtc atatgtggtg 2760 tctcctaggg tacggaggga tattcattca tttactcact cattttcatg tgtgtccatt 2820 cattcaccag atattgagtg cctctatgtc aggcactatg ttaggttaag gattcctgat 2880 gtttttgtgt atcagggatt ccttggagaa tctgcag 2917

<210> 19 <211> 21 <212> DNA <213> Artificial Sequence <400> gaacttcagg gtcagcttgc c 21

<210> 20 <211> 34 <212> DNA <213> Artificial Sequence <400> tgaacttgtg gccgttcacg tcgccgtcca gctc 34

<210> 21 <211> 33 <212> DNA <213> Artificial Sequence <400> gaccaggatg ggcaccaccc cggtgaacag ctc 33

<210> 22 <211> 36 <212> DNA <213> Artificial Sequence <400> ggccacgcgt cgactagtac gggiigggii gggiig 36

<210> 23 <211> 20 <212> DNA <213> Artificial Sequence <400> ggccacgcgt cgactagtac 20

[Brief description of the drawings]

FIG. 1 shows the outline of the method of the present invention.

FIG. 2 shows an example of a cell line showing intracellular localization. From left: GFP fluorescence image, nucleus fluorescence image by DAPI staining, phase contrast image

FIG. 3 shows an example of Western blot analysis of a protein extracted from a trap cell line.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) C07K 19/00 C12N 5/00 BF term (reference) 2G045 AA35 BB20 CB01 CB17 DA12 DA13 DA14 FB07 FB12 GC15 4B024 AA11 BA80 CA07 DA02 EA04 GA11 HA20 4B063 QA01 QA08 QQ08 QQ42 QR60 QR80 QS38 QX02 4B065 AA90X AA90Y AB01 AC14 BA02 CA24 CA46 4H045 AA10 BA10 CA40 EA50 FA72 FA74

Claims (9)

[Claims] 1. In order from the 5 ′ end to the 3 ′ end, (1) an intron and a splice acceptor sequence; (2) an N-terminal methionine and valine-deleted green fluorescent protein (GFP) A trap vector comprising: (3) an internal ribosome binding site (IRES); (4) a DNA sequence encoding a selectable marker; and (5) a polyadenylation signal sequence. 2. Transformation by introducing (1) the trap vector of claim 1 into mammalian cells, (2) culturing the cells in a solid medium to obtain colonies, and (3) each colony. Selecting a cell expressing the green fluorescent protein from the obtained cells, and (4) identifying a protein endogenous to the cell fused to the green fluorescent protein, including: a protein endogenous to the cell fused to the green fluorescent protein A method for producing an animal cell expressing the same. 3. An animal cell containing a protein endogenous to a cell fused to a green fluorescent protein. 4. The cell according to claim 3, wherein the endogenous protein is localized in the cell. 5. The cell according to claim 3, wherein the endogenous protein is localized in a cell nucleus. 6. The cell according to claim 3, wherein the endogenous protein is located in the cytoplasm. 7. The cell according to claim 3, wherein the endogenous protein is localized in mitochondria. 8. The cell according to claim 3, wherein the endogenous protein is localized in a nuclear membrane. 9. A method for observing the dynamic localization of an endogenous protein fused to a green fluorescent protein in cells by observing the fluorescence emitted by the green fluorescent protein.