Ratio type cAMP fluorescent probe RaCamp and construction method of transgenic mouse

Abstract

The invention belongs to the technical field of GPCR drug research and development, and discloses a ratio type cAMP fluorescent probe RaCamp and a construction method of a transgenic mouse, the probe RaCamp is formed by series fusion of G-Flamp2 fluorescent protein and mCherry fluorescent protein through a flexible linker, and the preparation method of the probe RaCamp comprises the following steps: S1, obtaining an mCherry fragment; s2, a G-Flamp2 linear carrier is obtained; s3, homologous recombination; s4, converting and screening; the construction method of the transgenic mouse comprises the following steps: T1, designing a carrier; t2, microinjection of fertilized eggs; t3, embryo transplantation and reproduction; the invention has the beneficial effects that mCherry is added as an internal reference protein on the premise of keeping the sensitivity of the original G-Flamp2 probe, and the selected connecting peptide (GGGGS) 2 enables the expression quantity of the two proteins in cells to be accurately controlled to be 1: 1, so that the problem of background interference in cell observation of in-vivo imaging is well solved. And the sequence is inserted into a Rosa26-LSL box, so that the construction of a Rosa26-LSL-RaCamp mouse is realized.

Description

Technical Field

[0001] This invention relates to the field of GPCR drug development technology, specifically to a ratio-type cAMP fluorescent probe RaCamp and a method for constructing transgenic mice. Background Technology

[0002] cAMP (cyclic adenosine monophosphate, 3′,5′-cyclic adenosine monophosphate) is a key intracellular second messenger, generated by the cyclization of ATP catalyzed by adenylate cyclase (AC), and can be hydrolyzed to 5′-AMP by phosphodiesterase (PDE). It is a crucial downstream messenger in the G protein-coupled receptor (GPCR) signaling pathway, and the development of fluorescent probes for cAMP and its dynamic imaging in live cells / in vivo are core technological directions in GPCR research.

[0003] Current gene-encoding probes have certain shortcomings. FRET probes rely on bimolecular energy transfer, have a small dynamic range, and are easily affected by environmental interference. While monochromatic probes offer advantages over FRET probes, such as ease of operation and a larger dynamic range, they suffer from the inability to eliminate in vivo motion artifacts in practical applications. Monochromatic probes lack internal control channels, making it impossible to distinguish between changes in fluorescence intensity originating from genuine cAMP signals or motion interference when cells or tissues shift (e.g., immune cell migration, cardiac pulsation), leading to a significant reduction in detection sensitivity and reliability. However, by fusing independently luminescent fluorescent proteins within a single molecule, simultaneous dual-channel acquisition under single-wavelength excitation can be achieved, combining high dynamic range with real-time ratio correction capabilities, completely eliminating signal distortion caused by cell migration or tissue deformation.

[0004] Currently, some studies have constructed ratio-type cAMP fluorescent probes, such as Naoto Saitoha's gCarvi probe, but their response rate to cAMP changes is far inferior to that of the monochromatic G-Flamp2 probe. Articles on G-Flamp2 also mention the construction of the pCAG-G-Flamp1-IRES-mCherry probe; however, IRES, as a linker peptide, leads to a non-strict 1:1 ratio of the two proteins expressed intracellularly, and inconsistencies exist in subcellular localization, making it unsuitable as a ratio-type probe. To overcome the bottlenecks in motion artifacts and signal quantification in in vivo studies and provide a powerful tool for studying dynamic processes such as immune cell migration and neurovascular coupling, constructing a G-Flamp2-based ratio-type probe is crucial.

[0005] Furthermore, current in vivo cAMP imaging research mainly relies on three technical pathways: local injection of viral vectors, random integration transgenes, and conditional activation systems. However, these methods all have certain drawbacks: viral vectors (such as AAV-cAMPr) exhibit fluctuating expression levels due to microenvironmental differences and are prone to triggering TLR9 / STING pathway-mediated inflammatory responses; random integration transgenes face the risk of insertional mutations and expression inactivation due to epigenetic silencing, failing to achieve spatiotemporally specific regulation. In contrast, the LoxP-Stop-LoxP (LSL) system based on the Rosa26 safe site, through hybridization with Cre mice, can precisely activate probe expression in target cells (such as neurons or macrophages), possessing the advantages of both genetic stability and in vivo compatibility. However, the field of cAMP imaging still lacks usable Rosa26-LSL-cAMP probe transgenic mouse strains, severely hindering the dynamic analysis of cAMP signals in deep tissues (such as brain regions or lymph nodes). Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a ratio-type cAMP fluorescent probe RaCamp and a method for constructing transgenic mice. This invention is achieved through the following technical solutions.

[0007] A ratio-type cAMP fluorescent probe, RaCamp, is formed by tandem fusion of G-Flamp2 fluorescent protein and mCherry fluorescent protein via a flexible linker with the sequence (GGGGS)2. It is used to detect changes in intracellular cAMP concentration and eliminate in vivo motion artifacts.

[0008] Compared to other linkers, (GGGGS)2 has been experimentally proven not to reduce the sensitivity of G-Flamp2 to the cAMP response and has a higher ratio value.

[0009] As a further embodiment of the present invention, the G-Flamp2 fluorescent protein and the mCherry fluorescent protein are transcribed into single-stranded mRNA by the same promoter. The single-stranded mRNAs transcribed from the G-Flamp2 fluorescent protein and the mCherry fluorescent protein are synthesized in a fixed ratio of 1:1 through a linker, so that the two fluorescent proteins can rotate freely while maintaining their relative positions.

[0010] A method for preparing a ratio-type cAMP fluorescent probe RaCamp includes the following steps:

[0011] S1, obtain the mCherry fragment. Using the pB-mCherry-ires plasmid as a template, obtain the mCherry fragment through PCR reaction. Use the obtained mCherry fragment as the DNA template in the new PCR cycle, and perform PCR cycle again to obtain the mCherry fragment with added linker.

[0012] S2, obtain the G-Flamp2 linear vector. Using the G-Flamp2 plasmid as a template, the plasmid is digested with EcoRI restriction endonuclease to obtain the linear vector.

[0013] S3, homologous recombination: The obtained mCherry fragment is ligated with the G-Flamp2 linear vector using homologous recombinase to form the RaCamp probe expression vector;

[0014] S4, Transformation and Screening: The recombinant RaCamp probe expression vector was transformed into competent cells, colonies that successfully expressed the RaCamp probe were screened, and plasmids were extracted from the bacterial culture.

[0015] As a further embodiment of the present invention, the primers for the PCR reaction include forward primer 1, forward primer 2 and reverse primer.

[0016] A method for constructing a transgenic mouse includes the following steps:

[0017] T1, Vector design: The RaCamp probe sequence was inserted into the Rosa26-LSL box to construct a transgenic vector;

[0018] T2, microinjection of fertilized eggs, involves microinjecting the transgenic vector into fertilized eggs;

[0019] T3, embryo transfer and reproduction: the injected fertilized eggs are transferred into pseudopregnant female mice to reproduce F0 generation transgenic mice;

[0020] T4, genotyping, identifies the genotype of F0 generation mice and screens out transgenic mice that successfully integrate the RaCamp probe sequence.

[0021] An application of a transgenic mouse, characterized in that: a transgenic mouse that has successfully integrated the RaCamp probe sequence is hybridized with different Cre mice to obtain offspring mice that express the RaCamp probe in specific cell types, for the purpose of visualizing the dynamic changes of cAMP signal in different cell types in vivo.

[0022] The beneficial effects of this invention are that, based on the G-Falamp2 probe and combined with mCherry fluorescent protein, a ratio-type cAMP probe, RaCamp, was designed. While maintaining the sensitivity of the original G-Falamp2 probe, mCherry was added as an internal control protein. The selected linker peptide (GGGGS)2 allows for precise control of the expression levels of the two proteins in cells at a 1:1 ratio, effectively solving the problem of background interference in in vivo cell observation. Furthermore, this sequence was inserted into the Rosa26-LSL cassette, enabling the construction of the Rosa26-LSL-RaCamp mouse. Attached Figure Description

[0023] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Schematic diagram of the RaCamp probe structure;

[0025] Figure 2 The effect of probe detection in HEK293T cells under confocal microscopy;

[0026] Figure 3 Δf / f0 and ratio values ​​before and after adding Forskolin to HEK293T cells;

[0027] Figure 4 Agarose gel image showing a band at 750bp;

[0028] Figure 5 Agarose gel image showing a band at 7600bp;

[0029] Figure 6 : 5' arm PCR identification diagram of transgenic mice;

[0030] Figure 7 : 3' arm PCR identification diagram of transgenic mice;

[0031] Figure 8 Comparison of the effects of different linker sequences on probe performance;

[0032] Figure 9 Flow cytometry analysis results;

[0033] Figure 10 Schematic diagram of RaCamp probe plasmid;

[0034] Figure 11 : Schematic diagram of the preparation process of gene modification constructs for conditional expression of exogenous genes;

[0035] Figure 12 : Donor vector map for homologous recombination knock-in at ROSA26 locus;

[0036] Figure 13 Sequencing results for the 5' arm and 3' arm ends. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] like Figure 1 As shown, a ratio-type cAMP fluorescent probe, RaCamp, is formed by tandem fusion of G-Flamp2 fluorescent protein and mCherry fluorescent protein via a flexible linker with the sequence (GGGGS)2. It is used to detect changes in intracellular cAMP concentration and eliminate in vivo motion artifacts.

[0039] Compared to other linkers, (GGGGS)2 has been experimentally proven not to reduce the sensitivity of G-Flamp2 to the cAMP response and has a higher ratio value.

[0040] G-Flamp2 fluorescent protein and mCherry fluorescent protein are transcribed into single-stranded mRNA by the same promoter. The single-stranded mRNAs transcribed from G-Flamp2 and mCherry fluorescent proteins are synthesized in a fixed 1:1 ratio through a linker, allowing the two fluorescent proteins to rotate freely while maintaining their relative positions.

[0041] This application modifies the G-Falamp2 probe, which exhibits excellent cAMP response, by binding mCherry fluorescent protein to the G-Falamp2 probe using a homologous recombinase method, and expressing it via tandem fusion through a flexible linker (GGGGS)2. As a fusion protein, this probe is transcribed into single-stranded mRNA by the same promoter, avoiding expression fluctuations caused by promoter strength differences and vector copy number imbalances in dual-vector transfection. The cAMP probe and mCherry, linked by the flexible linker, are synthesized in a fixed 1:1 ratio, with the fluorescence ratio directly reflecting changes in cAMP concentration without the need for additional correction for probe concentration differences. Furthermore, the flexible linker allows the two fluorescent proteins to rotate freely while maintaining their relative positions, ensuring synchronous localization in subcellular organelles (such as dendritic spines and endoplasmic reticulum) and avoiding signal shifts due to diffusion differences. The plasmid map of this probe is shown below. Figure 10 As shown.

[0042] While maintaining the original sensitivity of the G-Flamp2 probe, mCherry was added as an internal control protein. The selected linker peptide (GGGGS)2 allows for precise control of the expression levels of the two proteins in cells at a 1:1 ratio, effectively solving the problem of background interference in cell observation for in vivo imaging.

[0043] A method for preparing a ratio-type cAMP fluorescent probe RaCamp includes the following steps:

[0044] S1. Obtain the mCherry fragment. Using the pB-mCherry-ires plasmid as a template, obtain the mCherry fragment through PCR reaction. Use the obtained mCherry fragment as the DNA template in the new PCR cycle, and perform PCR cycle again to obtain the mCherry fragment with added linker.

[0045] The primers for PCR reactions include forward primer 1, forward primer 2, and reverse primer.

[0046] The sequences of each primer are shown in Table 1.

[0047] Table 1. Sequences of each primer

[0048]

[0049] First, using pB-mCherry-ires plasmid as a template, add the primers in Table 1 (primers synthesized by Qingke Biotechnology Co., Ltd.) to prepare the PCR reaction system (as shown in Table 2), and perform the first PCR reaction. After mixing with a pipette, obtain the target fragment through the PCR program.

[0050] Table 2. System for the first PCR reaction

[0051]

[0052] The first PCR reaction program was set as follows: pre-denaturation at 98℃ for 5 min, denaturation at 98℃ for 5 sec, annealing at 56℃ for 3.8 sec, extension at 72℃ for 3.8 sec for a total of 30 cycles, and a final extension at 72℃ for 5 min. After the PCR program was completed, 1 g of agarose was weighed and brought to 100 ml with 1×TAE. 10 μl of Glodview dye was added to prepare a 1% agarose gel. The PCR product was mixed with DNA and loaded onto the gel. The sample band was observed to see if it was at the correct position of 750 bp. If the sample band was indeed at 750 bp, then... Figure 4 As shown, gel extraction and recovery are then performed. First, the 750bp band of agarose gel is cut off with a knife and recovered using the Tiangen DNA Purification and Recovery Kit. After recovery, the DNA concentration is measured using a Nanodrop instrument. If the concentration reaches 50 ng / μl or higher, it can be used.

[0053] The obtained mCherry fragment was used as the DNA template in the new PCR cycle for a second PCR reaction. The system configuration for the second PCR reaction is shown in Table 3. The PCR cycle was repeated, followed by agarose gel electrophoresis, gel extraction, and recovery to obtain the mCherry fragment with (GGGGS)2 as the linker.

[0054] Table 3. System for the second PCR reaction

[0055]

[0056] S2. Obtain the G-Flamp2 linear vector. Using the G-Flamp2 plasmid as a template, the plasmid is digested with EcoRI restriction endonuclease to obtain the linear vector.

[0057] The enzyme digestion reaction system was prepared on ice. See Table 4 for the specific components.

[0058] Table 4 Components of the Enzyme Digestion Reaction

[0059]

[0060] Next, gently mix the system with a pipette and briefly centrifuge using a handheld centrifuge. In the PCR instrument, set the program to 37℃ for 10 min and 80℃ for 20 min. Then, stop the reaction and temporarily store the digestion product on ice. After the digestion reaction is complete, prepare a 0.8% agarose gel (0.8 g of agarose dissolved in 100 ml of 1×TAE, with 10 μl of Glodview added). Mix the digestion product with DNA 6× loading buffer and load the sample. Observe whether the sample band is at the correct position of 7600 bp. If the sample band is indeed at 7600 bp and has been digested into a linear fragment, such as... Figure 5 As shown, gel extraction and recovery are then performed. First, the agarose gel containing the 7600bp band is cut off with a knife, and then recovered using the Tiangen DNA purification and recovery kit. After recovery, the DNA concentration is measured using a Nanodrop instrument. If the concentration reaches 50 ng / μl or higher, it can be used. Alternatively, after recovery, the DNA concentration is measured using a Nanodrop instrument. If the concentration reaches 10 ng / μl or higher, it can be used.

[0061] S3, homologous recombination: The obtained mCherry fragment is ligated with the G-Flamp2 linear vector using a homologous recombinase to form the RaCamp probe expression vector.

[0062] The homologous recombination reaction system was prepared on ice in a PCR tube, as shown in Table 5.

[0063] Table 5. Homologous Recombination Reaction System

[0064]

[0065] After preparation, gently mix with a pipette, and briefly centrifuge to collect the reaction solution to the bottom of the tube. Then place the PCR tube into the PCR instrument and set it to 50℃ for 5 min, 4℃ ∞.

[0066] S4, Transformation and Screening: The recombinant RaCamp probe expression vector was transformed into competent cells, colonies that successfully expressed the RaCamp probe were screened, and plasmids were extracted from the bacterial culture.

[0067] Thaw DH5α chemocompetent cells on ice. Add 10 μl of the recombinant product to 100 μl of competent cells, gently tap the tube to mix, and incubate on ice for 30 min. Preheat a metal bath to 42°C, then heat-shock the competent cells containing the recombinant product at 42°C for 30 sec, followed immediately by cooling on ice for 2-3 min. Add 900 μl of LB liquid medium to the competent cells and incubate at 37°C for 1 h, while preheating the ampicillin-resistant LB solid medium plates in a 37°C incubator. After 1 h of incubation, centrifuge at 5000 rpm for 5 min and discard the 900 μl supernatant. Resuspend the cells in the remaining medium and gently spread them evenly on a plate containing the correct antibiotic using a sterile spreader. Incubate upside down at 37°C for 12-16 h. If colonies grow in the culture dish after 12 hours of adding ampicillin antibiotics, it is highly likely that bacteria expressing the RaCamp probe have grown. The colonies are then purified and sent to Qingke Biotechnology Co., Ltd. for sequencing verification.

[0068] After successful verification, the bacterial culture was expanded, and glycerol was added to a final concentration of 30% before being stored at -80℃. Plasmids were extracted from the bacterial culture using Tiangen's endotoxin-free plasmid extraction kit for subsequent transfection.

[0069] The specific procedure for in vitro live-cell validation of the RaCamp plasmid is as follows:

[0070] Experimental steps:

[0071] Preparation of DMEM complete medium: 5 ml fetal bovine serum + 500 μl penicillin-streptomycin-gentamicin + 45 ml DMEM high glucose medium

[0072] Preparation of DMEM antibiotic-free medium: 5 ml fetal bovine serum + 45 ml DMEM high glucose medium

[0073] Cell plating: The night before, HEK-293T cells were digested with 750 μl trypsin for 1 min, then added to 2250 μl of DMEM complete medium to terminate the process. Centrifugation was performed at 1000 rpm for 3 min. The old medium was discarded, and 1 ml of DMEM complete medium was added. The cells were resuspended using a pipette. 10 μl of the cell culture was added to a cell counting chamber for counting. HEK-293T cells were plated at a rate of 1 × 10⁻⁶ cells / mL. 4 The cells were seeded in 96-well plates with 100 μl of DMEM complete medium. The next day, after observing that the cell confluence reached 70%-80%, the medium in the wells was replaced with DMEM antibiotic-free medium.

[0074] Plasmid transfection: Prepare two clean 1.5 ml centrifuge tubes. Centrifuge tube 1: 25 μl Opti-Men medium + 0.6 μl Lipofectamine 2000; Centrifuge tube 2: 25 μl Opti-Men medium + 300 ng endotoxin-free RaCamp plasmid. After preparation, let stand for 5 min. Slowly add the liquid from centrifuge tube 1 to centrifuge tube 2, gently mix with a pipette, and let stand for 20 min. Add the prepared liposome-plasmid complex to the well plate. After 6 h, remove the old culture medium from the well plate and replace it with fresh DMEM complete medium.

[0075] Laser confocal microscopy imaging to assess probe effectiveness: A live-cell imaging system was set up on an Olympus rotary confocal microscope 48 hours after transfection. Time-lapse imaging of cells was performed with a 15-second frame capture, 8% laser efficiency, and a 500ms exposure time. During imaging, 60μM Forskolin reagent was added. Changes in fluorescence intensity were observed in successfully transfected cells upon excitation at 488nm. Figure 2 and Figure 3 As shown, where Figure 3 In the figure, the left side represents the Δf / f0 value, and the right side represents the ratio value. The Δf / f0 peak is at 1200%, and the fluorescence does not decrease due to the addition of mCherry. The ratio peak is at 1300%, indicating that mCherry fluorescence is stable and suitable as an internal control protein for cell localization in vivo.

[0076] Comparison of the effects of different linker sequences on probe performance:

[0077] To screen and determine the optimal linker sequence for connecting the G-Flamp2 probe to mCherry, this invention tested other linkers. The method for linking probes with different linkers was the same as the method for constructing the RaCamp probe, only the linker sequence in the probe was replaced. The probe with the linker sequence replaced by GGGGS was named RaCamp-GS, and the probe with the linker sequence replaced by AEAAAAKEAAAKEAAAKA was named RaCamp-EK. After transfecting HEK293T cells with RaCamp-GS, RaCamp-EK, and RaCamp probe plasmids for 48 h, imaging was performed under an Olympus laser disc confocal microscope. Time-lapse imaging was performed with one frame every 15 s, 8% laser efficiency, and an exposure time of 500 ms. During imaging, 60 μM Forskolin reagent was added to observe the changes in probe fluorescence intensity under 488 nm excitation. Subsequently, fluorescence intensity analysis was performed using Imaris and Graphpad software to determine the optimal linker.

[0078] like Figure 8 As shown, when the linker sequence is (GGGGS)2, the probe transfected into HEK293T cells showed the best response to stimulation with 60 μM Forskolin reagent, with the highest ratio value, which was significantly better than other control sequences.

[0079] A method for constructing a transgenic mouse, such as Figure 11 As shown, this invention systematically demonstrates the entire process of constructing a mutant allele conditionally expressing the RaCamp reporter gene from a wild-type allele. Specifically, the core of this invention lies in the precise integration of a designed targeting vector into the ROSA26 locus, a "safe harbor" site in the genome, through a CRISPR / Cas9-mediated homology-directed repair mechanism. The specific integration site is located in the first intron region downstream of exon 1, a site chosen to ensure no disruptive impact on the function of the endogenous ROSA26 gene. The targeting vector includes long homologous arms flanking the integration site in the genome, ensuring efficient and precise recombination. After successfully obtaining the targeted allele (i.e., F0 generation mice), the allele's structure contains a RaCamp reporter gene unit driven by a strong CAG promoter, but its expression is blocked by a "terminator" box flanked by loxP sites in the same direction. This "terminator" box consists of a neomycin resistance gene driven by a PGK promoter and its following SV40 polyadenylate signal. Its function is to prevent the expression of the downstream RaCamp reporter gene by strongly terminating transcription in the absence of Cre recombinase. When this transgenic mouse is mated with a mouse strain expressing Cre recombinase, in cells expressing Cre enzyme, the recombinase specifically recognizes the two loxP sites and precisely excises the PGK-Neo-pA selection marker box located between them. This excision event allows the CAG promoter to directly connect to the RaCamp reporter gene, thereby relieving expression inhibition and efficiently and specifically initiating the expression of the cAMP fluorescent biosensor RaCamp at specific cell types or time points, enabling real-time, in vivo monitoring of intracellular cAMP signals. The specific experiment includes the following steps:

[0080] T1, Vector Design: The RaCamp probe sequence was inserted into the Rosa26-LSL box to construct a transgenic vector. The mouse ROSA26 gene (NCBI reference sequence: NR_027008.1) is located on mouse chromosome 6. To ensure the correct insertion of the knock-in fragment into intron 1 of the mouse ROSA26 gene, a donor vector for homologous recombination repair was constructed in vitro, such as... Figure 12As shown, the 5' and 3' homologous arm sequences are listed in Table 1 below. The obtained donor vector is denoted as "CAG promoter-loxP-PGK-Neo-6×SV40 pA-loxP-Kozak-RaCamp-WPRE-BGH pA", containing the following elements or sequences arranged in sequence: CAG promoter, loxP element, PGK-Neo puromycin resistance gene, 6×SV40 pA transcription termination sequence, loxP element, Kozak sequence, RaCamp sequence (i.e., the constructed ratio-type cAMP probe sequence), WPRE sequence, and BGH ployA sequence. Based on the mouse Rosa26 gene intron 1 sequence and the CRISPR / Cas9 system, a gRNA was designed, with the gRNA sequence being CTCCAGTCTTTCTAGAAGAT-GGG.

[0081] T2, microinjection into fertilized eggs: The transgenic vector was microinjected into fertilized eggs. Using the C57BL / 6J mouse strain, a mixture containing a pre-assembled Cas9 / gRNA ribonucleoprotein complex and a donor vector was introduced into C57BL / 6J mouse fertilized eggs via microinjection; wherein the Cas9 / gRNA ribonucleoprotein complex was assembled in vitro from purified Cas9 protein and a single-stranded gRNA targeting the ROSA26 locus. The donor vector contained the expression cassette “CAG promoter-loxP-PGK-Neo-6×SV40 pA-loxP-Kozak-RaCamp-WPRE-BGH pA”, which was injected into mouse fertilized eggs via microinjection.

[0082] In T3, embryo transfer and reproduction: surviving fertilized eggs after injection are surgically transferred into the oviducts of pseudopregnant mice. Approximately 20 days later, F0 generation mice are born. Genotyping of the F0 generation mice is performed using polymerase chain reaction (PCR) to screen for positive individuals with the target element successfully knocked into the ROSA26 locus. Once these positive F0 generation mice reach sexual maturity, they are mated with wild-type C57BL / 6J mice. Genotyping of the resulting F1 generation offspring is performed. If positive heterozygous mice are confirmed to be born, this demonstrates that the knock-in element has been stably inherited through the germline, and a new transgenic mouse strain can be established based on this.

[0083] For T4 genotyping, after F0 generation mice were born and reached 3 weeks of age, the tail tips of the mice were surgically removed, marked, and placed in PCR tubes. 90 μl of 0.05 M NaOH was added to the collected tail tips, and lysis was performed in a PCR instrument at 98 °C for 1 h. Then, 10 μl of Tris-HCl (pH 8.0) was added to terminate the lysis, and the tubes were centrifuged at 3000 rpm for 5 min. Subsequently, the PCR reaction system was prepared, and the 5' arm identification primers are shown in Table 6.

[0084] Table 6

[0085]

[0086] The PCR system was prepared as shown in Table 7.

[0087] Table 7

[0088]

[0089] After gently mixing the system with a pipette, set up the following procedure in the PCR instrument to carry out the polymerase chain reaction, as shown in Table 8.

[0090] Table 8

[0091]

[0092] Subsequently, a 0.8% agarose gel was prepared, and the PCR products were verified by agarose gel electrophoresis. The verification results are as follows: Figure 6 As shown.

[0093] The identification method for 3'arm is the same as that for 5'arm, except that the primers involved are replaced with F2 and R2. The primer sequences are shown in Table 9. The 3'arm identification results are as follows: Figure 7 As shown.

[0094] Table 9

[0095]

[0096] The expected positive fragment is 2808 bp at the 5' end and 2481 bp at the 3' end, derived from... Figure 6 , Figure 7 The agarose gel electrophoresis results confirmed the presence of positive mice at birth. Furthermore, the fragment was sequenced, and the results are as follows. Figure 13 As shown, sequencing comparison confirmed that the transgenic mouse was successfully constructed.

[0097] An application of a transgenic mouse, characterized in that: a transgenic mouse that has successfully integrated the RaCamp probe sequence is hybridized with different Cre mice to obtain offspring mice that express the RaCamp probe in specific cell types, for the purpose of visualizing the dynamic changes of cAMP signal in different cell types in vivo.

[0098] Currently, RaCamp probe Rosa26-LSL mice have been hybridized with Csf1r-iCre tool mice, and the validation was performed by inducing reporter gene expression. The validation method included collecting peripheral blood samples from mice and detecting reporter gene expression using flow cytometry. The specific experimental steps are as follows:

[0099] Sample collection and anticoagulation: Aliquot 10 μL of 20 Mmol / L ethylenediaminetetraacetic acid (EDTA) solution into 1.5 mL centrifuge tubes. Fix the mice to be tested, and use surgical scissors to cut a small segment from their tails, collecting approximately 50 μL of tail blood into centrifuge tubes containing EDTA. Immediately and gently vortex or shake to thoroughly mix the blood with the anticoagulant and prevent coagulation.

[0100] Red blood cell lysis: Add 1 mL of ACK red blood cell lysis buffer to the centrifuge tube containing anticoagulated whole blood, mix thoroughly by pipetting or vortexing, and incubate at room temperature for about 10 minutes to fully lyse the red blood cells.

[0101] Cell washing and collection: Place the centrifuge tubes in a centrifuge and centrifuge at 400 ×g for 5 minutes. Carefully discard the supernatant after centrifugation. Add 1 mL of PBS buffer to the cell pellet, gently resuspend the cells, and centrifuge again at 400 ×g for 5 minutes. Discard the supernatant after centrifugation. Repeat this step once.

[0102] Cell resuspension and flow cytometry: 300 μL of phosphate-buffered saline (PBS, pH 7.4) was added to the final cell pellet, and the cells were gently resuspended by pipetting to prepare a single-cell suspension. The sample was then transferred to a flow cytometer-specific sample loading tube and immediately analyzed using a flow cytometer. The percentage of mCherry-positive cells and the average fluorescence intensity were recorded.

[0103] The detection was performed using a Cytoflex flow cytometer, with channels Y610-A (mCherry) and B525-A (EGFP) used. Figure 9 This indicates that, compared with the WT control group, a significant proportion of mCherry and EGFP fluorescently positive cells can be detected in the peripheral blood of the Csf1r-RaCamp transgenic mouse strain, confirming that the element was successfully activated and expressed in vivo.

[0104] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to any specific implementation. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A ratio-type cAMP fluorescent probe RaCamp, characterized by, The probe RaCamp is formed by tandem fusion of G-Flamp2 fluorescent protein and mCherry fluorescent protein through a flexible linker, which is used to detect the change of cAMP concentration in cells and eliminate the motion artifact in vivo. 2.The ratio type cAMP fluorescent probe RaCamp of claim 1, wherein, The G-Flamp2 fluorescent protein and the mCherry fluorescent protein are transcribed into single-stranded mRNA by the same promoter, and the single-stranded mRNA after transcription of the G-Flamp2 fluorescent protein and the mCherry fluorescent protein is synthesized in a fixed ratio of 1:1 through the linker, so that the two fluorescent proteins are free to rotate while maintaining the relative position.

3. A method for preparing the ratio type cAMP fluorescent probe RaCamp according to any one of claims 1-2, characterized in that, The method comprises the following steps: S1, obtaining an mCherry fragment, using a pB-mCherry-ires plasmid as a template, obtaining an mCherry fragment through a PCR reaction; taking the obtained mCherry fragment as a new DNA template in a PCR cycle, and performing the PCR cycle again, so that the mCherry fragment with the linker is obtained; S2, obtaining a G-Flamp2 linear carrier, taking a G-Flamp2 plasmid as a template, and using an EcoR I restriction endonuclease to perform single enzyme cutting on the plasmid to obtain a linear carrier; S3, homologous recombination, connecting the obtained mCherry fragment and the G-Flamp2 linear carrier through a homologous recombination enzyme to form a RaCamp probe expression vector; S4, transformation and screening, transforming the recombined RaCamp probe expression vector into a competent cell, and screening a colony successfully expressing the RaCamp probe, and extracting a plasmid in a bacterial liquid.

4. The method for preparing ratio type cAMP fluorescent probe RaCamp according to claim 3, characterized in that, The primers of the PCR reaction include a forward primer 1, a forward primer 2 and a reverse primer.

5. A method for the construction of a transgenic mouse based on the probe RaCamp prepared according to any one of claims 3-4, characterized in that, The method comprises the following steps: T1, vector design, inserting the RaCamp probe sequence into a Rosa26-LSL box to construct a transgenic vector; T2, microinjection of zygotes, microinjection of the transgenic vector into zygotes; T3, embryo transplantation and breeding, transplanting the injected zygotes into pseudopregnant female mice to breed F0 generation transgenic mice; T4, genotype identification, identifying the genotype of the F0 generation mice, and screening transgenic mice successfully integrating the RaCamp probe sequence.

6. Use of a transgenic mouse constructed by the method of claim 5, wherein: The transgenic mice successfully integrating the RaCamp probe sequence are crossed with different Cre mice to obtain offspring mice expressing the RaCamp probe in specific cell types, which are used to visualize the dynamic change of cAMP signal in different cell types in vivo.

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