Cre reporting system based on single-domain antibody labeling and application of Cre reporting system

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a Cre reporting system based on single-domain antibody labeling and application of the Cre reporting system. According to the shark single-domain antibody with Cre specificity, when a DNA sequence of dLox-EGFP is provided, Cre can be marked and reported in real time, and reporter genes can be expressed only when Cre exists in cells, so that a strictly regulated gene expression platform is provided. The mark is completely reversible and is completely compatible with the currently used Cre / loxp system.

Description

Technical Field

[0001] The present invention belongs to the field of gene engineering technology, and specifically relates to a Cre reporter system based on single-domain antibody labeling and its application. The reporter system can realize real-time labeling and reporting of Cre recombinase protein. Background Art

[0002] Cre recombinase is a DNA recombinase from bacterial phage P1, which is widely used in genetic engineering and molecular biology research. Cre recombinase can specifically recognize and cut loxP sites on DNA, resulting in rearrangement of inserted or deleted gene fragments. By binding Cre recombinase to the target genome at the loxP site, researchers can precisely control the knockout, activation or regulation of genes. The advantage of the Cre / loxP system is its spatiotemporal controllability, which can activate Cre recombinase in specific cells or tissues, thereby precisely manipulating the expression of target genes. This system has a wide range of applications in multiple research fields such as gene knockout, conditional gene expression, and cell tracking, especially in cancer research, neuroscience, developmental biology and other fields.

[0003] Many transgenic mice contain Cre driven by specific gene promoters; however, it remains challenging to sort target cells expressing Cre and the genes they drive for further study. Previous studies have shown that the LoxP-stop-LoxP strategy can mark cells expressing Cre, but this marking is irreversible and also marks daughter cells. For example, in Sox2-Cre mice, both Sox2-positive neural progenitor cells (NPCs) and their differentiated daughter cells exhibit EGFP signals using the LoxP-stop-LoxP method, preventing researchers from specifically isolating Sox2-positive NPCs.

[0004] Although Cre recombinase technology has made significant progress, there are still some challenges in practical applications, especially in real-time labeling. Currently commonly used Cre labeling methods include fluorescent protein labeling, immunohistochemistry (IHC), and immunofluorescence staining. By fusing Cre recombinase with fluorescent proteins (such as GFP, RFP, etc.), researchers can observe the localization and activity of Cre under a fluorescence microscope. However, this method may affect the function of Cre recombinase. Immunohistochemistry and immunofluorescence staining are labeled by specific anti-Cre antibodies, which can detect the expression and localization of Cre in tissue sections, but these methods are usually indirect and cannot monitor the activity of Cre in real time. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a Cre reporter system based on single-domain antibody labeling, aiming to solve one of the above technical problems to at least a certain extent or at least provide a technical option.

[0006] Therefore, the present invention discloses a Cre reporter system based on single domain antibody labeling, wherein the system comprises three plasmids, namely:

[0007] (1) Plasmid 1: a plasmid for expressing a Cre-specific single-domain antibody and VP64 fusion, the plasmid comprising the nucleotide sequences of the Cre-specific single-domain antibody and VP64;

[0008] (2) Plasmid 2: a plasmid for expressing dLox-EGFP, which includes the nucleotide sequence of dLox-EGFP;

[0009] (3) Plasmid 3: a plasmid for Cre expression, which includes the nucleotide sequence of Cre.

[0010] Preferably, the amino acid sequence of the variable region VNAR of the Cre-specific single-domain antibody of the present invention is as shown in SEQ ID NO.1, wherein the optimized nucleotide sequence of the Cre-specific single-domain antibody is as shown in SEQ ID NO.6.

[0011] Preferably, the amino acid sequence of VP64 described in the present invention is as shown in SEQ ID NO.8, wherein the nucleotide sequence of VP64 is as shown in SEQ ID NO.9.

[0012] Preferably, the nucleotide sequence of dLox in the dLox-EGFP of the present invention is as shown in SEQ ID NO.7.

[0013] Preferably, the nucleotide sequence of dLox-EGFP in the expression vector of the present invention further includes a CMV enhancer.

[0014] Preferably, when the system of the present invention is used in 293T cells, plasmid 1, plasmid 2 and plasmid 3 are transfected at a mass ratio of 1:1:1.

[0015] Preferably, when the system of the present invention is used in mice, only plasmid 1 and plasmid 2 need to be knocked into the mouse genome, and plasmid 3 is not required.

[0016] Preferably, the vectors of plasmid 1, plasmid 2 and plasmid 3 of the present invention are all pcDNA3.1.

[0017] In one aspect, the present invention also discloses an application of the system in Cre real-time expression marker reporting.

[0018] Beneficial effects of the invention: The present invention designs a single-domain antibody-linked VP64 method that marks cells only when Cre is expressed and is reversible. Our method involves the development of a new gene regulation system based on a dead-Lox (dLox) cascade sequence. Cre recombinase can bind to the dLox region, but it cannot cut it. In this system, we use a single-domain antibody that specifically binds to Cre. The advantages of this single-domain antibody are its small size, excellent stability, strong thermal stability and adaptability to extreme environments. This single-domain antibody-VP64 is recruited to the dLox site by Cre, thereby activating EGFP expression. In our system, EGFP is expressed only when Cre is present in the cell, providing a strictly regulated gene expression platform.

[0019] In the subsequent animal application process, we only need to construct a mouse containing the single-domain antibody-VP64 and dLox-EGFP genotype, and mate this genotype mouse with other Cre genotype mice to obtain offspring, so that our Cre labeling system can be fully compatible with the current Cre mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Cre-VP64 and mCherry-VP64 report the fluorescence of EGFP, verifying the feasibility and rigor of the system.

[0021] Figure 2 This is the SDS-PAGE result of Cre antigen purification.

[0022] Figure 3 This is a statistical analysis of shark-derived single-domain antibodies that were screened for Cre specificity by NGS.

[0023] Figure 4 This is the result of phage ELISA screening of Cre-specific shark-derived single-domain antibodies through phage display.

[0024] Figure 5 This is the result of monoclonal ELISA screening of Cre-specific shark-derived single-domain antibodies by phage display.

[0025] Figure 6 These are partial peptide fragments of Cre-specific shark-derived single-domain antibodies detected by mass spectrometry in shark serum after immunization.

[0026] Figure 7 Comparison between the Loxp-STOP-Loxp marking system (left) and the Cre marking system (right).

[0027] Figure 8 It is a schematic diagram of the structure of dLox.

[0028] Fig. 9 This is a fluorescent image of Cre intracellular labeling using a Cre-specific antibody.

[0029] Fig.10 This is a flow cytometric plot of Cre intracellular labeling using a Cre-specific antibody. DETAILED DESCRIPTION

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0031] The present invention is described or further illustrated in detail by the following examples, and those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the above. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.

[0032] In the embodiments of the present invention, conventional experimental methods are used without further explanation. The processes involved in the embodiments without further explanation are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field. The reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially, and therefore will not be described in detail.

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0034] The advantages of single-domain antibodies are their small size, excellent stability, strong thermal stability and adaptability to extreme environments, and they can be expressed intracellularly and exert functional activity. The present invention designs a single-domain antibody-linked VP64 method that can mark Cre-expressing cells in real time and is reversible. Our method involves the development of a new gene regulation system based on a dead-Lox (dLox) cascade sequence. The Cre recombinase can bind to the dLox region, but it cannot cut it. This single-domain antibody-VP64 is recruited to the dLox site by Cre, thereby activating EGFP expression. In our system, EGFP is expressed only when Cre is present in the cell, thus providing a strictly regulated gene expression platform that is compatible with the current Cre system and has great application value.

[0035] Example 1: System feasibility verification

[0036] To verify the rigor of our system, we transfected Cre-VP64 and dLox-EGFP into 293T cells as a test, and transfected mCherry-VP64 and dLox-EGFP as a control (mCherry is a red fluorescent protein that theoretically does not bind to Cre and is used as a negative control here). Co-transfection was performed using Lipofectamine 2000 (Thermo). One day before transfection, cells were passaged into a 6 cm dish to a cell density of 70%-90% before transfection.

[0037] For each transfection sample, prepare the complex in two EP tubes as follows: add 4 μg of each plasmid DNA to be transfected to 500 μl of serum-free Opti-MEM I reduced serum medium (or other serum-free medium) and mix gently. Dilute 12 μl of Lipo2000 in 488 μl of Opti-MEM I medium. Incubate at room temperature for 5 minutes.

[0038] After 5 minutes of incubation, the diluted DNA was gently mixed with diluted Lipo2000 (total volume = 1000 μl) and incubated at room temperature for 20 minutes. During the 20 minutes of incubation at room temperature, the cell medium was changed. 1000 μl of the complex was added dropwise to the dish. The dish was gently mixed by shaking it back and forth, and the cells were placed in a cell culture incubator and cultured for 48 hours. After 48 hours of transfection, the results were as follows Figure 1 As shown, it can be seen that the Cre-VP64 group can successfully report EGFP, while the mCherry-VP64 transfection group does not report EGFP.

[0039] Example 2: Antigen purification and immunization of bamboo sharks

[0040] 1. Construction of recombinant expression plasmid: The gene sequence (Gene ID: 2777477) was inserted into the expression vector pET-28a-TrxA-SUMO by double enzyme digestion to obtain the pET-28a-Cre recombinant plasmid, and then the recombinant plasmid was transformed into BL21Star (DE3) cells.

[0041] 2. Induced expression and purification of recombinant proteins

[0042] 1. Inoculate the recombinant expression bacteria BL21 Star (DE3)-pET-28a-Cre into LB medium containing Amp resistance at a ratio of 1:100, and culture at 37°C and 220 rpm for 4 hours.

[0043] 2. Add IPTG to make the final concentration of IPTG 0.1-0.5 mM, preferably 0.2 mM, and continue culturing at 37°C for 4-6 h (preferably 5 h) and 16°C for 14-16 h (preferably 16 h).

[0044] 3. After induction, centrifuge the bacterial pellet and resuspend it in 1×PBS (40ml PBS per 1L cells), and use a high-pressure cell disruptor to lyse it at a pressure of 800bar. After centrifugation, incubate the lysate supernatant with Ni-NTA beads at 4°C for 3 hours, wash 3 times with 50mM imidazole solution, and elute with 300mM imidazole solution. Add SUMO protease (Ulp1) and digest it at 30°C for 2h, and remove the digested solution using a Ni-NTA column. The SDS-PAGE test results of the purified Cre antigen are as follows: Figure 2 As shown, its purity is above 90%, which is suitable for subsequent immune experiments.

[0045] Cre antigen was diluted to 1 μg / ml and mixed with an equal volume of biphasic adjuvant to immunize bamboo sharks. The inoculation site was the intersection of the fins and abdomen on both sides. The control group used BSA to immunize sharks. Immunization was performed every two weeks, with the mass of the antigen being 100 μg each time, for a total of 8 times.

[0046] Example 3: Screening of Cre-specific shark-derived single-domain antibodies by next-generation sequencing (NGS)

[0047] The spleen and PBMC of the sharks were taken after immunization, and RNA from the spleen and PBMC of the sharks was extracted for RT-PCR to obtain cDNA libraries. The cDNA libraries were constructed by high-throughput sequencing (NGS) using the VNAR library construction primers. The sequencing libraries were prepared using the method recommended by the manufacturer. Ultra TM II DNA library preparation kit (NEB, USA). Indexes were added to both ends of each library sample for subsequent differentiation. The library was quantified by qPCR (concentration of 1.5 nM), and only qualified libraries were submitted to the Illumina platform for sequencing using the PE250 strategy based on the required effective library concentration and the required data volume.

[0048] The data from the machine were analyzed and processed to obtain the complete shark VNAR sequence obtained by the final splicing of each group ( Figure 3 ). About 5000-7000 full-length VNAR sequences were screened from the Cre and BSA immunization groups, and the classification of these VNARs revealed that most of them belonged to the IgNAR1 type. NGS can quickly obtain the sequence and abundance information of single-domain antibodies produced by sharks after immunization, greatly saving time and cost.

[0049] Example 3: Screening of Cre-specific shark-derived single domain antibodies by phage display

[0050] The VNAR library for phage display was prepared using a two-round PCR method using VNAR library construction primers. PCR products of 500 to 600 bp were recovered in the first PCR reaction, and products of 200 to 400 bp were recovered in the second PCR reaction. The recovered PCR products were digested with SfiI and connected to the p3RdV vector, and then transformed into SS320 cells. M13KO7 was used to assist phage packaging in SS320. The resulting phage library was screened and enriched for 3-4 rounds using Cre antigen. It can be seen from phage ELISA that the library showed significant enrichment from the first round of panning, and the degree of enrichment of the library tended to be stable from the second to the third round of panning ( Figure 4 ). A total of 88 single clones were selected from the third and fourth round panning plates with good panning enrichment for IPTG induction, and the supernatant after induction was tested by ELISA to detect the absorbance value of OD450. The red dotted line is the average absorbance value of all clones, and the green dots are the absorbance values ​​of the wells with absorbance values ​​higher than the average of the whole plate ( Figure 5 ).

[0051] Example 4: Detection of Cre-specific shark-derived single-domain antibody peptides from immunized shark serum using mass spectrometry

[0052] Serum was separated from shark blood, incubated with Avi-labeled TrxA-SUMO protein at room temperature for 2 hours, and then streptavidin magnetic beads were added for overnight incubation at 4°C. The next day, the magnetic beads were removed by a magnetic rack to remove the IgNAR bound to the TrxA-SUMO tag. The Avi-labeled TrxA-SUMO-Cre antigen was added to the supernatant and incubated at room temperature for 2 hours, then streptavidin magnetic beads were added again and incubated overnight at 4°C. The magnetic beads were washed 3-4 times with 1×PBS (pH 7.4) containing 0.05% Tween-20, the bound IgNAR was eluted, and mass spectrometry was performed after digestion. Subsequently, the obtained peptides were compared with the high-throughput sequencing results to find the corresponding complete IgNAR antibody sequence. The results are as follows. Figure 6 As shown in the figure, the diversity of VNAR is mainly composed of CDR1, HV2, HV4 and CDR3, and the sequences of the remaining peptides are relatively conservative. Among them, the diversity of the sequence at CDR3 is the richest, and it contributes the most to the types of VNAR.

[0053] Based on the above results, this study screened and obtained 5 better Cre-specific shark-derived single domain antibodies, which were labeled as Cre-specific shark-derived single domain antibody 1 to Cre-specific shark-derived single domain antibody 5, and the amino acid sequences of their variable regions VNAR are shown in SEQ ID NO.1 to SEQ ID NO.5, respectively.

[0054] Example 5: Construction of single domain antibody eukaryotic expression vector

[0055] The VNAR sequence of the Cre-specific shark-derived single-domain antibody 1 obtained by screening was optimized according to human codons (the optimized nucleotide sequence is shown in SEQ ID NO.6), and the optimized DNA sequence was synthesized. The synthesized DNA was cloned into the expression vector pcDNA3.1-VP64 using the Hieff Clone One Step Cloning Kit, and then transformed into DH5α cells, clones were picked and sequenced to obtain positive clones.

[0056] Inoculate DH5α-pcDNA3.1-VNAR-VP64 into 100ml LB medium and culture for 14-16h. Centrifuge at 5000×g for 10min at room temperature to collect the bacteria. Add 500μl of Solution I / RNase A mixture to the bacterial pellet and fully resuspend the bacterial pellet. Add 500μl of Solution II and gently mix 8-10 times. Add 250μl pre-cooled N3 Buffer and gently mix several times until a white flocculent precipitate is formed. Centrifuge at 13000×g for 10min at room temperature. Transfer the supernatant to a new centrifuge tube, add 0.1 times the volume of the supernatant ETR Solution to the lysate, mix upside down 10 times, and then ice bath for 10min. Place the lysate in a 42℃ water bath for 5min and centrifuge at 12000×g for 3min at room temperature. Transfer the supernatant to a new centrifuge tube, add 0.5 times the volume of anhydrous ethanol of the supernatant, turn it upside down 6-7 times, and let it stand at room temperature for 1-2 minutes. After the adsorption column is balanced, transfer all the mixed solutions to the centrifuge column one by one. Add 500μl HBC Buffer to the centrifuge column, centrifuge at 12000×g for 1min at room temperature, and discard the filtrate. Add 700μl DNA Washing Buffer, centrifuge at 12000×g for 1min at room temperature, and discard the filtrate. Repeat this three times. Add 50-100μl Endo-Free-Buffer to the adsorption column, let it stand at room temperature for 1min, and centrifuge at 12000×g for 1min to elute the plasmid.

[0057] Example 6: Design of Cre Marking System

[0058] The application schematic diagram of the Cre reporter system based on single domain antibodies designed in the present invention is as follows: Figure 7 As shown, in this system, a Cre-specific single domain antibody (the amino acid sequence of the variable region VNAR is shown in SEQ ID NO.1) will be recruited by Cre to the dLox site (the nucleotide sequence is shown in SEQ ID NO.7), and VP64 (wherein the amino acid sequence of VP64 is shown in SEQ ID NO.8 and the nucleotide sequence is shown in SEQ ID NO.9) expressed by fusion with the Cre-specific single domain antibody will drive the reporter gene to report Cre in real time.

[0059] In this system, Figure 7 We developed a novel gene regulation system based on the dead-Lox (dLox) cascade sequence. The dLox construct contains LoxP, Lox66, and Lox71 recognition sites, as well as a mutated spacer sequence that replaces the promoter of the EGFP reporter gene. The dLox sequence can be recognized and bound by the Cre recombinase, but cannot be cut. To enhance the reporter signal, we inserted the CMV enhancer before the dLox sequence.

[0060] For subsequent application in mice, we knocked the nucleotide sequence of Cre-specific single-domain antibody-VP64 fusion expression and the nucleotide sequence of dLox-EGFP into the mouse genome to obtain this strain of mice, and mated them with the Cre mice to be studied to obtain heterozygous mice. In this type of mice, this system can be used to specifically edit the Cre protein.

[0061] For example, in Sox2-Cre mice, when the traditional LoxP-stop-LoxP method is used to mark Cre, Sox2-positive neural progenitor cells and their differentiated daughter cells all show EGFP signals. Even if the daughter cells may not express Cre after differentiation, they will still report EGFP, resulting in false positives. However, the VP64 method connected to the nanoantibody we designed only marks cells when Cre is expressed and is reversible. If Cre is not expressed, it will not be reported, and there will be no false positives.

[0062] Example 7: Testing the application of this system in 293T cells

[0063] In order to detect the reporting ability of this system in mammalian cells, we co-transfected 293T cells (plasmid mass ratio of 1:1:1) with the Cre-specific single-domain antibody constructed in Example 5 and the VP64 fusion expression plasmid (pcDNA3.1-VNAR-VP64 plasmid), Cre expression plasmid (pcDNA3.1-Cre plasmid) and dLox-EGFP (pcDNA3.1-dLox-EGFP plasmid), and then detected the reporting status of the reporter gene 48 hours after transfection. Plasmid co-transfection was performed using Lipofectamine 2000 (Thermo). One day before transfection, cells were passaged into a 6 cm dish so that the cell density before transfection was 70%-90%.

[0064] For each transfection sample, prepare the complex in two EP tubes as follows: add 4 μg of each transfection plasmid DNA to 500 μl of serum-free Opti-MEM I reduced serum medium (or other serum-free medium) and mix gently. Dilute 30 μl of Lipo2000 in 488 μl of Opti-MEM I medium. Incubate at room temperature for 5 minutes.

[0065] After 5 minutes of incubation, the diluted DNA was gently mixed with diluted Lipo2000 (total volume = 1000 μl) and incubated at room temperature for 20 minutes. Cell culture medium was changed during the 20-minute incubation at room temperature. 1000 μl of the complex was added dropwise to the dish. The dish was gently mixed by shaking it back and forth, and the cells were placed in a cell culture incubator and cultured for 48 hours.

[0066] The results are as follows Fig. 9 As shown, after 48 hours of transfection, the cells were placed under a fluorescence microscope for observation, and it was found that the cells expressing Cre could successfully drive the expression of the reporter gene.

[0067] Example 8: Flow cytometry

[0068] 1. Preheat the cell culture medium and 1×PBS in a 37℃ water bath, and place them in a sterilized clean bench for later use. 2. Discard the culture medium in the cell culture dish, add 3mL 1×PBS to rinse the cells, and repeat once. 3. Add an appropriate amount of 0.25% trypsin solution, incubate at 37℃ until the intercellular gap of the adherent cells becomes larger, and when the cells tend to become round, add 1mL of cell culture medium, and shake the culture dish to quickly terminate the reaction. 4. Use a pipette to carefully blow the adherent cells. Transfer to a 15mL centrifuge tube, centrifuge at 1000rpm at room temperature for 3min, and discard the supernatant. 5. Add 1mL of 1×PBS containing 2% FBS to resuspend the cells, filter the cell suspension using a 200-mesh cell sieve, and place the filtered cell suspension in a flow tube for on-machine detection.

[0069] The results are as follows Fig.10 As shown, the flow cytometry results were consistent with the fluorescence observation results, and some cells reported EGFP.

[0070] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A Cre reporter system based on single domain antibody labeling, characterized in that: The system includes three plasmids, namely: (1) Plasmid 1: a plasmid for expressing a Cre-specific single-domain antibody and VP64 fusion, the plasmid comprising the nucleotide sequences of the Cre-specific single-domain antibody and VP64; (2) Plasmid 2: a plasmid for expressing dLox-EGFP, which includes the nucleotide sequence of dLox-EGFP; (3) Plasmid 3: a plasmid for Cre expression, which includes the nucleotide sequence of Cre.

2. The system according to claim 1, characterized in that The amino acid sequence of the variable region VNAR of the Cre-specific single-domain antibody is shown in SEQ ID NO.1, wherein the optimized nucleotide sequence of the Cre-specific single-domain antibody is shown in SEQ ID NO.

6.

3. The system according to claim 1, characterized in that The amino acid sequence of VP64 is shown in SEQ ID NO.8, wherein the nucleotide sequence of VP64 is shown in SEQ ID NO.

9.

4. The system according to claim 1, characterized in that The nucleotide sequence of dLox in the dLox-EGFP is shown in SEQ ID NO.

7.

5. The system according to claim 1, characterized in that The nucleotide sequence of dLox-EGFP in the expression vector also includes a CMV enhancer before it.

6. The system according to claim 1, characterized in that When the system is used in 293T cells, plasmid 1, plasmid 2 and plasmid 3 are transfected at a mass ratio of 1:1:

1.

7. The system according to claim 1, characterized in that When the system is used in mice, only plasmid 1 and plasmid 2 need to be knocked into the mouse genome, and plasmid 3 is not required.

8. The system according to claim 1, characterized in that The vectors of plasmid 1, plasmid 2 and plasmid 3 are all pcDNA3.

1.

9. Use of the system as claimed in claim 1 in Cre real-time expression marker reporting.

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