CN114790463A - Construction method and application of monoclonal cell strain of stable transfection CRISPR/dCas9 system - Google Patents
Construction method and application of monoclonal cell strain of stable transfection CRISPR/dCas9 system Download PDFInfo
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Abstract
The invention discloses a method for constructing a monoclonal cell strain of a stable transfection CRISPR/dCas9 system, which comprises the following steps: 1) lentivirus transfection; 2) performing ultracentrifugation; 3) cell infection and first flow fluorescence sorting; 4) and (4) performing flow-type fluorescence sorting for the second time. The invention belongs to the technical field of genetic engineering, and provides a construction method and application of a monoclonal cell strain for stably transfecting a CRISPR/dCas9 system by combining a lentivirus transfection method, an ultracentrifugation method and a flow fluorescence sorting method, so that the defects that the CRISPR/dCas9 system is difficult to transfect and cannot stably express for a long time are overcome, the stable expression of the CRISPR/dCas9 system is realized, and the targeted regulation and control on DNA methylation can be realized.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method and application of a monoclonal cell strain of a stably transfected CRISPR/dCas9 system.
Background
DNA methylation was the earliest epigenetic regulatory pattern studied and refers to the addition or removal of a methyl group at the cytosine position 5 carbon atom by the action of DNA methylation modifying enzymes (methyltransferases and demethyltransferases). DNA methylation is widely involved in the cellular processes of gene transcriptional regulation, cell differentiation, embryonic development, X chromosome inactivation, gene imprinting, tumorigenesis and development and the like. However, due to the limitation of the technical level, the existing DNA methylation regulation regulates the change of DNA methylation at the genome-wide level mainly by changing the activity of DNA methylation modification enzyme, and cannot meet the requirement of targeted regulation of DNA methylation of specific genes. Methylation modification is a highly refined and continuous process, so a specific DNA methylation editing technology is urgently needed, the function and action mechanism of DNA methylation of a single gene or even a specific CpG locus are researched under the condition that other genes are not influenced, and an effective tool is provided for further researching the function of DNA methylation modification and related medicine research and development.
The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated protein) system evolved from bacteria and archaea is a small RNA-mediated adaptive immune system, can recognize and destroy or silence exogenous DNA (viruses and plasmids) entering a host, and can be used for gene editing and epigenetic regulation after being engineered. Researchers have mutated the nuclease domain of Cas9 in streptococci to generate Cas9 with deleted endonuclease activity, called dead Cas9(dCas 9). dCas9 was no longer able to cleave DNA, but it could still target and bind DNA with the same precision under the guidance of the sgrnas. By designing a specific sgRNA complementary to a target DNA and fusing dCas9 with a DNA methyl modifying enzyme, CRISPR/dCas technology is expected to achieve specific DNA methylation regulation. Patent application CN 111073902A discloses a CRISPR/dCas9 vector for improving expression level of gliotoxin biosynthesis genes, a construction method and application thereof, establishes a CRISPR/dCas9 specific transcription regulation system suitable for erzia, and promotes transcription regulation of gliotoxin biosynthesis of erzia FS 110.
The lentivirus vector is transformed from HIV virus, and can mediate target gene into target cell for long-term stable expression. Compared with partial adenovirus vectors, the lentivirus transfection can greatly improve the mediating efficiency of target genes. However, for some plasmids with larger molecular weights, transfection using lentiviral vectors also suffers from low transfection efficiency. The CRISPR/dCas9 system can be used for methylation modification, but has the limitations of large plasmid, low transfection efficiency or difficulty in stable transfection. Therefore, it is of great significance to provide a monoclonal cell strain which stably expresses the CRISPR/dCas9-TET1CD system and can be stably cultured in vitro for a long time.
Disclosure of Invention
In order to solve the problems (the defect of low transfection efficiency or difficult stable transfection) in the prior art, the invention firstly provides a construction method and application of a monoclonal cell strain for stably transfecting a CRISPR/dCas9 system by combining a lentivirus transfection method, an ultracentrifugation method and a flow fluorescence sorting method, overcomes the defects of difficult transfection and long-time stable expression of the CRISPR/dCas9 system, realizes the stable expression of the CRISPR/dCas9 system, and can realize the targeted regulation and control of DNA methylation.
The objects of the present invention will be further explained by the following detailed description.
The invention provides a method for constructing a monoclonal cell strain of a stable transfection CRISPR/dCas9 system, which comprises the following steps:
1) lentivirus transfection: inoculating and culturing 293FT cells, transfecting a core plasmid and a lentivirus packaging plasmid system by using a transfection reagent when the cell fusion degree reaches 70-90%, and collecting virus liquid at 48h and 96 h;
2) ultracentrifugation: filtering the collected virus liquid by using a filter membrane, uniformly mixing and ultracentrifuging for 2-2.5h under the centrifugation condition of 4 ℃ and 22000-26000 rpm; centrifuging, removing supernatant, adding PBS solution for resuspension to obtain viral protein resuspension, and storing at 4 deg.C;
3) cell infection and first flow fluorescence sorting: inoculating and culturing a target cell A549 in a 6-well plate, culturing for 20-28h, then dropwise adding the virus protein heavy suspension for infection twice, carrying out passage amplification culture on the target cell for 2-4 generations, carrying out primary flow type fluorescence sorting, and recovering the cell by using a flow tube to obtain a cell strain expressing a CRISPR/dCas9 system;
4) and (3) second-time flow type fluorescence sorting: performing secondary flow-type fluorescence sorting on the cell strain expressing the CRISPR/dCas9 system, wherein the cell recovery mode is a 96-well plate recovery mode, and 1 cell is recovered in each well; observing cell adherence condition by using a microscope 4-8h after the second flow type fluorescence sorting, primarily screening monoclonal cells, digesting the cells by using trypsin when the cells grow over a 96-well plate, transferring, carrying out passage amplification for 2-3 generations, and then carrying out flow type fluorescence analysis verification to obtain the monoclonal cell strain of the stably transfected CRISPR/dCas9 system.
The invention takes CRISPR/dCas9-TET1CD as a core plasmid, adopts psPAX2 and pMD2.G with a specific mass ratio as a lentiviral packaging plasmid system for transfection, collects virus liquid at 48h and 96h, and carries out ultracentrifugation under specific conditions to obtain concentrated virus particles, thereby improving the transfection efficiency; because the molecular weight of the transfection plasmid is larger, the transfection efficiency is relatively lower, after the target cells are subcultured for 2-4 generations by passage and amplification, enough cells (the number of the cells is more than 250 ten thousand) are used for sorting, so that enough positive cells are obtained under the condition of lower positive rate, and the proliferation culture of the positive cells is facilitated. Meanwhile, a small amount of positive cells are sorted out by using the fluorescence label of the target gene through the first flow-type fluorescence sorting, and the cells are recovered by using a flow-type tube, so that the defect that positive cell strains cannot be obtained by drug screening under low transfection efficiency is overcome. Due to the recovery by the flow tube, the cells with fluorescence obtained by the first flow fluorescence sorting are not one hundred percent of positive cells, i.e. non-positive cells may be mistaken for positive cells, so that the proportion of positive cells will gradually decrease due to the cell growth advantage of the non-positive cells during the passage. On the basis of the discovery, the invention further develops second flow-type fluorescence sorting, adopts different cell recovery modes, overcomes the defect that the proportion of positive cells is gradually reduced due to the cell growth advantage in the passage process of non-positive cells obtained by the first flow-type sorting, selects the positive cells to be cultured independently, and finally obtains the stably expressed monoclonal cell strain through flow-type fluorescence analysis verification (only positive rate is analyzed, cells are not recovered) after passage culture.
Preferably, the transfection reagent is polyethylenimine (pei) MAX; the core plasmid is CRISPR/dCas9-TET1CD, the lentivirus packaging plasmid system comprises psPAX2 and pMD2.G, the mass ratio of the CRISPR/dCas9-TET1CD to the psPAX2 to the pMD2.G is 6:6:5, and the dosage ratio of the transfection plasmid to the transfection reagent is 1 mug: 3 muL. Transfection plasmids include core plasmids and lentiviral packaging plasmid systems.
Preferably, the viral titer of the viral protein resuspension is 1X 10 8 -1.68×10 8 TU/mL。
Preferably, the filter membrane is a Millipore filter membrane having a pore size of 0.45 μm.
Preferably, the channels of the first flow-type fluorescence sorting and the second flow-type fluorescence sorting are tagBFP respectively, the excitation wavelength is 400nm, and the emission wavelength is 450 nm.
Correspondingly, the invention also provides a monoclonal cell strain stably transfected with the CRISPR/dCas9 system, which is constructed by the construction method.
In addition, the invention also provides application of the monoclonal cell strain stably transfected with the CRISPR/dCas9 system in preparation of medicaments for treating DNA methylation related diseases.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a construction method and application of a monoclonal cell strain for stably transfecting a CRISPR/dCas9 system by combining a lentivirus transfection method, an ultracentrifugation method and a flow-type fluorescence sorting method and optimizing the operation conditions of the composition of a lentivirus packaging plasmid system, the mass ratio of the lentivirus packaging plasmid system, virus liquid collection, ultracentrifugation, virus titer and the like, overcomes the defects that the CRISPR/dCas9 system is difficult to transfect and cannot stably express for a long time, realizes the stable expression of the CRISPR/dCas9 system, and can realize the targeted regulation and control of DNA methylation. The invention provides a monoclonal cell strain for stably expressing CRISPR/dCas9-TET1CD, which can be used for carrying out DNA methylation modification change on a specific gene or site, provides a favorable tool for researching the DNA methylation function and action mechanism of the specific gene or site, and is expected to play an important role in preparing medicaments for treating DNA methylation related diseases.
Drawings
FIG. 1 is a schematic flow chart of a construction method provided by the present invention.
FIG. 2 CRISPR/dCas9-TET1CD plasmid map used in the present invention.
FIG. 3 is a graph showing the results of cell detection in the first flow fluorescence sorting according to the example of the present invention.
FIG. 4 is a graph showing the results of 7 generations of the first flow fluorescence-sorted cells of this example after passage expansion culture.
FIG. 5 is a graph showing the results of the verification of the monoclonal cell lines obtained in the examples of the present invention.
FIG. 6 is a graph showing the results of cell detection by flow fluorescent sorting according to comparative example 2 of the present invention.
FIG. 7 is a graph showing the results of cell detection by flow fluorescent sorting according to comparative example 3 of the present invention.
FIG. 8 is a graph showing the results of measuring the mRNA expression levels of the monoclonal cell strains dCas9 and TET1CD obtained in the examples of the present invention.
FIG. 9 is a chart showing the results of detecting the methylation of Line 1DNA of a monoclonal cell strain provided in the embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples.
In the present invention, the materials and reagents involved are all conventional commercial products, or can be obtained by conventional technical means in the field, such as CRISPR/dCas9-TET1CD from Addge, psPAX2 from Addge, pMD2.G from Addge, and polyethylene MAX from Polysciences.
Culture conditions for 293FT and a549 cells: DMEM medium (containing 10% volume percent fetal bovine serum), 5% CO 2 And culturing at 37 ℃.
EXAMPLE construction of a monoclonal cell line stably transfected with the CRISPR/dCas9 System
The schematic flow chart of the construction method of the monoclonal cell strain stably transfected with the CRISPR/dCas9 system is shown in FIG. 1, and the method specifically comprises the following steps:
1) lentivirus transfection: 293FT cells were seeded in 10-cm cell culture dishes at 5X 10 6 Per well, when the cell fusion degree reaches 70-90%, using polyethylene imine MAX transfection reagent, PEI application solution with concentration of 1mg/ml, transfecting CRISPR/dCas9-TET1CD core plasmid and lentivirus packaging plasmid system (including psPAX2 and pMD2.G), wherein the mass ratio of CRISPR/dCas9-TET1CD, psPAX2 and pMD2.G is 6:6:5, the amount of CRISPR/dCas9-TET1CD is 6 μ g, and the amount of transfection reagent is 51 μ L; fresh medium was changed at 8h after transfection, and virus fluid was collected at 48h and 96 h.
2) Ultracentrifugation: filtering the collected virus solution with Millipore filter membrane with pore diameter of 0.45 μm, mixing, distributing into 6 centrifuge tubes, ultracentrifuging for 2h at 4 deg.C and 25000 rpm; after centrifugation, supernatant is removed, 200 mu L of 1 XPBS is added into each centrifuge tube to resuspend virus protein, and virus protein resuspension is obtained, the virus titer of the virus protein resuspension is 1.05X 10 8 TU/mL, stored at 4 ℃.
3) Cell infection and first flow fluorescence sorting: the target cells A549 were seeded and cultured in 6-well plates at a cell seeding number of 2X 10 5 Performing cell/pore culture for 24h, dripping 100 mu L of virus protein resuspension for each infection, performing infection twice, changing liquid after 12h of infection, performing subculture amplification on target cells for 3 generations, performing first flow-type fluorescence sorting, recovering cells by using a flow tube, and obtaining a cell strain for expressing a CRISPR/dCas9 system, wherein the channel is tagBFP (excitation wavelength is 400nm, and emission wavelength is 450 nm); a small amount of positive cells are separated by primary flow fluorescence separation, a relatively high primary screening positive rate is obtained, the defect that positive cell strains cannot be obtained by drug screening under low transfection efficiency is overcome, and a cell detection result diagram is shown in figure 3. The detection result of the first flow fluorescence sorted cells after 7 passages of culture is shown in fig. 4. 53.0% is due to the technical disadvantages of the first flow sorting using a flow tube to recover the cells, and the increase in the number of passages after sorting, which reduces the positive rate, so that it is necessary to sort out the single clones to ensure 100% positive cells.
4) And (3) second-time flow type fluorescence sorting: performing secondary flow fluorescent sorting on the cell strain expressing the CRISPR/dCas9 system, wherein the cell recovery mode is 96-well plate recovery, and 1 cell is recovered in each well; and (3) observing the cell adherence condition by using a microscope and primarily screening monoclonal cells 6h after the second flow-type fluorescent sorting, digesting the cells by using trypsin and transferring the cells when the cells grow over a 96-well plate, and carrying out flow-type fluorescent analysis verification after passage amplification for 2 generations to obtain a monoclonal cell strain stably transfecting the CRISPR/dCas9 system. The verification result chart is shown in fig. 5, the step overcomes the defect that the flow sorting technology is difficult to sort out positive cells by 100 percent, the positive cells are individually sorted, and then the positive cells are individually selected for culture, and finally, the stably expressed monoclonal cell strain is obtained.
Comparative example 1
The inventor uses the existing calcium phosphate coprecipitation method to transfect 293FT cells according to the mass ratio of CRISPR/dCas9-TET1CD core plasmid VSVG:delta8.9 ═ 8:1:6, viruses produced by 293 cells in each dish infect target cells A549, virus solutions are collected twice 48h and 72h after transfection, infection is carried out twice, the selection method is purine selection, and the target cells stably transfected with the CRISPR/dCas9 system cannot be obtained due to the large plasmid and low transfection efficiency.
Comparative example 2
The inventor inoculates 293FT cells in 2 10cm cell culture dishes, uses Polyethylenimine (PEI) -MAX transfection reagent, PEI application solution concentration is 1mg/ml, carries out transfection on CRISPR/dCas9-TET1CD core plasmid and lentivirus packaging plasmid system (comprising psPAX2 and pMD2.G), the mass ratio of CRISPR/dCas9-TET1CD, psPAX2 and pMD2.G is 4:3:1, the mass ratio of transfection plasmid and transfection reagent is 1 mug: 3. mu.L, and virus solution is collected at 48h and 96 h; directly filtering the collected virus liquid into an Amicon Ultra-15ml Ultra-filtration tube, and centrifuging at 4000 Xg and 4 ℃ for 30min in an Allegra X-15R desk-top refrigerated centrifuge to concentrate the virus liquid; to the concentrated virus solution, 100. mu.l of complete medium containing 10% FBS by volume was added to prepare a virus protein suspension, which was stored at 4 ℃. 100 μ L of the viral protein resuspension was added dropwise to each infection, and the infection was performed twice.
The comparative example 2 differs from the first example mainly in that: CRISPR/dCas9-TET1CD, psPAX2 and pMD2.G were varied in mass ratio, concentrated using an ultrafiltration tube, and not ultracentrifuged. Comparative example 2 cell lines expressing CRISPR/dCas9 system were obtained by first flow fluorescence sorting, and the cell detection results are shown in fig. 6, with a positive sorting rate of only 0.071%.
Comparative example 3
The inventor inoculates 293FT cells in 5 10cm cell culture dishes, uses Polyethylenimine (PEI) -MAX transfection reagent, PEI application solution concentration is 1mg/ml, carries out transfection on CRISPR/dCas9-TET1CD core plasmid and lentivirus packaging plasmid system (comprising psPAX2 and pMD2.G), the mass ratio of CRISPR/dCas9-TET1CD, psPAX2 and pMD2.G is 6:6:5, the mass ratio of transfection plasmid and transfection reagent is 1 mug: 3. mu.L, carries out expanded culture on the transfected 293FT cells to 5 culture dishes, and collects virus solution at 48h and 96 h; filtering the collected virus solution with Millipore filter membrane with pore diameter of 0.45 μm, mixing, distributing into 4 centrifuge tubes, ultracentrifuging for 1.5h at 4 deg.C and 25000 rpm; after centrifugation, the supernatant was removed, 200. mu.L of 1 XPBS was added to each tube to resuspend the viral proteins, and a viral protein resuspension was obtained and stored at 4 ℃. 100 μ L of the viral protein resuspension was added dropwise for each infection, and the infection was performed twice.
The comparative example 3 differs from the first example mainly in that: less virus and shorter centrifugation times resulted in lower viral titers of the viral protein resuspension. Comparative example 3 a cell strain expressing CRISPR/dCas9 system was obtained by first flow fluorescence sorting, and the cell detection result is shown in fig. 7, with a positive sorting rate of only 0.337%.
Comparative example 4
The inventor inoculates 293FT cells in 10cm cell culture dishes, uses Polyethylenimine (PEI) -MAX transfection reagent, PEI application solution concentration is 1mg/ml, carries out transfection on CRISPR/dCas9-TET1CD core plasmid and lentivirus packaging plasmid system (including psPAX2 and pMD2.G), the mass ratio of CRISPR/dCas9-TET1CD, psPAX2 and pMD2.G is 6:6:5, the mass ratio of transfection plasmid and transfection reagent is 1 mug: 3 muL, and virus solution is collected at 48h and 96 h; filtering the collected virus solution with Millipore filter membrane with pore diameter of 0.45 μm, mixing, distributing to 6 centrifuge tubes on average, ultracentrifuging for 2h at 4 deg.C and 25000 rpm; after centrifugation, the supernatant was removed, 200. mu.L of 1 XPBS was added to each tube to resuspend the viral proteins, and a viral protein resuspension was obtained and stored at 4 ℃. 200 μ L of the viral protein resuspension was added dropwise for each infection, and the infection was performed twice.
Comparative example 4 differs from example one mainly in that: the viral protein resuspension per infection was added in 200. mu.L. As a result: the virus titer was too high, resulting in target cell death, and positive cells were not sorted.
Example two detection of mRNA expression level of A549 monoclonal cell strain stably expressing CRISPR/dCas9 system
Total RNA of A549 monoclonal cell line stably expressing CRISPR/dCas9 system obtained in example I was extracted by TRIzol method, and reverse transcription was performed using reverse transcription kit of TAKARA to generate cDNA, and TOYOBO was performed
The Green qPCR kit is used for qRT-PCR, and the sequences of primers used in the qRT-PCR are shown in Table 1.
TABLE 1 primers used in the present invention
The result is shown in fig. 8, the a549 monoclonal cell strain stably expressing CRISPR/dCas9 system was successfully constructed, and the mRNA expression levels of dCas9 and TET1CD were significantly higher than those of the control group.
Example three A549 monoclonal cell Line 1DNA methylation assay stably expressing CRISPR/dCas9 System
The detection condition of the genomic DNA of the A549 monoclonal cell strain of the stably expressed CRISPR/dCas9 system obtained in the first embodiment is extracted to examine the influence on the methylation of the whole genomic DNA, and the method specifically comprises the following steps:
digesting the constructed cell strain, and extracting cell genome DNA by using a TIANGEN blood/cell/tissue genome DNA extraction kit;
bisulfite modification:
bisulfite modification of genomic DNA using the QIAGEN EpiTect bisufite Kit (cat # 59104));
③ pyrosequencing:
the bisulfite-modified DNA was pyrosequenced using QIAGEN PyroMark PCRKit (cat # 978703), and the primers required for sequencing are shown in Table 1.
The results are shown in fig. 9, stably expressing CRISPR/dCas9-TET1CD monoclonal cell line did not alter the DNA methylation level of non-targeted genes.
The foregoing is a further detailed description of the invention in connection with specific preferred embodiments and it is not intended to limit the invention to the specific embodiments described. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
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Claims (7)
1. The construction method of the monoclonal cell strain of the stable transfection CRISPR/dCas9 system is characterized in that: the method comprises the following steps:
1) lentivirus transfection: inoculating and culturing 293FT cells, and when the cell fusion degree reaches 70-90%, transfecting a core plasmid and a lentivirus packaging plasmid system by using a transfection reagent, and collecting virus liquid at 48h and 96 h;
2) ultracentrifugation: filtering the collected virus liquid by using a filter membrane, uniformly mixing and ultracentrifuging for 2-2.5h under the centrifugation condition of 4 ℃ and 22000-26000 rpm; centrifuging, removing supernatant, adding PBS solution for resuspension to obtain viral protein resuspension, and storing at 4 deg.C;
3) cell infection and first flow fluorescence sorting: inoculating and culturing a target cell A549 in a 6-well plate, culturing for 20-28h, then dropwise adding the virus protein heavy suspension for infection twice, carrying out passage amplification culture on the target cell for 2-4 generations, carrying out primary flow type fluorescence sorting, and recovering the cell by using a flow tube to obtain a cell strain expressing a CRISPR/dCas9 system;
4) and (3) second-time flow type fluorescence sorting: performing secondary flow fluorescent sorting on the cell strain expressing the CRISPR/dCas9 system, wherein the cell recovery mode is 96-well plate recovery, and 1 cell is recovered in each well; 4-8h after the second flow-type fluorescent sorting, observing the cell adherence condition by using a microscope and primarily screening monoclonal cells, digesting the cells by using trypsin and transferring the cells when the cells grow over a 96-well plate, carrying out passage amplification for 2-3 generations, and then carrying out flow-type fluorescent analysis verification to obtain a monoclonal cell strain stably transfected with a CRISPR/dCas9 system.
2. The method of claim 1 for constructing a monoclonal cell strain stably transfected with CRISPR/dCas9 system, wherein the method comprises the steps of: the transfection reagent is Polyethylenimine MAX; the core plasmid is CRISPR/dCas9-TET1CD, the lentivirus packaging plasmid system comprises psPAX2 and pMD2.G, the mass ratio of the CRISPR/dCas9-TET1CD to the psPAX2 to the pMD2.G is 6:6:5, and the dosage ratio of the transfection plasmid to the transfection reagent is 1 mug: 3 muL.
3. The method for constructing a monoclonal cell strain stably transfected with CRISPR/dCas9 according to claim 1, wherein the method comprises the steps of: the virus titer of the virus protein resuspension is 1X 10 8 -1.68×10 8 TU/mL。
4. The method of claim 1 for constructing a monoclonal cell strain stably transfected with CRISPR/dCas9 system, wherein the method comprises the steps of: the filter membrane is a Millipore filter membrane, and the pore diameter is 0.45 mu m.
5. The method of claim 1 for constructing a monoclonal cell strain stably transfected with CRISPR/dCas9 system, wherein the method comprises the steps of: the channels of the first flow-type fluorescence sorting and the second flow-type fluorescence sorting are tagBFP respectively, the excitation wavelength is 400nm, and the emission wavelength is 450 nm.
6. A monoclonal cell strain stably transfected with CRISPR/dCas9 system, comprising: constructed by the construction method of any one of claims 1 to 5.
7. An application of a monoclonal cell strain of a stable transfection CRISPR/dCas9 system in preparing a medicament for treating DNA methylation related diseases.
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