CN114107386A - Method for preparing blood brain barrier defect mouse model - Google Patents
Method for preparing blood brain barrier defect mouse model Download PDFInfo
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- CN114107386A CN114107386A CN202111429800.1A CN202111429800A CN114107386A CN 114107386 A CN114107386 A CN 114107386A CN 202111429800 A CN202111429800 A CN 202111429800A CN 114107386 A CN114107386 A CN 114107386A
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Abstract
The invention discloses a method for preparing a blood brain barrier defect mouse model. The invention provides a method for preparing an animal model with blood brain barrier defect, which comprises the following steps: based on a CRISPR/Cas9 system, a cerebrovascular endothelial specific gonad-related virus vector is used as a delivery vector of sgRNA, and Ctnnb1 gene in a recipient animal cerebral vascular endothelial cell is knocked out in a targeted mode. The data of the results show that AAV-BR1 carrying sgRNAs in chimeric form results in disruption of target genes of cerebrovascular endothelial cells, where disruption of the chimeric pattern of BBB regulatory genes is sufficient to cause disruption of the BBB. Meanwhile, data show that the AAV-CRISPR system can be used as a useful tool for rapidly identifying genes with important functions for maintaining the integrity of a blood brain barrier.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for preparing a blood brain barrier defect mouse model.
Background
The Blood Brain Barrier (BBB), which consists of cerebrovascular endothelial cells, pericytes, perivascular astrocytes, is a selective interface that separates the central nervous system from peripheral tissues. The core component of BBB is specialized endothelial cell, which has the characteristics of continuous intercellular tight connection, specific expression of a series of transport proteins, extremely low frequency of transcytosis and the like. These properties allow endothelial cells to tightly control the paracellular and transcellular movement of molecules and ions through the blood-brain barrier, thereby maintaining a healthy environment for development and homeostasis of the central nervous system. In recent years, various efforts have been made to better understand how the BBB is formed and maintained. High throughput sequencing has yielded a large amount of data to explain the unique properties of the BBB and the mechanisms involved in BBB formation and maintenance. However, due to the lack of in vivo and in vitro BBB models, only a small portion of these data was validated for function in blood brain barrier formation and maintenance. Because brain microvascular endothelial cells lose certain BBB properties in vitro, to construct an in vitro BBB model that more accurately simulates the blood brain barrier in vivo, very complex culture systems must be used, requiring operators to have a rich experience, and such in vitro systems have poor stability. In contrast, genetically engineered mice remain an advantageous tool for studying target gene function, but generating genetically modified mice via transgenesis or gene targeting in embryonic stem cells is time consuming and costly.
The revolutionary progression of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated (Cas) proteins provides a simpler technique for genome editing that has been widely adopted. Generally, Cas9 nuclease and guide RNA are assembled together and are capable of recognizing, binding and cleaving DNA. DNA binding occurs at a DNA sequence that is complementary to a 20 base pair DNA sequence in the guide RNA, and the DNA recognition site must be adjacent to a Protospacer Adjacent Motif (PAM) that acts as a switch, which is then targeted to Double Strand Breaks (DSB) by Cas 9. In all multicellular organisms, such DSBs induce DNA repair through endogenous cellular pathways that may result in changes in DNA sequence, including changes in small sequences or insertion of genes. CRISPR-introduced targeted DSBs can be repaired by non-homologous end joining (NHEJ) which joins two cleaved DNA ends together at the cleavage site. The process of targeted cleavage and repair may be repeated until an insertion or deletion (insertion/deletion) occurs, thereby preventing further recognition of the target site by the nuclease. Indels in protein-encoding genes may cause frame shift mutations or exon skipping, thereby disrupting gene function. Systemic or local somatic gene editing using CRISPR has been achieved in mouse liver, lung, heart, skeletal muscle, and brain. However, no work has been reported on gene editing by CRISPR/Cas9 in adult mouse cerebrovascular endothelial cells.
Disclosure of Invention
The invention aims to provide a method for preparing a blood brain barrier defect mouse model.
In a first aspect, the invention claims a method for preparing an animal model with a defect in the blood-brain barrier.
The method for preparing the animal model with blood brain barrier defect, which is claimed by the invention, can comprise the following steps: based on a CRISPR/Cas9 system, a cerebrovascular endothelial cell specific gonad-associated virus vector is used as a delivery vector of sgRNA, and Ctnnb1 gene in a recipient animal cerebral vascular endothelial cell is knocked out in a targeted mode.
In a particular embodiment of the invention, the animal is in particular a mouse.
In a specific embodiment of the present invention, the cerebrovascular endothelial cell specific gonad-associated viral vector is an adeno-associated viral vector of serotype BR1 (AAV-BR 1).
In a specific embodiment of the invention, the target sequence of sgRNA is specifically SEQ ID No.1 by targeted knockout of Ctnnb1 gene in cerebral vascular endothelial cells of the recipient animal.
Further, the method may comprise the steps of: infecting a vascular endothelial cell-specific Cas9 transgenic mouse with BR1 serotype adeno-associated virus expressing the sgRNA, and obtaining a blood brain barrier defect mouse model after 4 weeks.
In a specific embodiment of the invention, the vascular endothelial cell-specific Cas9 transgenic mouse is obtained after crossing a heterozygous Tie2-Cre transgenic mouse and a homozygous R26-loxP-STOP-loxP-3xFLAG-Cas9-eGFP mouse.
Further, by expressing saidInfection of the vascular endothelial cell-specific Cas9 transgenic mice with BR1 serotype adeno-associated virus of sgRNA can be achieved by: single dose of 1.8 × 10 single dose injection into tail vein of the vascular endothelial cell-specific Cas9 transgenic mice11vg BR1 serotype adeno-associated virus expressing the sgRNA.
In the present invention, in BR1 serotype adeno-associated viral vectors expressing the sgrnas, the sgrnas are expressed under the U6 promoter. And the vector also expresses tdTomato fluorescent protein under the drive of a CMV promoter.
In a second aspect, the invention claims a kit for the preparation of a mouse model for blood brain barrier defects.
The kit for preparing a blood brain barrier deficient mouse model claimed in the present invention specifically can be composed of BR1 serotype adeno-associated virus expressing the sgRNA described above and a vascular endothelial cell specific Cas9 transgenic mouse described above.
In a third aspect, the invention claims the use of a kit as described hereinbefore for the preparation of a mouse model with a defect in the blood-brain barrier.
In a fourth aspect, the invention claims the use of an animal model prepared by the method described above for screening a medicament for treating or ameliorating a blood-brain barrier defect.
In a fifth aspect, the invention claims a method of identifying or aiding in the identification of a gene that is functional for maintaining the integrity of the blood brain barrier.
The method for identifying or assisting in identifying a gene having a function of maintaining the integrity of a blood brain barrier, which is claimed by the invention, can comprise the following steps:
(A1) infecting a vascular endothelial cell specific Cas9 transgenic mouse with BR1 serotype adeno-associated virus expressing sgRNA of a gene to be detected in a targeted mouse cerebral vascular endothelial cell;
(A2) determining whether the gene to be detected is a gene having a function of maintaining the integrity of the blood brain barrier according to the following steps: if after 4 weeks the mouse treated in step (a1) exhibits a blood brain barrier defect, then the gene to be tested is or is a candidate for a gene that is functional for maintaining blood brain barrier integrity; otherwise, the gene to be tested is not or is not a candidate gene having a function of maintaining the integrity of the blood brain barrier.
In a specific embodiment of the invention, the vascular endothelial cell-specific Cas9 transgenic mouse is obtained after crossing a heterozygous Tie2-Cre transgenic mouse and a homozygous R26-loxP-STOP-loxP-3xFLAG-Cas9-eGFP mouse.
Further, infection of the vascular endothelial cell-specific Cas9 transgenic mouse with the BR1 serotype adeno-associated virus expressing sgrnas targeting genes to be tested in mouse cerebral vascular endothelial cells can be achieved by: a single intravenous dose of 1.8 × 10 to the vascular endothelial cell-specific Cas9 transgenic mice11vg of the BR1 serotype adeno-associated virus that targets sgRNA of the gene to be tested in mouse cerebrovascular endothelial cells.
In the invention, in the BR1 serotype adeno-associated virus vector for expressing sgRNA of a gene to be detected in a targeted mouse cerebrovascular endothelial cell, the sgRNA of the gene to be detected in the targeted mouse cerebrovascular endothelial cell can be expressed under a U6 promoter. And the vector also expresses tdTomato fluorescent protein under the drive of a CMV promoter.
In the present invention, the blood brain barrier defect is specifically represented by: the Ctnnb1 gene has chimeric knockout in brain vascular endothelium, up-regulation of PLVAP expression in brain endothelial cells with deletion of CTNNB1 expression, inactivation of Wnt/beta-catenin pathway (expression of Claudin-5 in central nervous vasculature is limited), and increased blood brain barrier permeability.
Accordingly, the method of producing an animal model having a blood brain barrier defect may also be a method of producing an animal model having all or part of the features described above.
In the invention, the homozygous R26-loxP-STOP-loxP-3xFLAG-Cas9-eGFP mouse is specifically a Jackson laboratory 024857 mouse (Also Known As: Rosa26-LSL-Cas9 knock in, Rosa26-floxed STOP-Cas9 knock in).
The invention discovers that based on a CRISPR/Cas9 system, a cerebrovascular endothelial specific gonad-associated virus vector (AAV-BR1) is used as a delivery vector of sgRNA, and a cerebrovascular endothelial cell gene editing mouse can be quickly and effectively generated. The results data show that sgRNA-bearing AAV-BR1 in chimeric form resulted in disruption of cerebrovascular endothelial cell target genes. More importantly, disruption of the chimeric pattern of BBB regulatory genes in cerebrovascular endothelial cells is sufficient to cause disruption of the BBB. Meanwhile, data show that the AAV-CRISPR system can be used as a useful tool for rapidly identifying genes with important functions for maintaining the integrity of a blood brain barrier.
Drawings
Fig. 1 is sgRNA synthesis and in vitro gene editing using CRISPR/Cas 9. A is after transfection of control sgRNA (hereinafter abbreviated as sgCon) or candidate sgRNA (sgRNA 1, sgRNA2, sgRNA 3), the target sites of candidate sgRNA of NIH-3T3 cells are further verified by T7E1 analysis, and the intensity of the bands is determined to determine which group has the highest insertion/deletion frequency. sgRNA # 1, sgRNA # 2, and sgRNA # 3 in the figure represent three candidate sgRNAs — sgRNA1, sgRNA2, and sgRNA3, respectively, targeting Ctnnb1 gene. B is a T7E1 analysis of PCR amplification of the first 5 potential off-target DNA cleavage sites (OT 1 to OT5 in the figure) from sorted NIH-3T3 cells screened for sgRNA of Ctnnb1 (i.e., sgnnb 1 in the figure, which represents sgRNA1 targeting the Ctnnb1 gene, the same below). C is a mouse Ctnnb1 gene site schematic showing that the target of SpCas9 and the first 5 off-target sites of sgCtnnb1 are predicted by chopchopchop. Blue indicates target genomic site and red indicates PAM sequence. D is NIH-3T3 cells selected for transfection with sgCon or sgCtnnb1 and their mRNA expression was analyzed by qRT-PCR (mean ± SEM, n ═ 3). E Western blot analysis of Ctnnb1 transfection on sgCon and sgCtnnb1 in NIH-3T3 cells. F is the analysis of Ctnnb1 protein expression in NIH-3T3 cells after sgCon and sgCtnnb1 transfection by band intensity (mean ± SEM, n ═ 3).
FIG. 2 shows the use of BR 1-sgCnnb 1-tdTomato recombinant virus pair Tie2Cas9Mice underwent editing of the cerebrovascular endothelial Ctnnb1 gene. A is to produce Tie2Cas9Mouse form. B is a method for cloning SgCtnnb1 into BR 1; tie2 at 4 weeks of ageCas9Mouse tail vein injection BR1 (1.8X 10)11vg), killed 4 weeks post-injection and sampled for analysis. C is flow cytometry quantitative sorting tdTomato+And CD31+CellsPercentage of (c). D is measured by T7E1 test from Tie2 in comparison to controlCas9The mouse-isolated cerebrovascular endothelial cells showed a positive cleavage band at Ctnnb1 target gene site, which confirms that Cas9 cleaves the target gene site, resulting in insertion/deletion mutation. E is the result of immunofluorescence staining of brain sections 4 weeks after infection with BR1-sgCtnnb1-tdTomato virus, wherein tdTomato is purple, CTNNB1 is grey, CD31 is red, PLVAP is green, and the result shows that Tie2 is compared with the control groupCas9CTNNB1 expression decreased and PLVAP expression increased in mice. F is the statistical result of the infection efficiency of BR 1-sgCnnb 1-tdTomato virus to cerebrovascular endothelial cells, and shows that the virus is applied to control mice and Tie2Cas9The mice all have higher infection efficiency. G is a control group infected with BR 1-sgCnnb 1-tdTomato virus and Tie2Cas9Sorting the cerebral vascular endothelial cells of the mice, extracting RNA, detecting the change of the transcription level of a target gene Ctnnb1 by a qPCR method, and displaying the result of Tie2Cas9The mRNA level of the target gene in the brain endothelium of the mouse is obviously reduced compared with that of the control group. And H, analyzing CTNNB1 protein in cerebrovascular endothelial cells separated from editing mice by Western blot. I is the expression change of CTNNB1 in the editing mouse detected by the gray value in the statistical Western blot band.
FIG. 3 shows the editing efficiency of the target gene Ctnnb1 detected by DNA deep sequencing. A is the sequence classification after amplification at Ctnnb1 site. B is an image showing that most insertions/deletions cause shifts; for example: 3n +1 includes insertions/deletions of 1, 4 and 7bp, 3n +2 includes insertions/deletions of 2, 5 and 8bp, and 3n includes insertions/deletions of 3, 6 and 9 bp. C is based on CRISPRSO 2 analysis and summarizes Tie2 after BR 1-sgCnnb 1 infectionCas9The most frequent insertion/deletion reading in mouse brain endothelial cells. Horizontal dashed lines indicate CRISPR cleavage sites, dashed long lines indicate missing nucleotides, red rectangles indicate inserted nucleotides. D is the proportion of each base pair sequence that contains a particular mutation type (insertion or deletion) in all mutated sequences.
FIG. 4 shows that BR 1-sgCnnb 1-tdTomato mediated in vivo genome editing results in Tie2Cas9Mice are deficient in the blood brain barrier. A is Zhanyuanb BR 1-sgCnnb 1-tdTomato virus infected Tie2Cas9After injecting a Sulfo-NHS-LC-Biotin tracer, mice detect the leakage condition of the blood brain barrier, and BBB/BRB defects appear on olfactory bulbs, cerebral cortex, cerebellum and retina. B is Tie2 infected by BR 1-sgCnnb 1-tdTomato virusCas9Mice showed expression of PLVAP in the Sulfo-NHS-LC-Biotin permeable region. Scale bar, 50 μm. C is Tie2 infected with BR1-sgCtnnb1-tdTomato virus compared with control groupCas9More endothelial cells from PLVAP in mice-/Claudin5+ to PLVAP+/Claudin5-And (4) converting.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The sequence information of the primer of the present invention is shown in Table 1.
TABLE 1 primer sequence information according to the present invention
*NNNNNN＝index from Illumina TruSeq Small RNA Sample Prep Kit
Example 1 establishment of a method for preparing a blood brain barrier deficient mouse model
Materials and methods
1. Mouse
All experimental animal manipulations were performed according to the guidelines of the animal care and use committee (IACUC) of the beijing institute of life sciences and were maintained under specific pathogen-free conditions. All mice were housed on a C57BL/6 background, in a 12-hour backlight/dark cycle from 8 am to 8 pm. Food and water were freely available to all rats. Experiments were carried out using Cre-dependent R26-loxP-STOP-loxP-3xFLAG-Cas9-eGFP (stock number 024857 from Jackson Labs; Also Known As: Rosa26-LSL-Cas9 knockin, Rosa26-floxed STOP-Cas9 knockin, hereinafter Cas9 mouse) and Tie2 promoter-driven Cre recombinase-expressed Tie2-Cre transgenic mice (described in "Li et al, Essential role of endothial Smad4 in var remodelal and integration.2005", publicly available from the applicant, usable only for the experiments with duplicate copies, not otherwise) for obtaining tissue-specific expressed Cas9 mice, the invention generated by crossing Cas 4642 with hybrid Tie 3-Cre transgenic mice, so that Cas 4642 was producedCas9A mouse. The specific method comprises the following steps:
(1) mouse genomic DNA extraction
1) A small amount of tissue (ear rim, tail tip or toe) from the mouse to be identified was excised and placed in a centrifuge tube, and about 400. mu.L of tissue lysate was added and placed in a 55 ℃ water bath for lysis overnight.
2) 200. mu.L of saturated NaCl solution (6M) was added to the centrifuge tube, shaken vigorously 50-100 times, and then allowed to stand on ice for 10 min.
3) Centrifuge at 13000rpm for 10min at room temperature, pour the supernatant (about 500. mu.L) into a new centrifuge tube, add 800. mu.L of absolute ethanol to each tube, reverse the top and mix well.
4) Centrifugation was carried out at 13000rpm for 10min at room temperature, the supernatant was discarded, 500. mu.L of 75% ethanol was added to each tube, and the DNA was washed by inverting the top and bottom.
5) Centrifuging at 13000rpm for 5min at room temperature, discarding the supernatant, and inverting the centrifuge tube to completely dry at room temperature.
6) 100-200. mu.L of water was added to each tube, placed at 37 ℃ and vortexed to dissolve the DNA.
7) And (3) identifying the genotype by PCR.
(2) Mouse genotype identification: mouse genotype was identified by PCR.
1) Mouse genotype identifying primers are shown in Table 1 as Tie2 Cre fwd, Tie2 Cre rev, Cas9 mutant fwd, Cas9 mutant rev, Cas9 wild type fwd, Cas9 wild type rev.
2) Reaction System (20. mu.l)
3) PCR amplification procedure
4) Agarose gel electrophoresis
i. Weighing appropriate amount of agarose 2g, adding into 100ml of 1 XTAE microwave oven, heating and boiling, cooling, adding nucleic acid dye, pouring into a gel-making plate, inserting into a comb, and making into agarose gel.
And ii, after the agarose gel is solidified, placing the gel into an electrophoresis tank, dropping a PCR product into an agarose gel hole, placing 140V in the electrophoresis tank for 20min or more, and judging the genotype. The primers Tie2 Cre fwd and Tie2 Cre rev are used for PCR identification, the band obtained by a Tie2-Cre positive mouse is 487bp, and the band obtained by a Tie2-Cre negative mouse is not band. Cas9 knocks in positive, namely Cas9 mutant fwd and Cas9 mutant rev primers are used to obtain a 110bp band, if negative, no band is generated, and Cas9 wild type fwd and Cas9 wild type rev primers are used for PCR to obtain only a 296bp band. Tie2Cas9Refer to Tie2-Cre and Cas9 knock-in double positive mice.
The control mice were Tie2-Cre negative mice, and the mice used throughout the experiment were 4-8 weeks old. Only male Tie2 was used for the experimentCas9And Tie2-Cre negative mice, in Tie2-Cre negative mice and Tie2Cas9Mice were infected with AAV-BR1 at 4 weeks of age and cerebrovascular endothelial cells were isolated at 8 weeks of age.
2. Cell lines
Mouse embryo fibroblast (NIH-3T3) cells (CRL-1658, ATCC) were placed in DMEM (06-1055-57-1ACS, Biological Industries) containing 10% FBS (10099-141, GIBCO), 1% antibiotic-antifungal (15240-062, GIBCO) at 37 ℃ with 5% CO2Culturing in medium. Human embryonic kidney (HEK293T) cells (CRL-11268, ATCC) were placed in DMEM (06-1055-57-1ACS, Biological Industries) containing 10% FBS (10099-141, Gibco), 1% antibiotic-antifungal (15240-062, Gibco) at 37 ℃ with 5% CO2And culturing in an infiltrated incubator. These cell lines were obtained directly from ATCC.
3. sgRNA design
All SpCas9 targeting single guide RNAs (sgRNAs) are designed before CRISPR type II specific PAM sequence 5' -NGG by using CRISPR design tool (https:// chopchopchop. cbu. uib. no /), and off-target effect (OT) is systematically screened. Oligomers and sequences of candidate sgrnas are shown in table 1 as Ctnnb1sgRNA 1, Ctnnb1sgRNA 2, and Ctnnb1sgRNA 3.
4. sgRNA construction and transfection
After annealing the candidate single-stranded RNA sequence, it was cloned into the U6 promoter of PX459(addge plasmid #48139), cleaved with Bbs1(Thermo # FD0454), and confirmed by Sanger sequencing for correct cloning. In order to screen the functionality of sgRNA, PX459 vector for cloning candidate sgRNA is used according to the protocol of a manufacturer, and Lipofectamine is usedTM3000(Invitrogen, # L3000075) were transfected into 30% concentration NIH-3T3 cells in 6-well plates. Briefly, 2.5. mu.g of plasmid DNA was mixed with 5. mu. L P3000 and 2.5. mu.L of LipofectamineTM3000 mix, respectively diluted in 125. mu.L of Opti-Mem (gibco, #31985-0.70), and incubated for 5 min. These reagents were mixed and incubated for about 15min before being added dropwise to each well. After 24h, puromycin at 2.5. mu.g/mL was added for 48h and the selected transfected cells were used for subsequent studies.
5. T7E1 enzyme digestion method
The selected transfected cells were cultured at 37 ℃ for another 48 hours, and then genomic DNA was extracted. Total genomic DNA was amplified with Taq DNA polymerase (Toyobo, # KFX-201) according to the manufacturer's protocol (specific amplification primers were sgRNA1fwd/sgRNA1 rev, sgRNA2fwd/sgRNA2 rev, and sgRNA3fwd/sgRNA3 rev in Table 1). The PCR product was denatured and annealed using a thermal cycler. The PCR product was mixed with 2. mu.L of NEBuffer 2 for a total volume of 19m L to form a heteroduplex, programmed at 95 ℃ for 5 min; reducing the temperature to 85 ℃ at a speed of-2 ℃/s; then reduced to 25 ℃ at a rate of-0.1 ℃/s, then 1 μ L T7E1 enzyme (NEB # M0302S) was added and incubated at 37 ℃ for 25 minutes, and finally, the annealed product was run in a 2% agarose gel.
6. AAV-BR1 DNA vector construction
The targeted sgRNA and sgControl (sequence: sgCON in Table 1) were screened in an in vitro T7E1 experiment, cloned into AAV2 expression vector (described in "Korbelin et al, A broad microbial end-linked cell-specific viral vector with the possibility to use the molecular to treat neural and neurological diseases.2016. the public is available from the applicant, and can only be used in duplicate experiments, without other uses), followed by cloning 2A-tdTomato (SEQ ID No.2) into 2 expression vector before SV40 Poly-A, to construct pAAV-sgCtBn 1-tdnntro CMV plasmid. Whether each step was successful was verified by Sanger sequencing.
7. Synthesis of BR 1-sgCnnb 1-tdTomato recombinant virus
Cerebrovascular endothelial cell specific viral vector AAV-BR1 (Jakob) modified with AAV2
The professor provides text entitled "Korbelin et al, A broad microscopic endoscopic cell-specific viral vector with the potential to foot neurological and neurological diseases, 2016". Publicly available from the applicant, only available for repeated experiments of the invention, no other) to edit target genes in cerebrovascular endothelial cells. Briefly, HEK293T cells were cultured in DMEM medium containing 10% FBS, and AAV-BR1 DNA vector (i.e., pAAV-sgCtnnb1-CMV-tdTomato plasmid constructed in step 6) was transfected by the calcium phosphate (Macgene # Ctk001) method) Plasmid rAAV 2-retrohelper (addnne, cat 81070) encoding modified AAV2 capsid, and adenovirus helper plasmid pXX2-187-NRGTEWD (Jakob)
The professor provides that the plasmid is "modified pXX 2-187" modified to NRGTEWD "in" Korbelin et al, A broad microbial Another cell-specific viral vector with the possibility to reach neural and neural diseases, 2016 ". Publicly available from the applicant, can only be used to replicate experiments of the invention, and not others). After three days of transfection, supernatant and cell debris were collected separately, and BR1-sgCtnnb1-tdTomato recombinant virus was obtained by iodixanol density gradient ultracentrifugation.
At a rate of 1.8X 10 per mouse11Vg dose the above BR1-sgCtnnb1-tdTomato recombinant virus was injected into Tie2 through the caudal veinCas9And Tie2-Cre negative mice. For independent experiments, at least 3 mice with a specific genotype were used per group.
8. RNA extraction and real-time quantitative PCR
Total RNA was extracted using TRIzol reagent (Life Technologies) according to the manufacturer's procedure, and then mRNA was reverse-transcribed into cDNA using reverse Transcriptase (TOYOBO). According to SYBR Green Real-time PCR Master Mix (TOYOBO), Real-time quantitative PCR is carried out on a 7500fast Real-time PCR system (applied to a biological system) according to the instructions of manufacturers. The oligonucleotides used in the real-time quantitative PCR are listed in table 1.
9. Western blot analysis
NIH-3T3 cells in 6-well plates were lysed in RIPA supplemented with protease inhibitors (Roche) and the protein lysate samples were assayed using Pierce BCA protein assay reagent (Thermo Fisher Co.) according to the manufacturer's instructions. Protein samples were separated on a 10% polyacrylamide gel, boiled in Laemmli sample buffer, then transferred to PVDF membrane (Millipore), followed by blocking the PVDF membrane with 5% milk for 1h, and incubated with rabbit anti- β -catenin (β -catenin) (1:1000, Cell Signaling Technology, # D10A8) and GAPDH (1:1000, Zsbio, # TA-08) antibodies, respectively, overnight at 4 ℃ for the next day, the membrane was incubated with horseradish peroxidase-coupled secondary antibody for 1h at room temperature; protein signals were detected using Western Blotting Substrate (Engreen, #29100) and imaged by Image Quant LAS 4000Mini (GE healthcare).
10. Flow cytometer
Mouse brain tissue was isolated and digested in PBS containing 0.2% collagenase H (Roch #33278626) and 10% BSA (Sigma # WXBD0126V) for 1H at 37 ℃; after centrifugation, lipid is removed; after incubating the samples with CD31-APC antibody (eBioscience) at 4 ℃ for 30min in the absence of light, FACS analysis and sorting were performed using a FACS Aria III Caliber flow cytometer (BD Biosciences) following the manufacturer's procedure, dead cells were excluded by 7-amino-actinomycin D (7-AAD) staining.
11. Immunostaining
Brain tissue was fixed in 4% Paraformaldehyde (PFA) overnight and sections were paraffin embedded (6 μm) or embedded with OCT at optimal temperature (40 μm). After blocking with 10% goat serum/5% BSA/PBST (0.5% Triton X-100) for 30min at 37 deg.C, sections were incubated overnight at 4 deg.C with the following primary antibodies: CD31 (1:100, BD Biosciences, #550274), GFP (1:500, Cell Signaling Technology #2956s), RFP (1:500, Rockland, Limerick, PA # 600-401-; tdTomato can be recognized by RFP antibody), β -Catenin (1: 300, Cell Signaling Technology #8480), PLVAP/MECA-32 (1: 200, BD PharMingen #553849), PECAM1 (1:500, BD Biosciences #553370), CLAUDIN-5(1:500, Invitron #352588), and then coupled with corresponding Alexa Fluor coupled secondary antibodies (1:1000, Thermo Fisher Scientific) for 1h at room temperature. The image was taken with a Pro Long Gold (Invitrogen) coverslip, LSM 880 confocal microscope (Carl Zeiss AG).
12. BBB permeability assay
Mice were subjected to deep anesthesia and injected intravenously with Sulfo-NHS-LC-Biotin (0.5mg/g body weight, Thermo Scientific #21335), 5min later tissues were fixed in 4% paraformaldehyde overnight with 30% sucrose equilibration. After fixation of 40 μm thick sections in 4% PFA for 15min at room temperature, vessels were labeled with the corresponding Alexa Fluor-594 conjugated secondary antibody (1:1000, Thermo Fisher Scientific), CD31 primary antibody and 488-Alexa Fluor conjugated secondary antibody (1:1000, Invitrogen). The samples were then photographed by confocal microscopy (LSM 880, Carl Zeiss AG).
13. Deep DNA sequencing
For sequencing analysis of insertion/deletion mutations, cerebrovascular endothelial cell genomic DNA libraries were prepared and sequenced by first amplifying the targeted region by PCR using Taq DNA polymerase (primer sequences see Ctnnb1 fwd/Ctnnb1 rev in table 1), then taking 250ng from the PCR products using NEBNext Ultra DNA Library Prep Kit for Illumina (NEB) and Illumina HiSeq PE150 system (Genewiz). All data were performed according to the manufacturer's instructions and all sequencing reads were analyzed using crispreso 2 (element et al, 2019). Reads of insertion/deletion sequences are compared to reference values and the length and position of the insertion/deletion in the alignment is described, centered around the Cas9 predicted cleavage site of crispreso 2.
14. Statistical analysis
Data analysis was performed using GraphPad Prism 8/software. Data were analyzed using a two-sided independent sample t-test. Error bars on the graph represent mean ± Standard Deviation (SD),*P<0.05、**P<0.01 and***P<a difference of 0.001 is statistically significant.
Second, results and analysis
1. Design and in vitro validation of sgrnas
The invention selects Ctnnb1 gene which is important for BBB development to edit. The invention first designs three sgrnas (https:// chopchopchop. cbu. uib. no /) against Ctnnb1 using CRISPR design tool. All three sgRNA sequences were designed immediately before the 5' -NGG sequence (called PAM). The invention constructs sgRNA (named sgRNA1, sgRNA2 and sgRNA 3; corresponding to 'Ctnnb 1sgRNA 1, Ctnnb1sgRNA 2 and Ctnnb1sgRNA 3' in table 1) which is positioned in different areas of exon 1 or exon 2 near Ctnnb1 ATG, clones the sgRNA into a Cas9 expression vector (PX459), and then transfects the vector into NIH-3T3 cells. The targeting efficiency of the sgRNA was evaluated using a T7E1 detection method that can detect double strand breaks. As a result, the sgRNA1 and sgRNA2 groups generated cleavage bands, and the sgRNA1 was found to have the highest targeting efficiency and induce 74.9% insertion/deletion by gray value calculation (a in fig. 1). Therefore, the invention selects to further verify the AAV-CRISPR/Cas9 gene editing system in somatic cells by using sgRNA 1(the target sequence is shown as SEQ ID No. 1). The sgRNA design principle is to minimize the off-target effect (OT) of other genome sites, so the invention designs specific PCR primers aiming at the first 5 predicted off-target sites of sgRNA1 in the genome (see Sobp fwd/Sobp rev, Llgl2 fwd/Llgl2 rev, Gm10851 fwd/Gm10851 rev, Gtpbp3 fwd/Gtpbp3 rev, Gm26812 fwd/Gm26812 rev in Table 1), and then carries out T7E1 analysis to evaluate the off-target effect of sgRNA1 in NIH-3T3 cells. As a result, sgRNA1 was found to detect no off-target cleavage in the first five predicted off-target sites (B and C in fig. 1). To further evaluate the effectiveness of sgRNA1, the present invention also examined changes in the levels of Ctnnb1 transcription and translation in NIH-3T3 cells transfected with sgRNA 1-Cas9 plasmid, and the Ctnnb1 mRNA and protein expression was found to be significantly reduced in the sgRNA1 group compared to sgcontrol (sgcon) as assessed by real-time quantitative PCR analysis (D in fig. 1) and western blot (E and F in fig. 1). All these results indicate that sgRNA1 can direct efficient gene editing and can be used for further studies.
2. At Tie2Cas9Delivery of sgRNA by AAV-BR1 in mice results in reduced expression of brain endothelial Ctnnb1
To investigate whether endothelial-specific Cas9 expressing mice could be used to efficiently perform in vivo gene editing, we crossed heterozygous Tie2-Cre transgenic mice with homozygous R26-loxP-STOP-loxP-3xFLAG-Cas9-eGFP mice to obtain heterozygous Tie2Cas9Mice (a in fig. 2). We integrated sgRNA1 verified in vitro into the U6 driven AAV2 expression vector backbone and packaged into a BR1 capsid serotype virus (B in fig. 2) that efficiently transduces whole brain endothelial cells. Then Tie2-Cre negative and Tie2 of 30d (P30) bornCas9The single dose of intravenous injection of the mice is 1.8 multiplied by 1011Vg of BR 1-sgCnnb 1-tdTomato recombinant virus, 4 weeks later (P60) (FIG. 2B). To examine the transduction efficiency of BR 1-sgCnnb 1-tdTomato, we isolated primary cerebrovascular endothelial cellsCell, flow cytometry (FACS) was used to analyze endothelial cells expressing the endothelial markers CD31 and tdTomato, and we found that 65% of the brain endothelium was double positive for CD31 and tdTomato, indicating a higher infection efficiency of the virus (C in fig. 2). T7E1 analysis of DNA isolated from cerebrovascular endothelial cells by FACS revealed Tie2Cas9The target site DNA of mouse Ctnnb1 was disrupted, while sgCtnnb1 (i.e., sgRNA1 targeting Ctnnb1 gene) had no effect on control mice (D in fig. 2). Tie2 compared to control miceCas9Immunofluorescent staining results of mice 4 weeks after virus infection showed that CTNNB1 expression was reduced in tdTomato positive sections in a chimeric pattern. Meanwhile, PLVAP expression was up-regulated in brain endothelial cells with loss of CTNNB1 expression, indicating that chimeric knock-out of CTNNB1 gene occurred in brain endothelium (fig. 2E). Quantitative analysis proves that the BR 1-sgCnnb 1-tdTomato virus can infect cerebral vascular endothelial cells more efficiently (F in figure 2). We then found Tie2 by qRT-PCR detectionCas9mRNA expression of target gene Ctnnb1 in mouse cerebral vascular endothelial cells was down-regulated relative to control (G in fig. 2). Western blot detects the expression of CTNNB1 in cerebrovascular endothelial cells, and compared with a control group, Tie2Cas9The expression of CTNNB1 in mice was reduced by about 25% (H and I in fig. 2). Taken together, these results indicate that BR1-sgCtnnb1-tdTomato virus successfully induced Tie2Cas9Disruption of Ctnnb1 in mouse cerebral vascular endothelial cells.
3. Evaluation of the editing efficiency of the target Gene Ctnnb1 in cerebrovascular endothelial cells by DNA sequencing
In order to accurately detect the editing efficiency of a target gene in a cerebral vascular endothelial cell of an infected mouse, PCR amplification is carried out on a target site of a cerebral endothelial cell Ctnnb1 separated from an experimental mouse, and DNA deep sequencing is carried out on an amplification product. Non-homologous end joining (NHEJ) can repair Cas9 nuclease-induced double-stranded DNA breaks, eventually leading to target site mutations. By deep sequencing we are dealing with Tie2Cas9About 43.7% of the mutant DNA was detected in the mouse DNA (a in fig. 3), of which about 17.1% was in-frame (3n) mutations (B in fig. 3) but most were out-of-frame (82.9%, 3n +1 or 3n +2), indicating a higher DNA mutation rate (B in fig. 3). Based onAccording to CRISPRResso 2 analysis, we found in high-throughput sequencing results that mutations generated by BR 1-sgCnnb 1-tdTomato virus-mediated target gene editing are mainly insertion mutations and deletion mutations, and most of the mutated sequences are adjacent to a cleavage region of Cas9 (C in FIG. 3). In addition, we also calculated the ratio of each mutation type at each base pair position in all the mutation reads, and observed that the sgRNA showed the highest mutation rate of deletion and insertion at nucleotide positions 17 to 19 (D in fig. 3). The above results show that Tie2 was obtained after the treatment of BR 1-sgCnnb 1-tdTomato virusCas9Efficient gene editing occurred in mice.
4. Genomic editing of Ctnnb1 in vivo results in disruption of the BBB
Classical Wnt/β -catenin is a key signaling pathway for BBB regulation, so we examined the change in blood brain barrier permeability in mice following infection with BR1-sgCtnnb1-tdTomato virus. Tie2 infected with BR 1-sgCnnb 1-tdTomato virusCas9After the mice were subjected to the Sulfo-NHS-LC-Biotin perfusion experiment, the mice were found to have BBB leakage in olfactory bulb, cortex, cerebellum and retina, while no dye leakage was observed in the control mice (A in FIG. 4). The permeability-associated protein PLVAP is related to vesicle transport and abnormal development of blood brain barrier, and is regulated and controlled by a Wnt/beta-catenin pathway. During early development, PLVAP is initially expressed in immature cerebral vessels, and is later strongly down-regulated in the endothelial cells of the blood brain barrier; while in pathological conditions of the BBB, the expression of PLVAP is up-regulated. Therefore, we used the immunofluorescent staining method to detect Tie2Cas9The expression of PLVAP in mice revealed that the expression level of PLVAP in the Sulfo-NHS-LC-Biotin percolation region was significantly increased (B in FIG. 4). CLAUDIN-5 plays a crucial role in the formation of Tight Junctions (TJ) and BBB function, and knocking-out CLAUDIN-5 in mouse embryos can lead to early brain edema and even death in adult mice. Furthermore, CLAUDIN-5 is regulated by the Wnt/beta-catenin pathway and its expression is in contrast to PLVAP, BR1-sgCtnnb1-tdTomato virus-infected Tie2Cas9Immunofluorescence staining results of mouse brain sections show that expression of CLAUDIN-5 in brain vascular endothelial cells is limited and expression of PLVAP is up-regulated, which indicates that Wnt/beta-catenin pathway is in an inactivated state (C in figure 4). These results indicate that the mouse cerebrovascular endothelial Ctnnb1 gene is precisely edited and results in an increase in BBB permeability. In conclusion, the AAV-CRISPR/Cas9 technology is utilized to successfully construct a mouse blood brain barrier injury model.
Third, discuss
In this study, Ctnnb1 mutant mice were constructed in the present invention, and the sgRNA was mediated by AAV-BR1 for transport into endothelial-specific Cas9 mouse brain vascular endothelial cells, resulting in disruption of the BBB. Although several recent studies have extensively evaluated the feasibility of adult gene editing in several organisms using Cas9, the present studies have for the first time targeted genes in adult cerebrovascular endothelial cells. The results of the present invention indicate that systemic delivery of sgrnas results in chimeric gene knockouts. Finding an effective delivery vector is considered to be a barrier that hinders CRISPR from being most solved in a method of in vivo genome editing, the present invention uses AAV-BR1 to specifically target cerebrovascular endothelial cells with Cas9 expression; the results show that 30 days after intravenous injection, about 65% of cerebrovascular endothelial cells are successfully infected, and the insertion/deletion frequency is 43% by DNA sequencing; meanwhile, the protein expression of the target gene is obviously reduced by 25% in the research. More importantly, the present invention found that about 25% of CTNNB1 defects induced by AAV-CRISPR were sufficient to cause severe BBB disruption. Therefore, the CRISPR/Cas9 system may be a useful tool to screen genes essential for BBB development and maintenance by editing candidate genes in cerebrovascular endothelial cells. Based on the rapid development of CRISPR technology, a variety of editing strategies can be implemented. In the research of the invention, the AAV-CRISPR system can effectively realize the gene disruption in the cerebrovascular endothelial cells through NHEJ.
As mentioned previously, the improvement of existing BBB models is a significant challenge. The new generation of BBB models is expected to better study the uniqueness of BBB, the mechanisms involved in the development and maintenance of BBB and the occurrence of diseases. In addition, blood-brain barrier penetration is often associated with the development and progression of neurological diseases, such as Alzheimer's disease, Parkinson's disease, stroke, etc., and studies have shown that cerebrovascular endothelial cells may be a key regulator of the alleviation of these neurological diseases. Due to the lack of animal models consistent with patient-related mutations, the AAV-CRISPR adult genome editing system still provides a potential tool for exploring the possibility of reverting blood brain barrier function in neurological diseases.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> military medical research institute of military science institute of people's liberation force of China
<120> a method for preparing a blood brain barrier deficient mouse model
<130> GNCLN210847
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
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<212> DNA
<213> Artificial sequence
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gccacgaact tctctctgtt aaagcaagca ggagacgtgg aagaaaaccc cggtcctatg 60
gtgagcaagg gcgaggaggt catcaaagag ttcatgcgct tcaaggtgcg catggagggc 120
tccatgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 180
acccagaccg ccaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 240
ctgtcccccc agttcatgta cggctccaag gcgtacgtga agcaccccgc cgacatcccc 300
gattacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 360
gacggcggtc tggtgaccgt gacccaggac tcctccctgc aggacggcac gctgatctac 420
aaggtgaaga tgcgcggcac caacttcccc cccgacggcc ccgtaatgca gaagaagacc 480
atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag 540
atccaccagg ccctgaagct gaaggacggc agccactacc tggtggagtt caagaccatc 600
tacatggcca agaagcccgt gcaactgccc ggctactact acgtggacac caagctggac 660
atcacctccc acaacgagga ctacaccatc gtggaacagt acgagcgctc cgagggccgc 720
caccacctgt tcctggggca tggcaccggc agcaccggca gcggcagctc cggcaccgcc 780
tcctccgagg acaacaacat ggccgtcatc aaagagttca tgcgcttcaa ggtgcgcatg 840
gagggctcca tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac 900
gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc cttcgcctgg 960
gacatcctgt ccccccagtt catgtacggc tccaaggcgt acgtgaagca ccccgccgac 1020
atccccgatt acaagaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac 1080
ttcgaggacg gcggtctggt gaccgtgacc caggactcct ccctgcagga cggcacgctg 1140
atctacaagg tgaagatgcg cggcaccaac ttcccccccg acggccccgt aatgcagaag 1200
aagaccatgg gctgggaggc ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag 1260
ggcgagatcc accaggccct gaagctgaag gacggcggcc actacctggt ggagttcaag 1320
accatctaca tggccaagaa gcccgtgcaa ctgcccggct actactacgt ggacaccaag 1380
ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga gcgctccgag 1440
ggccgccacc acctgttcct gtacggcatg gacgagctgt acaagtaa 1488
Claims (10)
1. A method of making an animal model deficient in the blood-brain barrier comprising the steps of: based on a CRISPR/Cas9 system, a cerebrovascular endothelial cell specific gonad-associated virus vector is used as a delivery vector of sgRNA, and Ctnnb1 gene in a recipient animal cerebral vascular endothelial cell is knocked out in a targeted mode.
2. The method of claim 1, wherein: the animal is a mouse.
3. The method according to claim 1 or 2, characterized in that: the specific gonad-related virus vector of the cerebral vascular endothelial cells is a BR1 serotype adeno-associated virus vector.
4. A method according to any one of claims 1-3, characterized in that: the target sequence of the sgRNA is SEQ ID No. 1.
5. The method according to any one of claims 1-4, wherein: the method comprises the following steps: infecting a vascular endothelial cell-specific Cas9 transgenic mouse with BR1 serotype adeno-associated virus expressing the sgRNA, and obtaining a blood brain barrier defect mouse model after 4 weeks.
6. The method of claim 5, wherein: the vascular endothelial cell-specific Cas9 transgenic mouse is obtained by hybridizing a heterozygous Tie2-Cre transgenic mouse and a homozygous R26-loxP-STOP-loxP-3xFLAG-Cas9-eGFP mouse.
7. The method according to claim 5 or 6, characterized in that: infection of the vascular endothelial cell-specific Cas9 transgenic mice with BR1 serotype adeno-associated virus expressing the sgRNA was achieved by: a single intravenous dose of 1.8 × 10 to the vascular endothelial cell-specific Cas9 transgenic mice11vg BR1 serotype adeno-associated virus expressing the sgRNA.
8. A kit for preparing a blood-brain barrier deficient mouse model consisting of BR1 serotype adeno-associated virus expressing the sgRNA described in claim 5 and a vascular endothelial cell-specific Cas9 transgenic mouse described in claim 5 or 6.
9. Any of the following applications:
use of P1, the kit of claim 8, for the preparation of a blood brain barrier deficient mouse model;
use of P2, the animal model prepared by the method of any one of claims 1 to 7, for screening a medicament for treating or ameliorating a blood brain barrier defect.
10. A method for identifying or assisting in identifying whether a gene to be tested has a function of maintaining the integrity of a blood brain barrier or not comprises the following steps:
(A1) infecting a vascular endothelial cell specific Cas9 transgenic mouse with BR1 serotype adeno-associated virus expressing sgRNA of a gene to be detected in a targeted mouse cerebral vascular endothelial cell;
(A2) determining whether the gene to be detected is a gene having a function of maintaining the integrity of the blood brain barrier according to the following steps: if after 4 weeks the mouse treated in step (a1) exhibits a blood brain barrier defect, then the gene to be tested is or is a candidate for a gene that is functional for maintaining blood brain barrier integrity; otherwise, the gene to be tested is not or is not a candidate gene having a function of maintaining the integrity of the blood brain barrier.
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