CN110714057A - Universal FISH probe for detecting exogenous gene integration and positioning and application thereof - Google Patents
Universal FISH probe for detecting exogenous gene integration and positioning and application thereof Download PDFInfo
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Abstract
The invention relates to a universal FISH probe for detecting exogenous gene integration and positioning and application thereof. The universal FISH probe is prepared and labeled by taking a non-target gene sequence of a recombinant expression plasmid, such as a selective marker gene sequence, as a template through a PCR method, a nick translation method, a random primer method and a DNA synthesis method, and the obtained labeled probe can be complementary with the sequence of the recombinant expression plasmid and is used for indirectly detecting the integration of endogenous and exogenous genes in mammalian cells or confirming the positioning of the exogenous genes on chromosomes; or combined with a reagent of an in-situ hybridization reaction system to be used as a detection kit for detecting the integration and positioning of exogenous genes of scientific research recombinant cells and cells of biological medicine engineering.
Description
Technical Field
The invention relates to the field of genetic engineering of biotechnology, in particular to a universal FISH probe for detecting exogenous gene integration and positioning and application thereof.
Background
Fluorescence In Situ Hybridization (FISH) technology is a nonradioactive in situ hybridization technology developed by combining cytogenetics and molecular biology, and the basic principle is that a target gene sequence is used as a template, a nucleic acid probe is prepared by using biotin, digoxin, fluorescein and other labels by a direct or indirect method, the nucleic acid probe is hybridized with a denatured cell chromosome according to a base complementary pairing principle, a hybrid of target DNA and the nucleic acid probe can be formed after denaturation-annealing-renaturation, and the integration and positioning of the target gene can be detected and analyzed through an immunofluorescence system. Interphase cell FISH and chromosomal FISH have been widely used for diagnosis or prenatal diagnosis of chromosomal disorders, as well as for qualitative and localized analysis of malignant oncogenes.
In the field of genetic engineering, some progress has been made in detecting the integration and localization of foreign genes on cells and chromosomes and analyzing the effectiveness and stability of target proteins by using FISH, but the application of the FISH technology in the field of biopharmaceutical genetic engineering is very limited. New drug legislation or related guidelines (Points to Consider,1997 and ICH Q5D,1998) require that cell clones originate from single progenitor cells and provide evidence of monoclonality of recombinant cells or cell bank screen cells to ensure purity and homogeneity of engineered cell lines and thus product homogeneity and consistency in the production process. Ensuring the monoclonality of recombinant cell lines is a key point for obtaining regulatory approval of biopharmaceutical approval, but health authorities or supervising agencies have recognized that the clonality (consistency) of production cell lines in pharmaceutical enterprises currently lacks sufficient research details and evidence (Frye C, et al. industry view on the relative evaluation of "consistency" of biochemical-production cells. biological loads.2016, 44: 117-.
The most effective means for identifying the monoclonality of recombinant cells is FISH technology, and the application of FISH can not only directly determine the location of the exogenous gene on the chromosome, but also predict the influence of the chromosome position effect on the expression of the exogenous gene (Derouazi M, et al. genetic characterization of CHO production host DG44 and innovative recombinant cells lines. biochem Biophys Res Commun.2006,340(4):1069-1077.), especially by detecting the integration of the exogenous gene at the same site on the same chromosome, the monoclonality of the cell line can be directly analyzed. In the FISH technology for detecting genetically engineered cell lines, the most classical method for preparing probes is to directly use an expression plasmid containing a target gene sequence as a template, prepare and label probes by a Nick translation method (Nick translation) or a Random Primer (Random Primer) method, and a PCR or RNA reverse transcription method is also a commonly used method for preparing and labeling probes. Although the plasmid is very simple and easy to obtain as a template, the labeled probe must be prepared again for recombinant cell lines expressing different proteins or monoclonal antibodies, especially for a large number of recombinant cell lines, namely the existing labeled probe has no universality.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention aims to provide a universal probe for detecting the integration and positioning of exogenous genes of different expression cell lines, analyzing the monoclone source and solving the problem of complicated procedures for repeatedly preparing plasmid probes.
At present, recombinant expression plasmid vector structures for mammalian expression systems have similar characteristics, and all contain prokaryotic replication initiation sequences, selection marker genes, antibiotic resistance genes, promoters, enhancers, terminators, polynucleotide signals and the like, and some plasmid vectors contain special sequences, such as Ubiquitin Chromatin Opening Elements (UCOEs), which can make the chromatin environment of the integration sites in a state of being favorable for the transcription of target genes.
Based on the self characteristics of the expression vector, the invention provides a universal FISH probe for detecting exogenous gene integration and positioning and application thereof.
The universal FISH probe is prepared by taking a non-target gene sequence of a eukaryotic expression plasmid as a template, wherein the non-target gene sequence is selected from one or more of the following genes:
the SV40 promoter has the gene sequence of SEQ ID NO. 1;
the gene sequence of the pCMV promoter is SEQ ID NO 2;
a pan-chromatin opening element (UCOE) gene having a gene sequence of SEQ ID NO: 3;
ampicillin resistance gene, the gene sequence of which is SEQ ID NO 4;
kanamycin resistance gene, the gene sequence of which is SEQ ID NO. 5;
hygromycin resistance gene, the gene sequence is SEQ ID NO 6;
the gene sequence of the bleomycin resistance gene is SEQ ID NO. 7;
puromycin resistance gene, the gene sequence of which is SEQ ID NO 8;
the chloramphenicol resistance gene has the gene sequence of SEQ ID NO 9;
the terminator poly A sequence with the gene sequence of SEQ ID NO 10;
a dihydrofolate reductase (DHFR) selection marker gene, the gene sequence of which is SEQ ID NO: 11;
a Glutamine Synthetase (GS) selective marker gene, the gene sequence of which is SEQ ID NO: 12;
human IgG1 constant region gene sequence, which is SEQ ID NO 13;
human IgG2 constant region gene sequence, which is SEQ ID NO. 14;
human IgG4 constant region gene sequence, which is SEQ ID NO. 15.
Further, the universal FISH probe of the present invention is prepared by using a combination of non-target gene sequences of eukaryotic expression plasmids as a template, wherein the non-target gene sequences are selected from one or more of the following sequence combinations:
the combination of the AMP and pCMV gene sequences,
the combination of the AMP and DHFR gene sequences,
the combination of the AMP and GS gene sequences,
AMP and human IgG constant region gene sequences,
AMP, pCMV and SV40poly A gene sequence combination,
the gene sequence combination of AMP, pCMV and SV40 promoters,
AMP, pCMV, and DHFR gene combinations,
the AMP, pCMV and GS gene combinations,
AMP, pCMV, and UCOE genes,
AMP, pCMV, SV40poly A and DHFR gene combinations,
AMP, pCMV, SV40poly A and GS gene combinations.
Further, the invention provides a method for preparing the universal FISH probe. The preparation method comprises but is not limited to the following steps:
(1) preparing a universal FISH probe by taking an antibiotic resistance gene sequence as a template:
the antibiotic resistance gene is resistance genes such as ampicillin, kanamycin, hygromycin, chloramphenicol, bleomycin and the like, the ampicillin resistance gene is used as the most extensive selection marker in gene operation, and the gene sequence can be independently used for preparing a probe or can be used as a template together with other resistance genes such as kanamycin resistance gene sequences for preparing and marking the probe;
(2) preparing a universal FISH probe by taking a screening marker gene sequence as a template:
the screening marker gene is Glutamine Synthetase (GS) and dihydrofolate reductase (DHFR) screening genes, the two screening genes are selective marker genes commonly used by recombinant expression plasmids, and the expression plasmids containing GS or DHFR are used for constructing recombinant cells, so that an expression cell line of stable and high-yield protein or monoclonal antibody can be rapidly screened.
(3) A labeled universal FISH probe is prepared by using a strong promoter sequence (such as pCMV) as a template.
(4) And preparing a labeled probe by taking the UCOE sequence as a template, and selecting a corresponding universal FISH probe.
The invention provides a preparation and labeling method of a universal FISH probe, which comprises the following steps: PCR, nick translation, random primer, DNA sequence synthesis, etc. Wherein the content of the first and second substances,
the PCR method comprises the following steps: in a high-fidelity polymerase system, any universal DNA sequence or combination thereof, reaction buffer solution, specific upstream and downstream primers, high-fidelity DNA polymerase, dNTP, biotin, digoxin or fluorescent molecules FAM, FITC, Cy3, Cy5 and dATP/dCTP/dUTP marked by OBEA are sequentially added, and a PCR instrument is put into the high-fidelity polymerase system for expansion to obtain a DNA fragment with the product length of 200-1000 bp.
The random primer method comprises the following steps: taking any universal DNA sequence or combination thereof with the amount of 100-2000 ng by using a random primer DNA labeling kit, placing the universal DNA sequence or combination thereof in a water bath with the temperature of 95-100 ℃ for denaturation for 5min, immediately placing the universal DNA sequence or combination thereof in an ice bath, adding dNTP, a random primer (random primer), a reaction buffer solution, a DNA polymerase solution, biotin, digoxin or fluorescent molecules FAM, FITC, Cy3, Cy5 and OBEA labeled dATP/dCTP/dUTP, and placing the mixture at the temperature of 37 ℃ for reaction for 1-24 h.
The notch translation method comprises the following preparation steps: using any one or a combination of universal DNA double-stranded sequences as a template, dATP, dTTP, dCTP and dGTP are added under the condition of a Nick Translation Enzyme (Nick Translation Enzyme) system, biotin, digoxin and the like or dATP/dCTP/dUTP labeled with fluorescent molecules FAM, FITC, Cy3, Cy5, OBEA and the like are added, and the mixture is placed at 37 ℃ for reaction for 1-24 hours.
The invention also provides application of the universal FISH probe. Preferably, the universal FISH probe is complementary to a recombinant expression plasmid sequence comprising the exogenous gene, and is used to detect exogenous gene integration in mammalian cells, or to confirm chromosomal localization of the exogenous gene.
The invention also provides application of the universal FISH probe. Preferably, the universal FISH probe can be combined with an in-situ hybridization reaction system or a reagent to be used as a detection kit for detecting the integration and positioning of exogenous genes of scientific research recombinant cells and biological medicine engineering cells.
The invention also provides a FISH kit for detecting exogenous gene integration, positioning and analyzing monoclonality source, which consists of the universal FISH probe and reaction reagents obtained by commercial means.
The universal FISH probe provided by the invention has the same effect with a probe prepared by taking a full-length plasmid as a template, avoids the complicated procedure of repeatedly preparing plasmid probes by different cell lines, does not need to consider the target gene sequence of transfected cells, and can be used for detecting the integration and positioning of exogenous genes of a recombinant cell line and analyzing the monoclonality by selecting the proper universal FISH probe according to the genetic background of the plasmid.
Drawings
FIG. 1 is a diagram showing the FISH detection results of the universal FISH probe of the present invention applied to interphase cell FISH detection of a DG44 recombinant cell line;
FIG. 2 is a diagram showing the detection result of the universal FISH probe of the present invention applied to FISH detection of interphase cells of CHOK1GS-KO recombinant cell line;
FIG. 3 is a diagram showing the detection result of the universal FISH probe of the present invention applied to the chromosome FISH detection of the DG44 recombinant cell line;
FIG. 4 is a diagram showing the result of applying the universal FISH probe of the present invention to the chromosome FISH detection of CHOK1GS-KO recombinant cell line;
FIG. 5 is a diagram showing the detection result of the universal FISH probe kit of the present invention in FISH detection.
Detailed Description
The following examples are provided to further illustrate the invention. It should be understood that these examples are only for illustrating the present invention and are not to be construed as limiting the present invention in any way. Any variations that may be made in the practice of the invention by those skilled in the art in light of the teachings herein will fall within the scope of the appended claims.
Example 1 preparation of Universal FISH Probe
1. Primer and Gene Synthesis
The sequence required by the preparation of the universal FISH probe is derived from UCOE-dhfr plasmid or pEE6.4 plasmid. Primers and genes were synthesized by Shanghai Bioengineering Co., Ltd.
TABLE 1 primer information Table
2. Universal FISH probe gene preparation
And amplifying by a PCR method by using the synthesized specific primer to obtain the gene fragment. PCR reaction system as shown in Table 2 (total volume 50. mu.l): kappa HiFi HotStart Hi-Fi enzyme 0.5 ul, 5 XBuffer 10 ul, dNTP 1 ul, primer 1 ul, template 1 ul, finally make up to 50 ul with double distilled water; reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, 15s at 98 ℃, 30s at 62 ℃, 60s at 72 ℃, 32 cycles, and extension at 72 ℃ for 5 min. The gene products were detected by agarose gel electrophoresis.
TABLE 2 primer and template information
3. Probe preparation
3.1 random primer method for preparing Universal FISH Probe
Labeling the FISH probe by using a Random primer DNA Labeling Kit (Random primer DNA Labeling Kit, Roche), and mainly comprising the following steps: 500ng of the prepared DNA fragments of AMP, dhfr, GS, pCMV, SV40poly A, UCOE, etc., and linearized UCOE-dhfr plasmid (Pvu I), linearized pEE6.4 plasmid (Pvu I) were taken, and DEPC water was added to the mixture to reach 24.8. mu.l, denatured at 95 ℃ for 10min, immediately placed on ice and cooled for 15min, 2. mu.l each of dATP, dCTP, and dGTP, 1.6. mu.l of dTTP, 1.6. mu.l of Dioxigenin-11-dUTP, and 4. mu.l of Reaction mix, and 2. mu.l of Klenozyme were added, incubated at 37 ℃ for 16h, and inactivated at 65 ℃ for 10min and stored at-20 ℃ for further use.
TABLE 3 Probe information Table
3.2 preparation of Universal FISH Probe by PCR
FISH probes were prepared by PCR amplification of inserts of Digoxin-11-dUTP or Biotin-14-dCTP. PCR reaction system as shown in Table 3 (total volume 50. mu.l): kappa HiFi HotStart Hi-Fi enzyme 0.5 u l, 5 x Buffer10 u l, dNTP (10mM)1 u l, Biotin-14-dCTP (10mM)1 u l, primer 1 u l, template 1 u l, finally double distilled water to make up to 50 u l; reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, 15s at 98 ℃, 30s at 62 ℃ and 30s at 72 ℃ for 33 cycles, and extension at 72 ℃ for 5 min. The probes were detected by agarose gel electrophoresis.
TABLE 4 primers and template information 2
Example 2 preparation of Universal FISH Probe kit
The universal probe kit is composed of a group of universal FISH probes or groups of universal FISH probes selected from those prepared in example 1 of the present invention and commercially available detection reagents.
The kit comprises the following components: 300ng of the single or combination universal FISH probe prepared in example 1, 220. mu.l of probe hybridization solution (50% deionized formamide, 10% dextran sulfate, 5 XSSC, 10% Denhardts, 100ng/ml Salmonon DNA), 330ml of DNA denaturation solution (70% SSC solution of deionized formamide), 330ml of chromosomal peripheral tissue digest (0.01% pepsin solution, pH 2.0), 1.2ml of hybridization blocking solution (2 XSSC solution of 1 XSSalmonon Testes), 330ml of hybridization renaturation solution (2 XSSC solution of 50% formamide), 100ml of 20 XSSC wash (20 XSSC wash: 6M NaCl, 0.6M sodium citrate), 1.5ml of antibody blocking solution (3% Tween, 2 XSSC solution of 0.05% Tween-20), 15. mu.l of probe-labeled molecular antibody (FITC-conjugated IgG France monoclonal antibody-lectin concentration 0.7mg/ml or FITC-Streptavidin, concentration 1mg/ml), 200. mu.l of cell nucleus/chromosome fluorochrome (DAPI/antipade solution, 1.5. mu.g/ml).
Example 3 use of Universal FISH Probe for detecting interphase cell exogenous Gene integration
1. Interphase cell sample preparation
Respectively carrying out amplification culture on a monoclonal recombinant cell line JLK004DG44 (CHO DG44 cells transfected by expression plasmid of polypeptide-Fc fusion protein gene inserted into UCOE-dhfr vector) and a monoclonal recombinant cell line JLK004CHOK1 (CHOK 1GS-KO cells transfected by expression plasmid of polypeptide-IgG 1Fc fusion protein gene inserted into pEE6.4 vector) until the cell concentration is about 1 × 106Per ml; collecting cells in a 15ml centrifuge tube, collecting cell precipitates, resuspending the cell precipitates by 8ml of 0.075mol/L potassium chloride solution, and swelling the cells in a water bath at 37 ℃ for 25 min; directly adding 2mL of stationary liquid, mixing, centrifuging for 10min at 1000g, discarding part of supernatant, leaving about 0.5mL of supernatant, adding 4mL of stationary liquid, mixing, fixing at room temperature for 20min, centrifuging for 10min at 1000g, and removing supernatant; repeatedly fixing once, centrifuging, discarding part of supernatant, reserving about 0.5mL of supernatant, and re-suspending the cells; take 20. mu.l of cellsThe suspension is smeared on a glass slide and dried at room temperature for standby.
2. Universal FISH probe detection interphase cell exogenous gene integration
Placing the cell slide prepared in the step 1 in a denaturing solution (a 2 XSSC solution of 70% deionized formamide) at 78 ℃ for incubation for 6min, then sequentially dehydrating the cell slide for 3min by precooled 70%, 90% and 100% ethanol respectively, and airing the cell slide at room temperature; incubating in 0.01% pepsin (pH 2.0) solution at 37 deg.C for 5min, dehydrating with 70%, 90%, and 100% ethanol for 3min, and air drying at room temperature. 20-30 ng of the probe prepared in example 2 (22. mu.L of probe hybridization solution) was denatured at 98 ℃ for 10min, immediately placed on ice to cool for 5min, then placed at 37 ℃ for prehybridization for 30min, dropped on the treated cell slide, mounted, and incubated at 37 ℃ and saturated humidity for 16 hr. Then, the slide is put into a 2 XSSC/50 percent formamide solution at the temperature of 43 ℃ for renaturation for 15min, and the slide is washed for 3 times by 2 XSSC and dried at room temperature; dripping 100 μ L of 2 XSSC solution containing 3% BSA and 0.05% Tween-20 on the glass slide, incubating for 30min at 37 deg.C in dark, washing with 2 XSSC solution containing 0.05% Tween-20 for 2 times, sequentially adding 70%, 90% and 100% ethanol, dehydrating for 3min, and air drying at room temperature; then 100 μ L of FITC-Streptavidin (1: 50) or FITC-conjugated IgG Fraction monoclonal mouse anti-Digoxin (1: 50) is added dropwise, the mixture is incubated for 60min at 37 ℃ in the dark, the washing with 2 XSSC solution is carried out for 4 times, then the mixture is respectively removed by 70%, 90% and 100% ethanol for 3min, and then the mixture is placed in the dark and dried at room temperature; and finally, adding DAPI/antipade Solution to counterstain the cell nucleus, covering a slide, and placing the slide on an upright microscope for observation and image acquisition.
FIG. 1 shows that the universal FISH probe is applied to FISH detection of JLK004DG44 recombinant cell line interphase cells: A-H are respectively probes UCOE-dhfrrp (whole plasmid probe), AMPrp, dhfrrp, pCMVrp, UCOErp, SV40rp, IgG1FCrp, AMPrp and pCMVrp which are used for detecting cell integration results of exogenous target genes at JLK004CHOK1 interval;
FIG. 2 shows the application of universal FISH probe in JLK004CHOK1 recombinant cell line interphase cell FISH detection: a to G are respectively the results of detecting the cell integration of exogenous target genes in JLK004CHOK1GS-KO interval by probes pEE6.4rp (whole plasmid probe), AMPrp, GSrp, pCMVrp, SV40rp, IgG1FCrp, AMPrp and pCMVrp.
The results of detecting the integration of the exogenous gene in the interphase DG44 recombinant cell line and the interphase CHOK1GS-KO recombinant cell line by the universal FISH probe are shown in figures 1 and 2, the fluorescence detection signal of the universal FISH probe is consistent with that of a whole plasmid probe, the exogenous gene detected by the single or multiple combined universal FISH probe is integrated at multiple points on the DG44 recombinant cell, the exogenous gene detected by the single or multiple combined universal FISH probe is integrated at single points and double points on the CHOK1GS-KO recombinant cell, and the chromosome doubling period can be judged according to the position of the fluorescence signal integrated at the double points.
Example 4 Universal FISH Probe for detecting integration and location of exogenous Gene on chromosome
1. Chromosome preparation
Monoclonal recombinant cell line JLK004DG44 (polypeptide-Fc fusion protein gene inserted into UCOE)-Expression plasmid of dhfr vector transfects CHO DG44 cell) and monoclonal recombinant cell line JLK004CHOK1 (expression plasmid of polypeptide-IgG 1Fc fusion protein gene inserted into pEE6.4 vector transfects CHOK1GS-KO cell) are respectively enlarged and cultured until the cell concentration is about 1 x 106Adding colchicine (final concentration of 0.5 μ g/ml) and incubating for 3 hr; collecting cells in a 15mL centrifuge tube, collecting cell precipitates, resuspending the cell precipitates by 8mL of 0.075mol/L potassium chloride solution, and swelling the cells in a water bath at 37 ℃ for 25 min; directly adding 2mL of stationary liquid, mixing, centrifuging for 10min at 1000g, discarding part of supernatant, leaving about 0.5mL of supernatant, adding 4mL of stationary liquid, mixing, fixing at room temperature for 20min, centrifuging for 10min at 1000g, and removing supernatant; repeatedly fixing once, centrifuging, discarding part of supernatant, reserving about 0.5mL of supernatant, and re-suspending the cells; 20 mul of cell suspension was smeared on the slide and dried at room temperature for further use.
2. Universal FISH probe for detecting integration and location of exogenous gene on chromosome of recombinant cell line
Placing the chromosome slide prepared in the step 1 in a denaturing solution (a 2 XSSC solution of 70% deionized formamide) at 78 ℃ for incubation for 6min, then sequentially dehydrating for 3min by precooled 70%, 90% and 100% ethanol respectively, and airing at room temperature; incubating in 0.01% pepsin (pH 2.0) solution at 37 deg.C for 5min, dehydrating with 70%, 90%, and 100% ethanol for 3min, and air drying at room temperature. Taking 20-30 ng of the probe prepared in the embodiment 2 of the invention (supplementing the probe hybridization solution to 22 mu L), denaturing at 98 ℃ for 10min, immediately placing on ice for cooling for 5min, then placing at 37 ℃ for prehybridization for 30min, dropwise adding to the treated chromosome slide, sealing, and incubating at 37 ℃ and saturation humidity for 16 hr. Then, putting the chromosome slide in a 2 XSSC/50% formamide solution at 43 ℃ for renaturation for 15min, washing the chromosome slide for 3 times by 2 XSSC, and airing at room temperature; dripping 100 μ L of 2 XSSC solution containing 3% BSA and 0.05% Tween-20 on the glass slide, incubating for 30min at 37 deg.C in dark, washing with 2 XSSC solution containing 0.05% Tween-20 for 2 times, sequentially adding 70%, 90% and 100% ethanol, dehydrating for 3min, and air drying at room temperature; then 100 μ L of FITC-Streptavidin (1: 50) or FITC-conjugated IgG Fraction monoclonal mouse anti-Digoxin (1: 50) is added dropwise, the mixture is incubated for 60min at 37 ℃ in the dark, the washing with 2 XSSC solution is carried out for 4 times, then the mixture is respectively removed by 70%, 90% and 100% ethanol for 3min, and then the mixture is placed in the dark and dried at room temperature; and finally, adding DAPI/antipade Solution to counterstain the cell nucleus, covering a slide, and placing the slide on an upright microscope for observation and image acquisition.
FIG. 3 shows the application of universal FISH probe to JLK004DG44 recombinant cell line chromosome FISH detection: a to H are respectively probes UCOE-dhfrrp(Whole plasmid Probe) AMPpcr-1～2、dhfrpcrDetecting the integration of the exogenous target gene in the chromosome of the JLK004CHOK1 cell line by combining-1-2, pCMV pcr-1-2, UCOE pcr-1-5, SV40pcr-1, IgG1 FCpcr-1-2 and probes AMP pcr-1-2 and dhfr pcr-1-2;
FIG. 4 shows the application of universal FISH probe in JLK004CHOK1 recombinant cell line chromosome FISH detection: a to G are probes pEE6.4rp (full plasmid probe), AMPpcr-1-2, GS pcr-1-3, pCMV pcr-1-2, SV40pcr-1, IgG1 FCpcr-1-2, and probes AMP pcr-1-2 and GS pcr-1-3 respectively, and are combined to detect integration of exogenous target genes in JLK004CHOK1GS-KO cell line chromosome.
The results of detecting the integration and localization of exogenous genes on chromosomes of a DG44 recombinant cell line and a CHOK1GS-KO recombinant cell line by using the universal FISH probe are shown in FIGS. 3 and 4, the fluorescence signals detected by using the universal FISH probe are consistent with those of a whole plasmid probe, and exogenous genes detected by using a single universal gene probe or a plurality of combined universal gene probes are integrated at the proximal plasmid end of a chromosome in a multi-point integration manner on a chromosome of a DG44 recombinant cell and are integrated at the proximal centromere of a chromosome in a point integration manner on a chromosome of a CHOK1GS-KO recombinant cell.
Combining the results of example 3, it was demonstrated that the universal probe of the present invention can be used in FISH assay to detect the integration and localization of a gene of interest on the chromosome of a recombinant cell instead of a whole plasmid probe.
Example 5 Universal FISH Probe kit for detecting integration and localization of foreign genes on chromosomes
Placing JLK004DG44 recombinant cell line interphase cells and chromosome slides prepared in examples 3 and 4 in 30ml DNA denaturation solution preheated to 78 ℃ for incubation for 6min, then dehydrating for 3min respectively by precooled 70%, 90% and 100% ethanol, and drying at room temperature; and then placing the chromosome in 30ml of chromosome peripheral tissue digestive juice to incubate for 5min at 37 ℃, dehydrating for 3min by 70%, 90% and 100% ethanol respectively in sequence, and airing at room temperature for later use. Taking 20-30 ng of the probe in the kit (supplementing the probe hybridization solution to 22 mu L), denaturing at 98 ℃ for 10min, immediately placing on ice for cooling for 5min, then placing in a prehybridization chamber at 37 ℃ for 30min, dripping into the treated cells and chromosome slides, sealing, and incubating at 37 ℃ and saturation humidity for 16 hr. Then, putting the slide into 30ml hybridization renaturation solution at 43 ℃ for renaturation for 15min, washing the slide for 3 times by 2 Xhybridization cleaning solution, and airing at room temperature; dripping 100 μ L antibody blocking solution on the slide, incubating at 37 deg.C in dark for 30min, washing with 2 × hybrid washing solution for 2 times, sequentially adding 70%, 90%, and 100% ethanol, respectively dehydrating for 3min, and air drying at room temperature; dripping 100 μ L probe labeled molecular antibody (1: 50), incubating at 37 deg.C in dark for 60min, washing with 2 × hybrid washing solution for 4 times, removing with 70%, 90%, and 100% ethanol for 3min, standing in dark, and volatilizing at room temperature; and finally, adding a cell nucleus/chromosome fluorescent staining solution, covering a slide, and placing the slide in an upright microscope for observation and image acquisition.
FIG. 5 is a universal FISH probe kit for FISH detection: a is interphase cell FISH, B is chromosome FISH.
The results of detecting the integration and location of the exogenous gene on the chromosome of the CHOK1 recombinant cell line by using the universal probe kit are shown in FIG. 5, and the results of detecting that the exogenous gene of the JLK004CHOK1 recombinant cell line is integrated on cells and cell chromosomes in a multi-point integration manner by using the universal FISH probe kit are located at the proximal plasmid end of a chromosome in the chromosome integration and location. It is demonstrated that the universal probe kit of the present invention has practical and practical applicability.
Sequence listing
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taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca 120
ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg 180
gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg 240
ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc 300
ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg 360
atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca 420
agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt 480
ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg 540
gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagaa 590
<210>3
<211>3051
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tttaccacta agccatctct ccagccctga gtcatggttt tagtgtgaga ggcatcattg 60
aattttctga gcacggccat cagggtagct ggcacaggtc ttcagataca aggagatagt 120
tataagaagg cagccatggc tgtggtgcac tagaaatgga gaaacagctt catcaggtga 180
cagaccagtc tgactctgtc ccatgattag aagccatctt gttacaaggt caaaataagt 240
tcattcctgt tttctgtaac acttgggttt gatcctgtcg tcaacccatt ttctggaatt 300
tgacatgttc catactccat tataccctga cttccaccct gataagatgt tctgccaagt 360
tcctgtgtag ccaacattcc cctggaaatc tctcttccct tggaaaccac ctagtcttag 420
aaattttgag ttatataaat tccacttcta tgtttgatgc tattctttaa aactccactt 480
tagggagata gccctgtctg atagaaaata aaacttgctt aatttgtcta aaagatttta 540
agtaatagtt tttacttttg ttccgtggga ttagtacagg gtgaaacaga ctcccgtgtt 600
tccagtgtga agtgagccac acactgcagt acaagttata tcagcaggtt ctgcctctgc 660
gcaatgaact tttgcttgtg tggacatcag ggtctgtgtg aagggaaggt cctatggcct 720
agttttatac tattcaacag tctgtccccg aagccctggt gctttattat tttgacaagc 780
ccctgctgct ggtattccac cctgctgcga gtcaaaaaag ttcctgtctc ggaaaaacaa 840
aacaaaacaa aacaaccaaa aaataaattt ttttttccca caggttctag tggaggtgct 900
cactaccaga aatcctacaa ataagcccat ctcatggatc agggtttacc tttgtaataa 960
tattaaatct gtgtgcatgt gcgcacgcat gtgttttatg cttgcatata tgtatacgca 1020
gccatggttt tctactgtcc cactcactct gtaacttact gagccatcca gctggtcctc 1080
taaatacatt tcaatgaaag ttttcattag cgtgaacgtg aaggtggtaa aatctgttag 1140
tgtgtgctta tgcctgtggt ttgcacctct agtctgaagg ttgctctttt caaatttttt 1200
atttatttac gtttttactt ctgagtcaga aactcataaa ggccatggcc tcgaattcgc 1260
tatgtagtca acgatgacct taaacttgtg accctctact tcgttagtgc tggaacccca 1320
agcttgctga gtacagagca ctttcagacc ggaactagat gtctacttcc tgttccgcct 1380
acattacagg ttgctaggtt acaccccccc tacgccgttt tagacgcaaa acttcatttc 1440
ccatgcaaaa cttcatttcc catgaacact tgcaagggtc gccgcgctgc gcggcgtcat 1500
tgctcccgcc ctatatacct acttccgccc gcgagccact tcctttcctt tcagcggcgc 1560
gcggctgcaa gatggcggtg cagatttcca agaagaggaa ggtaagcgtc tgggcccggt 1620
tcgggagtcc gccgcgggtt ctacaagtgc cagggaggcc tgtggctccg taatcagtcc 1680
tgtggagcgt ctggggccgc ctgccgtctc ttcgagcctc ggatggccgt agattgtgta 1740
ttgggccgga gccgggcgag tgctgtgtgc ctgggcaagg gagggacaaa ctcctcgagt 1800
tctggaccga ctcgaacacc gggcgcctcc agttccggac tagacacctt tgagcgtttc 1860
ttggtctcca taatagtaat cctgtggcac agttagaggg cgtgtgccat cagatctagt 1920
ccagtctctt tagtaagtga agtttagcag tcccttctct tagtcgcgtg atcctgcaag 1980
tggccatagt tgaaagccta cttactgact gctgccgtgt tcactcggga cccggagctg 2040
cagcgtccct gtggttatca tttcatgggg gaaaagtgtg caggttgcca ggtttagaaa 2100
tagatggtct gtcgtttgtg cttatgcaca cagatgataa acctgttttg agtcaggatt 2160
cctctcctat ccgaggtaca acttacagtc ccagctgtac atgtgctact tggagacaga 2220
tttttctttg tctcttgggt gtagattatg ccgtagagcc cttcgatgaa gaggtgatga 2280
cgagtctgag taggaagtgt tgtctttgtc caagatgcct cactatgctg cgttctgtgg 2340
cacagctgaa agcactgtgg tcaaaagaaa cttcctaaag atgaccaaga ggcatttgtc 2400
tgagaagggt tgctgctttt ctgtagggcc attgggcttg ctctgactaa ccctgtcttc 2460
acctcagagg taacttgttt cctttggttc agtttgtagc tgatggcatc ttcaaagctg 2520
agctgaatga atttctcact cgggagctgg ctgaagatgg ctactctgga gttgaagtcc 2580
gagttacacc aaccaggaca gaaatcatta ttttagccac caggtagaaa taccattgat 2640
tgtcacctgt aaatattgtg tgtactgaga tgctgtgtaa acttgggcca accaagcagt 2700
aaatctggcc tcagtgggtg taactgcttt gttagaactg catttgggaa gaacttacct 2760
tccatttaac ctgtgtgctg gcgttgtggt gggcggcagg tgggatcttg agtaaatggt 2820
tgcgcttccc ctctacagga cacagaatgt tcttggggag aagggtcgtc ggatcagaga 2880
gttgaccgca gttgtccaga agcgctttgg cttccctgaa ggcagcgtag aggtgagttc 2940
ctctgcttta tctcccgggg gttttagact gagttgggat gtggcttctg ctatagaatt 3000
gtacttctga aaacctgaca tggccagtga cagtcacagg tacttgatgc t 3051
<210>4
<211>858
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ccaatactta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 60
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 120
tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 180
gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 240
tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 300
tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 360
ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 420
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 480
ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 540
gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 600
ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 660
cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 720
ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 780
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 840
gaaatgttga atactcat 858
<210>5
<211>792
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
aaacaattca tccagtaaaa tataatattt tattttctcc caatcaggct tgatccccag 60
taagtcaaaa aatagctcga catactgttc ttccccgata tcctccctga tcgaccggac 120
gcagaaggca atgtcatacc acttgtccgc cctgccgctt ctcccaagat caataaagcc 180
acttactttg ccatctttca caaagatgtt gctgtctccc aggtcgccgt gggaaaagac 240
aagttcctct tcgggctttt ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa 300
tggagtgtct tcttcccagt tttcgcaatc cacatcggcc agatcgttat tcagtaagta 360
atccaattcg gctaagcggc tgtctaagct attcgtatag ggacaatccg atatgtcgat 420
ggagtgaaag agcctgatgc actccgcata cagctcgata atcttttcag ggctttgttc 480
atcttcatac tcttccgagc aaaggacgcc atcggcctca ctcatgagca gattgctcca 540
gccatcatgc cgttcaaagt gcaggacctt tggaacaggc agctttcctt ccagccatag 600
catcatgtcc ttttcccgtt ccacatcata ggtggtccct ttataccggc tgtccgtcat 660
ttttaaatat aggttttcat tttctcccac cagcttatat accttagcag gagacattcc 720
ttccgtatct tttacgcagc ggtatttttc gatcagtttt ttcaattccg gtgatattct 780
cattttagcc at 792
<210>6
<211>1026
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
ctatttcttt gccctcggac gagtgctggg gcgtcggttt ccactatcgg cgagtacttc 60
tacacagcca tcggtccaga cggccgcgct tctgcgggcg atttgtgtac gcccgacagt 120
cccggctccg gatcggacga ttgcgtcgca tcgaccctgc gcccaagctg catcatcgaa 180
attgccgtca accaagctct gatagagttg gtcaagacca atgcggagca tatacgcccg 240
gagtcgtggc gatcctgcaa gctccggatg cctccgctcg aagtagcgcg tctgctgctc 300
catacaagcc aaccacggcc tccagaagaa gatgttggcg acctcgtatt gggaatcccc 360
gaacatcgcc tcgctccagt caatgaccgc tgttatgcgg ccattgtccg tcaggacatt 420
gttggagccg aaatccgcgt gcacgaggtg ccggacttcg gggcagtcct cggcccaaag 480
catcagctca tcgagagcct gcgcgacgga cgcactgacg gtgtcgtcca tcacagtttg 540
ccagtgatac acatggggat cagcaatcgc gcatatgaaa tcacgccatg tagtgtattg 600
accgattcct tgcggtccga atgggccgaa cccgctcgtc tggctaagat cggccgcagc 660
gatcgcatcc atagcctccg cgaccggttg tagaacagcg ggcagttcgg tttcaggcag 720
gtcttgcaac gtgacaccct gtgcacggcg ggagatgcaa taggtcaggc tctcgctaaa 780
ctccccaatg tcaagcactt ccggaatcgg gagcgcggcc gatgcaaagt gccgataaac 840
ataacgatct ttgtagaaac catcggcgca gctatttacc cgcaggacat atccacgccc 900
tcctacatcg aagctgaaag cacgagattc ttcgccctcc gagagctgca tcaggtcgga 960
gacgctgtcg aacttttcga tcagaaactt ctcgacagac gtcgcggtga gttcaggctt 1020
tttcat 1026
<210>7
<211>385
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
atcatcatga tcagtcctgc tcctcggcca cgaagtgcac gcagttgccg gccgggtcgc 60
gcagggcgaa ctcccgcccc cacggctgct cgccgatctc ggtcatggcc ggcccggagg 120
cgtcccggaa gttcgtggac acgacctccg accactcggc gtacagctcg tccaggccgc 180
gcacccacac ccaggccagg gtgttgtccg gcaccacctg gtcctggacc gcgctgatga 240
acagggtcac gtcgtcccgg accacaccgg cgaagtcgtc ctccacgaag tcccgggaga 300
acccgagccg gtcggtccag aactcgaccg ctccggcgac gtcgcgcgcg gtgagcaccg 360
gaacggcact ggtcaacttg gccat 385
<210>8
<211>600
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt ccttcgggca cctcgacgtc 60
ggcggtgacg gtgaagccga gccgctcgta gaaggggagg ttgcggggcg cggaggtctc 120
caggaaggcg ggcaccccgg cgcgctcggc cgcctccact ccggggagca cgacggcgct 180
gcccagaccc ttgccctggt ggtcgggcga gacgccgacg gtggccagga accacgcggg 240
ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt tgctgcgcgg ccagccggga 300
accgctcaac tcggccatgc gcgggccgat ctcggcgaac accgcccccg cttcgacgct 360
ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc gcgacccaca ccttgccgat 420
gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg gtgacccgct cgatgtggcg 480
gtccgggtcg acggtgtggc gcgtggcggg gtagtcggcg aacgcggcgg cgagggtgcg 540
tacggcccgg gggacgtcgt cgcgggtggc gaggcgcacc gtgggcttgt actcggtcat 600
<210>9
<211>658
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg 60
gaagccatca cagacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc 120
ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac 180
gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc 240
aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata 300
tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc 360
agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc 420
gtctttcatt gccatacgga attccggatg agcattcatc aggcgggcaa gaatgtgaat 480
aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc 540
cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc 600
tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatt 658
<210>10
<211>226
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc 60
tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag 120
cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt 180
cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atctta 226
<210>11
<211>565
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
catggttcga ccattgaact gcatcgtcgc cgtgtcccaa aatatgggga ttggcaagaa 60
cggagaccta ccctggcctc cgctcaggaa cgagttcaag tacttccaaa gaatgaccac 120
aacctcttca gtggaaggta aacagaatct ggtgattatg ggtaggaaaa cctggttctc 180
cattcctgag aagaatcgac ctttaaagga cagaattaat atagttctca gtagagaact 240
caaagaacca ccacgaggag ctcattttct tgccaaaagt ttggatgatg ccttaagact 300
tattgaacaa ccggaattgg caagtaaagt agacatggtt tggatagtcg gaggcagttc 360
tgtttaccag gaagccatga atcaaccagg ccacctcaga ctctttgtga caaggatcat 420
gcaggaattt gaaagtgaca cgtttttccc agaaattgat ttggggaaat ataaacttct 480
cccagaatac ccaggcgtcc tctctgaggt ccaggaggaa aaaggcatca agtataagtt 540
tgaagtctac gagaagaaag actaa 565
<210>12
<211>1122
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
atggccacct cagcaagttc ccacttgaac aaaggcatca agcaaatgta catgtccctg 60
ccccagggtgagaaagtcca agccatgtat atctgggttg atggtaccgg agaaggactg 120
cgctgcaaga cccgtaccct ggactgtgag cccaagtgtg tggaagagtt acctgagtgg 180
aactttgatg gctctagtac ctttcagtct gaaggctcca acagcgacat gtacctccat 240
cctgttgcca tgtttcgaga ccccttccgc aaagacccca acaagctggt gctatgtgaa 300
gttttcaagt ataaccggaa acctgcagag tccaacttga ggcacatctg taaacggata 360
atggacatgg tgagcaacca gcacccctgg tttggaatgg agcaggaata tactcttatg 420
ggaacagacg gccacccatt tggttggcct tccaatggct tccctggacc ccaaggcccg 480
tattactgcg gtgtgggagc agacaaggcc tacggcaggg acatcgtgga ggctcactac 540
cgggcctgct tgtatgctgg agtcaagatt acggggacaa atgcggaggt tatgcctgcc 600
cagtgggaat tccagatagg accctgtgag gggatccgaa tgggagatca tctttggata 660
gcccgtttta tcttgcatcg ggtgtgcgaa gactttgggg tgatagcaac ctttgacccc 720
aagcccattc cagggaactg gaatggtgca ggctgccaca ccaacttcag caccaaggcc 780
atgcgggagg agaatggtct gaagtggatt gaggaggcca ttgacaaact gagcaagagg 840
caccagtacc acattcgcgc ctacgatccc aaggggggcc tggacaacgc ccggcgtctg 900
actggattcc acgaaacctc caacatcaac gacttttctg ccggtgttgc caaccgcggt 960
gccagtatcc gcattccccg gactgtcggc caggagaaga agggctactt tgaagaccgt 1020
cggccttctg ccaattgtga cccctatgcg gtgacagaag ccatcgtccg cacgtgtctc 1080
ctcaacgaaa caggcgacga gcccttccag tacaaaaact aa 1122
<210>13
<211>687
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
gagtccaaat atggtccccc atgcccatca tgcccagcac ctgagttcct ggggggacca 60
tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 120
gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 180
gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 240
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 300
tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 360
gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 420
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 480
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 540
gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 600
gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 660
aagagcctct ccctgtctcc gggtaaa 687
<210>14
<211>684
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc aggaccgtca 60
gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 120
acgtgcgtgg tggtggacgt gagccacgaa gaccccgagg tccagttcaa ctggtacgtg 180
gacggcgtgg aggtgcataa tgccaagaca aagccacggg aggagcagtt caacagcacg 240
ttccgtgtgg tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac 300
aagtgcaagg tctccaacaa aggcctccca gcccccatcg agaaaaccat ctccaaaacc 360
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga ggagatgacc 420
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga catcgccgtg 480
gagtgggaga gcaatgggca gccggagaac aactacaaga ccacacctcc catgctggac 540
tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 600
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 660
agcctctccc tgtctccggg taaa 684
<210>15
<211>687
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
gctgagtcca agtatggccc tccctgccct ccttgccctg ctcctgaggc tgctggaggc 60
cctagcgtgt tcctgttccc ccctaagcct aaggacaccc tgatgatttc ccggaccccc 120
gaggtgacct gtgtggtggt ggatgtgtcc caggaggacc ctgaagtgca gttcaactgg 180
tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgggaaga gcagttcaac 240
agcacctaca gggtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaaa 300
gagtacaagt gcaaggtgag caataagggc ctgccctcct ccatcgagaa gaccatttcc 360
aaggccaagg gccagcccag ggaaccccag gtgtacaccc tccctcccag ccaggaggag 420
atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc ctccgacatt 480
gccgtcgagt gggaaagcaa cggccagccc gagaacaatt acaagaccac accccccgtg 540
ctggacagcg atggcagctt tttcctgtac tccaggctga ccgtcgacaa gtccaggtgg 600
caggagggca acgtcttctc ctgctccgtg atgcatgagg ccctgcacaa ccactacacc 660
cagaagtccc tgtccctgag cctgggc 687
Claims (8)
1. The universal FISH probe for detecting the integration and the location of a foreign gene is characterized in that the universal FISH probe is prepared by taking a non-target gene sequence of a eukaryotic expression plasmid as a template, and the non-target gene sequence is selected from one or more of the following genes:
the SV40 promoter has the gene sequence of SEQ ID NO. 1;
the gene sequence of the pCMV promoter is SEQ ID NO 2;
a gene of the open element of the pan chromatin, the gene sequence of which is SEQ ID NO 3;
ampicillin resistance gene, the gene sequence of which is SEQ ID NO 4;
kanamycin resistance gene, the gene sequence of which is SEQ ID NO. 5;
hygromycin resistance gene, the gene sequence is SEQ ID NO 6;
the gene sequence of the bleomycin resistance gene is SEQ ID NO. 7;
puromycin resistance gene, the gene sequence of which is SEQ ID NO 8;
the chloramphenicol resistance gene has the gene sequence of SEQ ID NO 9;
the terminator poly A sequence with the gene sequence of SEQ ID NO 10;
a dihydrofolate reductase selective marker gene, the gene sequence of which is SEQ ID NO. 11;
a glutamine synthetase selective marker gene, the gene sequence of which is SEQ ID NO 12;
human IgG1 constant region gene sequence, which is SEQ ID NO 13;
human IgG2 constant region gene sequence, which is SEQ ID NO. 14;
human IgG4 constant region gene sequence, which is SEQ ID NO. 15.
2. The universal FISH probe for detecting integration and localization of foreign genes according to claim 1, wherein the universal FISH probe is prepared using a combination of non-target gene sequences of eukaryotic expression plasmids as templates, wherein the non-target gene sequences are selected from one or more of the following combinations of sequences:
the combination of the AMP and pCMV gene sequences,
the combination of the AMP and DHFR gene sequences,
the combination of the AMP and GS gene sequences,
AMP and human IgG constant region gene sequences,
AMP, pCMV and SV40poly A gene sequence combination,
the gene sequence combination of AMP, pCMV and SV40 promoters,
AMP, pCMV, and DHFR gene combinations,
the AMP, pCMV and GS gene combinations,
AMP, pCMV, and UCOE genes,
AMP, pCMV, SV40poly A and DHFR gene combinations,
AMP, pCMV, SV40poly A and GS gene combinations.
3. The universal FISH probe for detecting integration and localization of a foreign gene according to claim 1 or 2, wherein the universal FISH probe is labeled by PCR, nick translation, random primer and/or DNA sequence synthesis.
4. The universal FISH probe for detecting integration and localization of foreign genes according to claim 3, wherein the PCR method is performed by the following steps:
in a high-fidelity polymerase system, any universal DNA sequence or combination thereof, reaction buffer solution, specific upstream and downstream primers, high-fidelity DNA polymerase, dNTP, biotin, digoxin or fluorescent molecules FAM, FITC, Cy3, Cy5 and dATP/dCTP/dUTP marked by OBEA are sequentially added, and a PCR instrument is put into the high-fidelity polymerase system for expansion to obtain a DNA fragment with the product length of 200-1000 bp.
5. The universal FISH probe for detecting integration and localization of foreign genes according to claim 3, wherein the random primer method is performed by the following steps:
a random primer DNA labeling kit is used, any one or a combination of 100-2000 ng of universal DNA sequences is taken and placed in a water bath or a PCR instrument at 95-100 ℃ for denaturation for 5min, the mixture is immediately placed in an ice bath, dNTP, a random primer, a reaction buffer solution, a DNA polymerase solution, biotin, digoxin or fluorescent molecules FAM, FITC, Cy3, Cy5 and OBEA labeled dATP/dCTP/dUTP are added, and the mixture is placed at 37 ℃ for reaction for 1-24 h.
6. The universal FISH probe for detecting integration and localization of foreign genes according to claim 3, wherein the nick translation method is performed by the following steps:
any universal DNA double-stranded sequence or the combination thereof is used as a template, dATP, dTTP, dCTP and dGTP are added under the condition of a nick translation enzyme system, biotin, digoxin or fluorescent molecules FAM, FITC, Cy3, Cy5 and OBEA labeled dATP/dCTP/dUTP are added, and the mixture is placed at 37 ℃ for reaction for 1-24 h.
7. Use of a universal FISH probe according to any of claims 1 to 6 for detecting integration of a foreign gene in a mammalian cell or for confirming chromosomal localization of a foreign gene, complementary to a recombinant expression plasmid sequence comprising a foreign gene.
8. Use of a universal FISH probe according to any of claims 1 to 6 in combination with an in situ hybridization reaction system or reagents as a detection kit for detecting integration and localization of foreign genes in research recombinant cells and in cells of biopharmaceutical engineering.
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