TW201248152A - Dual-fluorescent MSI reporter plasmid and cell model harboring this MSI reporter plasmid - Google Patents

Abstract

A dual-fluorescent MSI reporter plasmid and a cell model that harbors this MSI reporter are disclosed. From the promoter the dual-fluorescent MSI reporter plasmid of the present invention, the plasmid sequentially comprises a microsatellite sequence (or a random sequence as a control), a first fluorescent gene, an internal ribosome entry site, and a second fluorescent gene. In the cell model that harbors the MSI reporter plasmid, the cells emitting the second fluorescent protein is used for normalizing the cells emitting the first fluorescent protein achieving an accurate measurement of the MSI frequency.

Description

201248152 VI. Description of the Invention: [Technical Field] The present invention relates to a dual-fluorescence microsatellite reporter plastid and a cell model containing the microsatellite unstable reporter plastid, especially an effective screening for causing or inhibiting micro The dual-fluorescence microsatellite of satellite instability-related drugs reports plastid and cell models. [Prior Art] Cancer is a complex genetic disease in which 25% is from chronic inflammatory patients. In terms of clinical treatment of cancer patients, in addition to surgery, it is necessary to use chemotherapy to achieve the best therapeutic effect. β Studies have indicated that chronic inflammatory patients, cancer patients, and cancer patients after chemotherapy all have microsatellites. Stability (microsatellite instability, MSI). However, patients with microsatellite instability are not only more likely to have a mutation rate, but are also prone to resistance to chemotherapy. Microsatellite instability occurs mainly because the function of the DNA mismatch repair (MMR) does not function properly, and thus it is impossible to repair the errors often caused by microsatellites during DNA replication. For example, if the repeating unit of the microsatellite in the coding region is changed, the frame shift is abruptly changed. If the repeating unit of the microsatellite on the intron changes, it is easy to cause genetic translation or transcription disorder.

MMR also recognizes and repairs 8_oxo-guanine DNA (8_〇x〇 guanine DNA) caused by reactive 〇xygen species (ROS). In addition to H202 being one of the major factors responsible for the production of R〇s, it is known that 5FU 201248152 and cisplatin anticancer drugs also produce R〇S, which can also cause in-frame sequence shift mutations and microsatellite instability. The genotoxic stress produced by drugs can damage DNA and cause apoptosis in cancer cells. However, if the MMR function of the fine cells is lost, it is impossible to go to apoptosis and thus develop drug resistance. Therefore, if a microsatellite instability reporting system that can be quickly and highly sensitive can be developed, an anticancer drug that is prone to microsatellite instability can be found. Furthermore, this reporting system can be further used to screen foods in large quantities, and to find foods that inhibit the instability of microsatellites caused by ROS or anticancer drugs. The anti-ROS-induced microsatellite instability of foods is suitable for patients with chronic inflammation, thereby reducing the incidence of cancer. In addition, the use of anti-cancer drugs to prevent the microsatellite instability of food and anti-cancer drugs, will inevitably increase the success rate of cancer treatment, that is, reduce the death rate of cancer 〇 [Summary]

Cell-type derived cell line.

A factor or compound associated with instability.

Unstable dual-fluorescence microsatellite reporter plastids for use in reporting microsatellites from the promoter region of the dual-fluorescence microsatellite reporter 201248152, including: microsatellite sequence, first fluorescence The protein sequence, the internal ribosome insertion site sequence, and the second fluorescent protein sequence, and the fluorescent color of the first fluorescent protein and the second light-emitting protein are different. Among them, the reading sequence of the first fluorescent protein is shifted out of the reading frame, that is, the instability of the microsatellite. Furthermore, the present invention also provides a cell model comprising the above-described dual fluorescent microsatellite reporter. The cell model system comprises: a double-fluorescence microsatellite reporter plastid as described above, and a cell strain, wherein the dual-fluorescence microsatellite reporter system is transferred to the cell strain, and the cell line exhibits the double fluorescence The microsatellite reports the first fluorescent protein sequence in the plastid and the second fluorescent protein sequence. Therefore, the cell model of the present invention can express a double-fluorescent fluorescent protein (ie, a first fluorescent protein and a second fluorescent protein) as described above for the microsatellite, wherein the second fluorescent protein is used as a certain amount. protein. When the fluorescent light emitted by the first fluorescent protein is detected, it indicates that the reading frame sequence shift (ie, the microsatellite instability) occurs; and when the fluorescent light emitted by the first fluorescent protein cannot be detected, Indicates the occurrence of a frameless sequence shift (ie, 'microsatellite stabilization'). In addition, the second fluorescent protein can be used as a quantity of protein to normalize the number of cells that have the first fluorescent protein, and to improve the accuracy of microsatellite instability reporting. u in the double-fluorescent microsatellite reporter plastid provided by the present invention and the cell model containing the double-fluorescent microsatellite reporter plastid, the frame sequence of the first fluorescent protein is designed to be shifted to the position, ie, reading Outside the box (〇ut_〇f frame). Therefore, when there is no reading frame sequence displacement, the first fluorescent protein is not expressed. When oxidative stress or anticancer drugs cause microsatellite instability, the reading frame sequence I shifts 6 201248152 to in-frame to make the first fluorescent protein behave, so the first fluorescent protein can be observed. Fluorescent light emitted. In addition, the internal ribosome entry site (IRES) allows the second fluorescent protein to be unaffected by the first fluorescent protein, and can continue to express the second fluorescent protein for cell counting. use. In the dual-fluorescence microsatellite reporter plastid of the present invention and the cell model containing the double-fluorescent microsatellite reporter plastid, the 'microsatellite sequence system includes one (匸8^ sequence) and η may be 5-40. The 'η system is 10-16 in the (CA)n sequence included in the microsatellite sequence. More preferably, it is included in the (CA)n sequence included in the microsatellite sequence, and the η system is 13. In addition, The cell model containing the double-fluorescent microsatellite reporter plastid may further comprise a control group plastid, and the control group plastid is also transferred into the cell strain to obtain a human cell model containing the plastid of the control finger. Starting from the promoter region of the control plastid, the sequence includes: a control group sequence, a first Lloyd protein sequence, an internal ribosome insertion site sequence, and a second fluorescent protein sequence, wherein the first fluorescent protein The color of the fluorescent light emitted by the second fluorescent protein is different, and the second fluorescent protein is used as a certain amount of protein. Since the control group does not include a microsatellite sequence, it is not susceptible to drug or external oxidative stress. Producing microsatellite instability. Therefore, when the present invention contains When the fluorescent microsatellite report plastid cell model uses a cell line transfected with a double-fluorescence microsatellite to report plastids and a cell line transfected with a control plastid, the control group plastid can be used as a comparative control group. Therefore, the difference in the influence of the analyte on the fluoroplasts of the dual-fluorescence microsatellite and the control group can be clearly observed, and the accuracy of the cell model containing the dual-fluorescence microsatellite to report the plastid is further improved. t, the control group sequence comprises a (N)n sequence, each n line is independently selected from the group consisting of A, T, C, G, and n is 20-80. Preferably, in control In the sequence of (Ν)η included in the sequence of the group, the η system is preferably 2〇_3〇β, which is included in the (Ν)η sequence included in the control group sequence, and the η system is 16. In the present invention In the fluorescent microsatellite reporter plastid and the cell model containing the dual-fluorescence microsatellite reporter plastid, the first fluorescent protein may be a red light camping light protein' and the first fluorescent protein is preferably RFP. In addition, the second fluorescent protein may be a green fluorescent luminescent protein, and the second fluorescent protein is preferably GFP 〇 In the cell model containing the double-fluorescent microsatellite reporter plastid of the present invention, the cell strain may be a human cell strain. Preferably, the cell strain is a human cell model of the -dna mismatch repair deletion (MMR_deficient human eeli model) And the dual-fluorescence microsatellite reporter system is transferred to the human cell model in which the DNA mismatch repair is deleted. The human cell model in which the DNA mismatch repair is deleted can repair the missing cells for any known DNA mismatch, and Preferably, the cell having the DNA mismatch repair deletion is, for example, the human colon cancer cell line HCT116. [Embodiment] Hereinafter, embodiments of the present invention will be described by way of specific embodiments, and those skilled in the art can be disclosed by the present specification. The contents are readily understood to provide additional advantages and benefits of the present invention. The present invention may be implemented or applied by other specific embodiments of the present invention. The details of the present specification can be applied to various viewpoints and applications, and various modifications and changes can be made without departing from the spirit of the present invention. Construction of dual-fluorescence microsatellite reporter plastids First, DsRed cDNA (which can express RFP red fluorescent protein, the sequence is shown in SEQ ID NO: 5) is extracted from pDsRedl-Nl plastid (Clontech), and SacII and Notl are used. The DsRed cDNA was inserted into pIRES-hrGFP-la plastid (Stratagene) to prepare a pRFP-IRES-GFP vector, wherein the sequence of GFP is shown in SEQ ID NO: 6, and the sequence of IRES is as SEQ ID NO: 7 is shown. In order to confirm whether the pRFP-IRES-GFP vector constructs a work, in addition to the use of restriction enzymes (such as SacII, NotI), the RFP primer was used as a sequencing primer (SEQ ID NO: 3, SEQ ID NO: 4). DNA gene sequencing. Then, a microsatellite sequence ATG_(CA)I3 (SEQ ID NO: 1) or a control group sequence ATG-(N)16 (SEQ ID NO: 2) is inserted into the SacI and Agel cleavage sites. The upstream region of the DsRed promoter of the pRFP-IRES-GFP vector described above, and the reading frame of the RFP (i.e., DsRed) was shifted to the -1 position. Next, through Cre-mediated recombination, B. j. can be inserted into the hygromycin resistance gene of pExchange Module EC-Hyg (Stratagene) to construct a pair of fluorescent microsatellite reporters. The body p(CA)l3RFP-IRES-GFP-Hyg, and a control group plastid p(N)16RFP-IRES-GFP-Hyg. The dual-fluorescence microsatellite report of the plastid p(CA)i3RFP-IRES-GFP-Hyg is shown in Fig. 1A, Fig. 1B, 201248152, and the control group plastid p(N)丨6RFP-IRES-GFP-Hyg The schematic diagram is shown in Figures 2A and 2B. 1A and FIG. 2A are schematic diagrams of the dual-fluorescence microsatellite report plastid and control group plastid; and FIG. 1B shows the microsatellite sequence region of the double-fluorescence microsatellite report plastid, and FIG. 2B shows the double Fluorescence control group plastid control group sequence region" Also, restriction enzyme and DNA gene sequencing (sequence primers as shown in SEQ ID NO: 3, SEQ ID NO: 4) were also used to confirm the above double fluorescence Microsatellite reports whether the plastid and control group plastid construction is successful. Then, the pRFP-IRES-GFP vector, the dual-fluorescence microsatellite reporter plastid, and the control group plastid constructed above were transfected into human colon cancer cell line HCT116 (American Type Culture Collection), or HCT116+chr3. In order to express the above-mentioned performance plastids. Among them, the HCT116 cell line has a homozygous mutation, so it is a DNA mismatch repairing cell (MMR-deficient cells); and the HCT116+chr3 cell line has a band The chromosome 3 of the normal gene is a DNA mismatch repairing cell (M'MR-proficient cells). In addition, culture of HCT116 cell line was carried out in DMEM/F-12 medium containing 10% fetal bovine serum and 2 mM L-glutamine at 5% CO 2 and 37 ° C. Culture; the culture line of HCT116+chr3 cell line was similar to HCT116 cell line except that 400 pg/ml of G418 was added to the medium. Here, how to construct the pRFP-IRES-GFP vector, the dual-fluorescence microsatellite reporter plastid, and the control group plastid constructed above can be transfected into the HCT116 cell line. First, 6M 05 cells were planted in each well in a six-well plate; after 16 hours of planting the cells in 201248152, the liposome transfection reagent (Lip〇fectamine 20〇〇τΜ) was used, and the pRFP-IRES-GFP vector, double Fluorescent microsatellite reported plastid and control group plastid transfection to HCTU6 blister. Here, the transfected Shuangying PRFIMRES-GFP vector was transfected with Hctu6 cell line called HCTU6-V, and the HCTU6 cell line transfected with Shuangyingguang microsatellite reported plastid was called HCT116-(CA). n, and the HCT116 cell line transfected with the double-fluorescence control group plastid is called HCT116-(N)16. Two days after the transfection reaction, HCT116_(CA)n and HCTlie-CN) were transfected with 200 pg/ml of antihygromycin; 6 transfected cell lines and sequenced using dna gene (sequence primer such as SEQ ID NO) : 3, SEQ ID NO: 4) Confirm whether the transfection is successful. Further, the transfected cell line obtained by transfecting the double-fluorescence microsatellite reporter plastid into the HCT116+chr3 cell line' in the same manner as above was referred to as HCT116+chr3_(CA)13. The reading frame sequence was judged by high-throughput fluorescence microscopy. The cells were fixed with 4% paraformaldehyde and the nuclei were stained with 1 pg/ml of Hochest 33258 reagent. Here, the image was detected using an ImageXpressMicro system (Molecular Devices) at 1 Ox magnification. When using the DAPI filter set, the exposure is 35 ms; when using the FITC filter set, the exposure is 175 ms; and when using the TexRed filter set, the exposure is 1500 ms to obtain the fluorescent image. The resulting fluorescent image was then analyzed using MetaXpress® V3.1 software (Molecular Devices). When green fluorescein emitted by GFP fluorescent protein is detected, it means that cells can be detected; and when green fluorescent light emitted by GFP fluorescent protein and red fluorescent light emitted by RFP fluorescent protein are simultaneously detected , which indicates that the original double-fluorescence microsatellite reported that there was a shift in the reading frame sequence in the plastid or control group of the 201248152, and the RFP fluorescent protein was again exhibited. Therefore, when the above-mentioned HCT116-V, HCT116-(CA)13 and HCT116-(N)16 cell lines were observed using a fluorescence microscope, only HCT116-(CA)i3 and HCT116-(N)16 cell lines were present. The RFP design has a reading frame sequence shift, so red fluorescence can only be detected in the HCT116-V cell line. On the contrary, as shown in FIG. 1C and FIG. 2C, the HCT116-(CA)n and HCT116-(N)16 cell lines were not observed by a fluorescence microscope, and only the green fluorescence emitted by GFP was observed. Red fluorescent light emitted by the RFP. Experimental Example 1 - Evaluation of H2〇2 on cell viability and reading frame sequence shift HCT116 and HCT116+chr3 cell lines were seeded in 96-well plates at 1 Χίο4 cells per well. After one day of planting, η2〇2 The cells were treated for 1 hour, and the reaction medium contained H202 with final concentrations of 〇, 〇·25, 0.5, 0.75, 1.0 mM, and 1 PBS buffer solution. Then, the cell survival rate was analyzed by the MTT assay. After treating cells with h2〇2, add 25 μM of 5 mg/ml MTT to 100 μM of cell-containing medium in each well. After 4 hours of reaction, add sputum lysate to each well. Buffer) 'Through the overnight reaction. Finally, the absorbance of the cells was determined by measuring the absorbance at an absorbance of 595 nm using an enzyme immunoassay analyzer. The relative survival rate is determined by taking the absorbance of the untreated cell line as 100%, and calculating the relative absorbance of the treated cell strain relative to the untreated cell strain, and the results are shown in FIG. 3A (in the figure) Shows the mean soil standard deviation). 12 201248152 As shown in the results of Fig. 3A, h202 of the above dose is the cell non-lethal dose' and the above dose is used to evaluate whether H2〇2 produces a shift in the reading frame sequence, resulting in microsatellite instability. For the evaluation of the displacement of the reading frame sequence, the above HCT116-V, HCT116-(CA)13, HCT116-(N)16, and HCT116+chr3-(CA)1:^ cells were firstly 1 x 1 〇5 per well. The cells were planted in 12-well plates. One day after planting, the cells were treated with the above concentration of Η202 solution for 1 hour; then Η2〇2 was removed and the cells were washed and placed in the medium for further growth for three days. The flow cytometry is then used to analyze the displacement of the reading sequence in the cell. First, the cells were treated with trypsin and resuspended in PBS buffer containing 1 mM EDTA; then transitioned with a 40 μη transition and then flow cytometry (QuantaTM sc_mpL, Beckman Coulter) for analysis. . At the same time, HCT116 cells containing pIRES-hrGFP-la (GFP single fluorescent cells), pDsRedl-Nl (RFP single fluorescent cells) and pRFP-IRES-GFP (GFP/RFP double fluorescent cells) were used as The calibration was analyzed, and HCT116 cells that were not transfected with any vector or plastid were used as background values. Here, the readout sequence displacement rate is the relative value of the number of cells expressing Gfp/rfp double light to the number of cells expressing G F P single fluorescence in the same cell sample. The result of the displacement rate of the reading frame sequence is shown in Fig. 3B (the average soil standard deviation is also shown in the figure), where * represents the p<〇.〇2 of the student t-test. As shown in Fig. 3B, as the dose of the sputum 2 increases, the displacement rate of the reading frame sequence also increases; and the ΗΟΓΠΙδ-γΑ)^ containing the microsatellite sequence has a displacement rate of the reading frame sequence compared with the control group HCTIMJN),6 high. Prove that the dual-fluorescence system 201248152 faithfully reports the instability of the microsatellite caused by H2〇2 even in the absence of DNA mismatch repair in HCT116 cells. In addition, in the HCT116+chr3-(CA)13 cell line, almost no reading frame sequence shift was detected, because HCT116+chr3 cells were DNA mismatch repairing healthy cells and repairing the frame sequence caused by H202. Displacement, which reduces the instability of microsatellites. Therefore, it is proved that the dual-fluorescence microsatellite instability reporting system is more sensitive in DNA mismatch repair of damaged cells than in DNA mismatch repairing healthy cells. In addition, if high-flux fluorescence microscopy system is used to analyze cell firefly In the light image, the dose of H2〇2 from 0 (no treatment) to 0.5 mM was observed, and the expression of RFP red fluorescent protein increased from 1.1% to 3.6%, indicating that H202 increased the displacement rate of the reading frame sequence. 4A is shown. This result is consistent with the results of the flow cytometry analysis (as shown in Figure 4B). It has also been demonstrated that this dual-fluorescence microsatellite instability reporting system enables rapid and large-scale screening of drugs through high-throughput fluorescence microscopy systems. Experimental Example 2 - Evaluation of the effect of methotrexate (MTX) anticancer drugs on cell viability and reading sequence shifts The above HCT116-(CA)I3& HCT116-(N)16 cell lines were seeded at 96 wells at 1 x 104 cells per well. In the plate. One day after planting, cells were treated with methotrexate (MTX, purchased from Sigma-Adrich) for three days, and the reaction medium contained a final concentration of 〇, 5, 10, 25, 50, 100 nM, And 2.5 η Μ NaOH. Then, cell viability was analyzed using the same MTT assay as in Experimental Example 1, and the results are shown in Fig. 5A (also shown as mean standard deviation in 201248152), which shows that the non-lethal dose of MTX cells is about 25 η Μ. For the evaluation of the displacement of the reading frame sequence, the above HCT116-(CA)13 and HCT116-(N)16 cell lines were first planted in a 12-well plate at ΙχΙΟ5 cells per well. One day after planting, the cells were treated with MTX at final concentrations of 0, 6.3, 12.5, and 25 η for three days; then the sputum was removed and the cells were washed and placed in the medium for further three days. Next, using the same flow cytometer as in Example 1, the displacement of the reading frame sequence in the cells was analyzed, and the results are shown in Fig. 5A (the average soil standard deviation is also shown in the figure), wherein *** indicates the student t- Test ρ < 0.0001. As shown in Fig. 5A, the HCT116-(CA)13 containing the microsatellite sequence has a higher reading rate of the reading frame than the control group HCT116-(N)16; and especially when the MTX dose is 25 ηΜ, the reading frame sequence shift The frequency produced is more pronounced. Experimental Example 3 - Evaluation of the faithfulness of the dual-fluorescence microsatellite instability reporting system In addition, the HCT116-(CA)13 cell line was treated with MTX at a final concentration of 25 η 三 for three days, then MTX was removed and the cells were washed, and It was then placed in the medium and continued to grow for three days. After the above steps were cycled four times, about 80% of the double-fluorescent cells were collected, and the fluorescence images of the cells were analyzed by flow cytometry and high-throughput fluorescence microscopy. The results are shown in Figures 6A and 6B. Shown. The same cells were subjected to PCR amplification of the DNA in HCT116-(CA)13, followed by DNA gene sequencing (sequence primers as shown in SEQ ID NO: 3, SEQ ID NO: 4). The results of DNA gene sequencing showed that the cell line treated by MTX had a group of CA repeats of 201248152 units in the microsatellite sequence than the cell line without MTX treatment, that is, the sequence result was (CA) I2, as shown in Fig. 6C. Show. The upper panel of Figure 6C shows the sequencing results of the cell lines without MTX treatment, while the bottom panel of Figure 6C shows the sequencing results of the cell lines treated with four times of 25 nM MTX. Due to the absence of a set of CA repeating units, the RFP reading sequence can be shifted from outside the reading frame to the reading frame. According to this, the RFP protein can be expressed again, so that the red fluorescence emitted by the RFP fluorescent protein can be observed, as shown in Fig. 6B. Internationally, five microsatellites in the genome are currently used to test whether patients with colorectal cancer have microsatellite instability. Therefore, the above MTX treatment and the untreated cell DNA were amplified by five pairs of fluorescent PCR primers for the five microsatellites for the detection. The analysis showed that MTX increased the six sequences in the D17S250 microsatellite (as shown in Figure 6D). Since the sequence of the D17S250 microsatellite contains (TA)7/(CA)24, the MTX substantially adds three sets of repeating units of TA or CA. The above experimental results demonstrate that the dual-fluorescence system can faithfully report that MTX anticancer drugs cause microsatellite instability of exogenous (CA) 13 and endogenous D17S250. In addition, since the dual-fluorescence reporter system of the present invention does not require separation of DNA and PCR, it is possible to detect each cell in a timely and sensitive manner whether or not microsatellites are unstable. The above-described embodiments are merely examples for the convenience of the description, and the scope of the claims is intended to be limited by the scope of the claims. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A is a schematic diagram of a double-fluorescent microsatellite reporter of the present invention. 201248152 FIG. 1B is a schematic diagram of a double-fluorescent reporter plastid containing ((:) 13 microsatellite sequence of the present invention. FIG. 1C is a HCT116 cell line containing a double-fluorescence microsatellite reporter of the present invention under a fluorescence microscope. Figure 2A is a schematic diagram of the plastid body of the dual-fluorescence control group of the present invention. Figure 2B is a schematic diagram of the plastid of the double-fluorescence control group of the present invention. Fig. 3 is a graph showing the results of cell viability of the H2116 cell line of the fluorescent control group plastid under the fluorescence microscope. Fig. 3 is a graph showing the results of cell viability of the experiment 1 of the present invention treated with h2 〇 2. Fig. 3B is an experimental example 1 of the present invention. The results of the displacement rate of the reading frame sequence were analyzed by flow cytometry on the cells treated with h2〇2. Fig. 4A is a high-throughput firefly of the untreated or treated 〇5 mM H2〇2 cells in Experimental Example 1 of the present invention. The light microscopy system analyzes the sequence shift of the frame to result in the expression of RFP luciferin. Figure 4B shows the displacement rate of the reading frame sequence by flow cytometry of the cells treated with 〇.5 mM H2〇2 in Experimental Example 1 of the present invention. Figure 5A is an experimental example 2 of the present invention via methotrexate (MTX) Fig. 5B is a graph showing the results of analyzing the displacement rate of the reading frame sequence by the flow cytometry of the MTX-treated cells of the experimental example 2 of the present invention. Fig. 6A is a total of 25 nM of MTX of the experimental example 3 of the present invention. The processed cells were analyzed by flow cytometry for the results of the displacement rate of the reading frame sequence. 201248152 Fig. 6B is the experimental example 3 of the present invention. A total of four 25 nM MTX-treated cells were analyzed by high-throughput fluorescence microscopy system for frame sequence displacement. Figure 6C shows the results of DNA sequencing of cells in the experimental example 3 of the present invention which were not sputum-treated or subjected to a total of four 25 Μ MTX treatments. Figure 6D is an experimental example 3 of the present invention. A total of four 25nM MTX-treated cells were amplified by a fluorescence PCR primer to amplify five microsatellites for international testing. The results showed that MTX was increased by six in the D17S250 microsatellite containing (TA)7/(CA)24. Sequences, ie, three groups of repeating units. [Main component symbol description] 201248152 Sequence Listing <110〉National Success University/National Cheng Kung University <120>Double-fluorescence microsatellite report plastids and including this microsatellite instability Reporting plastid The cell model /Dual-fluorescent MSI reporter plasmid and cell model harboring this MSI reporter plasmid <130> 100-053BP_S4254 <160> 4 <170> Patentln version 3.3 <210> 1 <211> 65 <212> DNA <213> Artificial <220><223> Artificail primer for cloning <400> 1 ctggagctca tgcacacaca cacacacaca cacacacagt acgcgtaccg gtcgccacca 60 tggtg 65 <210> 2 <211> 47 <212> DNA <213 〉 Artificial <220〉 <223> Articifial primer for cloning <400> 2 ctggagctca tggatatcat tactagtaac cggtcgccac catggtg 47 <210> 3 19 201248152 <211> 21 <212> DNA <213> Artificial <220&gt <223> PCR primer for sequencing <400> 3 gttttggcag tacatcaatgg 21 <210> 4 <211> 20 <212> DNA <213> Artificial <220><223> PCR primer for sequencing <;400> 4 gtccttatca tcgtcgtctt 20

≪ 210 > 5 < 211 > 681 < 212 > DNA < 213 > Plasmid DsRedl-Nl < 400 > 5 atggtgcgct cctccaagaa cgtcatcaag gagttcatgc gcttcaaggt gcgcatggag 60 ggcaccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120 ggccacaaca ccgtgaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180 atcctgtccc cccagttcca gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc 240 cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg ctgcttcatc 360 tacaaggtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat gcagaagaag 420 20 201248152 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480 gagatccaca aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagtcc 540 atctacatgg ccaagaagcc cgtgcagctg cccggctact actacgtgga ctccaagctg 600 Gacatcacct cccacaacga ggactacacc atcgtggagc agtacgagcg caccgagggc 660 cgccaccacc tgttcctgta g 681

≪ 210> 6 < 211 > 723 < 212 > DNA < 213 > Plasmid IRES-hrGFP-la < 400> 6 atggtgagca agcagatcct gaagaacacc ggcctgcagg agatcatgag cttcaaggtg 60 aacctggagg gcgtggtgaa caaccacgtg ttcaccatgg agggctgcgg caagggcaac 120 atcctgttcg gcaaccagct ggtgcagatc cgcgtgacca agggcgcccc cctgcccttc 180 gccttcgaca tcctgagccc cgccttccag tacggcaacc gcaccttcac caagtacccc 240 gaggacatca gcgacttctt catccagagc ttccccgccg gcttcgtgta cgagcgcacc 300 ctgcgctacg aggacggcgg cctggtggag atccgcagcg acatcaacct gatcgaggag 360 atgttcgtgt accgcgtgga gtacaagggc cgcaacttcc ccaacgacgg ccccgtgatg 420 aagaagacca tcaccggcct gcagcccagc ttcgaggtgg tgtacatgaa cgacggcgtg 480 ctggtgggcc aggtgatcct ggtgtaccgc ctgaacagcg gcaagttcta cagctgccac 540 atgcgcaccc tgatgaagag caagggcgtg gtgaaggact tccccgagta ccacttcatc 600 Cagcaccgcc tggagaagac ctacgtggag gacggcggct tcgtggagca gcacgagacc 660 gccatcgccc agctgaccag cctgggcaag cccctgggca gcctgcacga gtgggtgtaa 720 tag 723 21 201248152

≪ 210 > 7 < 211 > 575 < 212 > DNA < 213 > Plasmid IRES-hrGFP-la < 400 > 7 acccccctct ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg 60 gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc 120 ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa 180 aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag 240 acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg 300 cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg 360 ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa 420 caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg 480 gtgcacatgc tttacatgtg tttagtcgag gttaaaaaac gtctaggccc cccgaaccac 540 ggggacgtgg ttttcctttg aaaaacacga tgata 575 22

Claims (1)

201248152 VII. Patent application scope: 1. A dual-fluorescence microsatellite report plastid for reporting microsatellite instability, in which the promoter region of the plastid is reported by the dual-fluorescence microsatellite, including: micro The satellite sequence, the first fluorescent protein sequence 'internal ribosome insertion site sequence, and the second fluorescent protein sequence, and the first fluorescent protein is different from the fluorescent color emitted by the second fluorescent protein. 2. A dual-fluorescence microsatellite reporter as described in claim 1 wherein the microsatellite sequence comprises a (CA)n sequence and the n is 1〇4〇. 3. The double-fluorescent microsatellite reporter as described in claim 1 wherein the first fluorescent protein is a red-emitting fluorescent protein. 4. The dual-fluorescence microsatellite reporter plastid as described in claim 4, wherein the first fluorescent protein is RFpe 5. The dual-fluorescence microsatellite report as described in claim 1 The second fluorescent protein is a fluorescent protein that emits green light. 6. The dual-fluorescence microsatellite reporter according to claim 5, wherein the second fluorescent protein is Gfp. 7. A cell model comprising a dual-fluorescence microsatellite reporter plastid, comprising: a pair of Laoguang microsatellite reporter plastids 'from the promoter region of the fluoroplast reported by the dual-fluorescence microsatellite' a satellite sequence 4, a sir-denacle protein sequence, an internal ribosome insertion site sequence, and a second fluorescent protein sequence, wherein the first-fluorescent protein is different from the fluorescent color of the second luminescent protein. And the second fluorescent protein is used as a -quantitative protein; and 23 201248152 a cell line, the double-fluorescent microsatellite reporter system is transferred into the cell strain, and the cell line expresses the double-fluorescent microsatellite Reporting a first fluorescent protein sequence ' and a second fluorescent protein sequence in the plastid; wherein 'when the fluorescent light emitted by the first fluorescent protein is detected, indicating that a sequence of reading frames is displaced; and when When the fluorescence emitted by the first fluorescent protein is detected, it indicates that the displacement of the in-frame sequence is not generated. 8. The reporting system of claim 7, further comprising a control group plastid, which is transferred to a cell line, from the promoter region of the control group, comprising: Controlling a sequence, a first fluorescent protein sequence, an internal ribosome insertion site sequence, and a second fluorescent protein sequence, wherein the first fluorescent protein is different from the fluorescent color emitted by the second fluorescent protein And the second fluorescent protein is used as a certain amount of protein. 9. The reporting system of claim 7, wherein the microsatellite sequence comprises a (CA)n sequence and the η system is 5_4〇. 10. The reporting system of claim 8, wherein the control group sequence comprises a sequence of (Ν)η, each of which is independently selected from the group consisting of Α, τ, C, G, and The η system is 2〇·8〇. 11. The reporting system of claim 7, wherein the first fluorescent protein is a red fluorescent luminescent protein. 12. The reporting system of claim 11, wherein the first fluorescent protein is RFP. 13. The reporting system of claim 7, wherein the second luminosity protein is a fluorescent protein that emits green light. The method of claim 14, wherein the second fluorescent protein is GFP, as described in claim 7, wherein the cell line is one DNA mismatched human cell model (MMR-deficient human cell model). 16. The reporting system of claim 15, wherein the DNA mismatch repairing defective cell line is human colon cancer cell line HCT116. Eight, schema (see next page): 25