Construction method of Cd47 knockout rat model and verification of knockout effect of Cd47 knockout rat model
By designing highly active gRNAs on both ends of the second exon of the rat Cd47 gene, and using the CRISPR/Cas9 system to construct a Cd47 knockout rat model, the lack of rat Cd47 gene knockout model in the prior art was solved, and a specific phenotype suitable for neural, immune and tumor research was provided.
Patent Information
- Application Number
- CN202510379919.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-08-08
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing technology lacks a rat Cd47 knockout model, and the mouse model has problems such as small body size, few blood samples, and high surgical technical requirements, which are difficult to meet the needs of preclinical pharmacological and toxicological experiments.
A pair of highly active gRNAs were designed on both ends of the second exon of the rat Cd47 gene by using the CRISPR/Cas9 system. A rat model of Cd47 knockout was constructed by microinjection of fertilized eggs, and the knockout effect was identified by PCR and Western blot.
The efficient knockout of the endogenous Cd47 gene isomer in rats was achieved, providing a specific phenotype of slow growth, larger spleen, smaller thymus, fewer peripheral red blood cells and reduced T cells, and is suitable for neural, immune and tumor research.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology and relates to a method for constructing a genetically modified animal model, and in particular to a method for constructing a Cd47 knockout rat model and verification of the knockout effect thereof. Background Art
[0002] CD47 is a heavily glycosylated transmembrane protein belonging to the immunoglobulin (Ig) superfamily. Its N-terminus houses a V-type Ig-like extracellular domain, with a central hydrophobic five-transmembrane segment and a short cytoplasmic domain at its C-terminus. CD47 interacts with αvβ3, αIIbβ3, and α2β1 integrins, thereby regulating cellular responses to extracellular matrix proteins containing arginine-glycine-aspartate. Therefore, it was originally named integrin-associated protein (IAP). CD47 interacts not only with its associated integrins but also with proteins such as thrombospondin and signal regulatory protein α (SIRPα), transducing signals into cells and participating in biological activities such as cell spreading, migration, phagocytosis, and apoptosis. The CD47-SIRPα "don't eat me" signaling pathway plays an important role in the central nervous system and blood system. Due to the negative regulatory effect of this signaling pathway on initial immunity (macrophages and dendritic cells, etc.), CD47 has been used as an important immune checkpoint in clinical research on immunotherapy of blood and solid tumors.
[0003] To study the functions of CD47 in various tissues and organs within individual animals, it is necessary to establish an animal model in which the gene is knocked out. Due to technical limitations, the only available CD47 knockout mouse model is currently available. However, mice have disadvantages such as their small size, limited blood sample collection, and demanding surgical techniques. While maintaining a cost-effective approach, rats offer an important alternative animal model. Rats are 10 times the size of mice and are physiologically closer to humans. They also offer the advantages of accommodating a larger sample size and facilitating orthotopic transplantation. Furthermore, rats are widely used in preclinical pharmacology and toxicology research.
[0004] There are multiple isoforms of rat Cd47 mRNA. When constructing a Cd47 gene knockout rat model, it is necessary to consider how to ensure that all isoforms are effectively knocked out, thereby constructing a complete Cd47 knockout rat model. Summary of the Invention
[0005] The present invention aims to provide a method for constructing and validating a Cd47 knockout rat model. The first exon of rat Cd47 is shorter than that of mouse Cd47 (NCBI) and does not encode important protein domains. This makes it more likely that the protein retains activity after knockout, making it unsuitable for target site design. Conversely, the second exon is longer and encodes a crucial extracellular domain, which is identical across all isoforms. Knocking out the second exon completely abolishes protein activity (even if a possible mechanism preserves the C-terminus). This method utilizes CRISPR gene editing technology to design a pair of gRNAs at either end of the second exon of the rat Cd47 gene, thereby deleting the identical fragment encoded by all isoforms of the Cd47 gene. This establishes a Cd47 knockout rat model that efficiently knocks out all isoforms of the endogenous rat Cd47 gene. In this model, the inventors verified the Cd47 knockout results by PCR and Western blot. Compared to wild-type rats, Cd47 knockout rats showed slower growth, larger spleens, and smaller thymuses. Cd47 knockout rats have fewer peripheral red blood cells and a reduced number of T cells. Due to species differences, the timing and extent of these phenotypes in Cd47 knockout rats differ from those in mice (Lab Anim Res 2018: 34(4), 302-310).
[0006] In order to achieve the above objectives, the first aspect of the present invention discloses a method for constructing a Cd47 knockout rat model, comprising the following steps: (1) For all isoforms of the rat Cd47 gene, a pair of target site recognition gRNA1 and gRNA2 were designed and synthesized at both ends of the second exon; the nucleotide sequence of the gRNA1 is shown in SEQ ID NO: 1, and the nucleotide sequence of the gRNA2 is shown in SEQ ID NO: 2; (2) The Cd47 gene of rats was knocked out using CRISPR gene editing technology to construct a Cd47 knockout rat model.
[0007] In step (2), Cas9 mRNA and gRNA are mixed and microinjected into fertilized eggs. The injected fertilized eggs are briefly cultured in an incubator and then transplanted into the oviduct of the recipient mother mouse to obtain the F0 generation of gene-modified rats, which are then subcultured to obtain recombinant positive rats.
[0008] Preferably, the method further comprises an identification step: using a primer pair P3 and P4 specific for both ends of the insertion site to identify whether knockout has occurred in the genomes of the obtained F0 generation of genetically modified rats and the recombinant-positive mice; The nucleotide sequences of primers P3 and P4 are shown in SEQ ID NOs: 8 and 9, respectively.
[0009] The second aspect of the present invention discloses a Cd47 knockout rat model constructed by the above method.
[0010] The third aspect of the present invention discloses the application of the above-mentioned Cd47 knockout rat model in the fields of neurology, immunity, and tumor research. The Cd47 knockout rat model is used to study the function of Cd47 in various tissues / organs in individual animals.
[0011] The applications specifically include: a) Development of products involving immune processes in human cells, and applications in the manufacture or screening of human antibodies; b) Application as a model system in medical research such as pharmacology, immunology, microbiology and oncology; c) Production and use of animal experimental disease models involving human cells for the study of immune processes and their application in etiology research; d) In vivo screening, efficacy testing, efficacy assessment, validation or evaluation of human CD47-related signaling pathway modulators; or e) Research on CD47 gene function, human CD47 antibodies, drugs targeting human CD47 target sites and their efficacy, and the use of drugs for immune-related diseases as well as anti-tumor or inflammatory drugs.
[0012] Compared with the prior art, the present invention has the following advantages: The present invention provides a method for constructing a Cd47 knockout rat model. The model is constructed based on the CRISPR / Cas9 system using screened highly active gRNA target sites. The inventors identified the gene knockout and protein expression, growth, immune organ growth and development, and red blood cell and T cell phenotypes of the model. It was demonstrated that the model achieves the knockout of all isoforms of the endogenous Cd47 gene in rats and can be applied to the research of Cd47 protein in the fields of neurology, immunity, and tumor-related fields. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the existing methods and experiments, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0014] Figure 1 Schematic diagram of the rat Cd47 gene structure and the strategy for constructing a Cd47 knockout rat model. E1-E9 represent nine exons; two gRNAs are designed at either end of the second exon (E2).
[0015] Figure 2 Figure 3: Agarose gel electrophoresis of T7EI digestion. Compared to the control (-) without T7EI, T7EI showed better cleavage efficiency in samples with gRNAs 1 and 2 (indicated by arrows); however, the cleavage efficiency of gRNAs 3 and 4 was lower.
[0016] Figure 3 The structure of the gene mutation and identification of gene knockout in the Cd47 knockout rat model. A shows the structure of the Cd47 gene in Cd47 knockout rats; B shows the electrophoresis pattern of F2 generation rats; numbers indicate rat numbers; M indicates a 100-bp DNA marker; C shows Western blot analysis results; D shows flow cytometry analysis of CD47 expression in rat peripheral blood cells. CD47+ / - represents heterozygous controls, and CD47- / - represents homozygous knockout.
[0017] Figure 4 Growth and development of key immune organs in Cd47 knockout rats. A, Body weight changes from weeks 4 to 32 in Cd47 knockout rats; B, Relative spleen and thymus weights (per body weight) in Cd47 knockout rats at week 8. CD47+ / - represents heterozygous controls, and CD47- / - represents homozygous knockout.
[0018] Figure 5 Figure 3: Peripheral blood erythrocyte and T cell phenotypes in Cd47 knockout rats. A: Peripheral blood erythrocyte count in Cd47 knockout rats. B: Peripheral blood T cell percentage in Cd47 knockout rats. DETAILED DESCRIPTION
[0019] The present invention is further described below with reference to specific examples. Experimental methods and techniques in the following examples, where specific conditions are not specified, are generally performed in accordance with conventional conditions in the art or conditions recommended by the manufacturer.
[0020] Example 1 Construction of Cd47 knockout rat model To knock out all isoforms of the Cd47 gene in Sprague Dawley (SD) rats, the inventors designed a gRNA at each end of the second exon of rat Cd47. The wild-type Cd47 gene structure and the strategy for constructing the Cd47 knockout rat model are as follows: Figure 1 shown.
[0021] 1) gRNA target site selection According to the experimental strategy, two pairs of gRNAs were designed (gRNA 1 and 3 at the 5' end of the second exon, gRNA 2 and 4 at the 3' end of the second exon). Figure 2), gRNA 1 (SEQ ID NO 1) and gRNA 2 (SEQ ID NO 2) demonstrated better cleavage efficacy than gRNA 3 (SEQ ID NO 3) and gRNA 4 (SEQ ID NO 4) in vitro. Therefore, gRNAs 1 and 2 were used for targeting experiments.
[0022] gRNA 1: CAGATTTGACTTTACTAAGCAGG (SEQ ID NO 1) gRNA 2: ATAGAGCTGAAAAACCGCCCAGG (SEQ ID NO 2) gRNA 3: CCAATTAAGGACTTTGCAAGGG (SEQ ID NO 3) gRNA 4: GATACCCATGAGGCTGTGGTGGG (SEQ ID NO 4) After transfection of Cas9 mRNA and gRNA in rat cell lines, DNA was extracted, amplified by PCR, and then digested with T7EI endonuclease. The designed PCR primers are shown in Table 1: Table 1 PCR amplification primers Primer name Primer sequence (5'—3') P1 ctatagtctactagctggtgtgc(SEQ ID NO 5) P2 tgagtgtcctgtttctctcca(SEQ ID NO 6) The reaction system is shown in Table 2: Table 2 Target gene PCR amplification reaction system 5 μL 5X, Q5 Reaction Buffer 0.5 μL 10 μM dNTP 1.25 μL 10 μM P1 primer 1.25 μL 10 μM P2 primer 0.25 μL Q5 Polymerase 50 ng DNA template X μL <![CDATA[ddH2O]]> 25 μL Total The reaction conditions are shown in Table 3: Table 3 PCR amplification reaction conditions
[0023] After the PCR reaction was completed, the PCR tube was quickly placed on ice and the reaction system was prepared. The results are shown in Table 4: Table 4 T7EI endonuclease reaction system 2 μL 10X, Buffer 2 0.5 μL 10 U / μL, T7 Endonuclease I 7.5 μL <![CDATA[ddH2O]]> 10 μL PCR Product 20 μL Total Set up a control group without T7EI. Gently pipette and quickly mix, then incubate at 37°C for 15 minutes. Finally, test the T7EI cleavage effect by agarose gel electrophoresis. Figure 2 shown) Example 2: Using amino acid sequence analysis software (https: / / www.novopro.cn / tools / translate.html) to predict the amino acids encoded by the DNA of the mutant Cd47, it was found that only 54 amino acids (SEQ ID NO 7) would be translated after the mutation, losing important functional domains such as the transmembrane domain, thereby completely eliminating the protein's function. (This avoids the situation where only partial function is eliminated.) Figure 3 Western blot analysis at C and flow cytometry analysis at D confirmed the above conclusions. )MWPLAAALLLGSCCCGSAQLLLSKVKSVEFTSCNDTVVIPCKVLAKPSFVVFYK* (SEQ ID NO 7) Example 3: The gRNA target sequence determines the efficiency and potential off-target activity of inducing Cas9 to cut the target gene. Efficient and specific target sequence selection and design are prerequisites for the successful construction of the Cd47 knockout rat model.
[0024] According to the recombination plan, software analysis was performed on potential gRNA sites near the insertion site, and sites with low off-target risks were preferentially selected as gRNA target sites gRNA1 and gRNA2 (SEQ ID NO: 1 and SEQ ID NO: 2) for subsequent SD rat fertilized egg injection experiments.
[0025] 3) Construction of Cd47 knockout rats: Fertilized eggs from SD rats were obtained and, following the literature, Cas9 mRNA and gRNA were mixed and microinjected into the fertilized eggs. After a short incubation period, the injected fertilized eggs were transplanted into the oviducts of recipient female mice to obtain the F0 generation of gene-modified rats. After birth, the tails of the F0 generation rats were cut and the genome was extracted. The presence of knockout was determined using primer pairs specific for both ends of the second exon (primers P3 and P4). The primer positions are shown in the figure below. Figure 1 As shown in P3 and P4 in, the sequence information is shown in Table 5: Table 5 The PCR reaction system of primer pair P3 / P4 is shown in Table 6: Primer name Sequence information (5'→3') P3 CAGTAAGCAGACATGATTACTTCAGAGC (SEQ ID NO: 8) P4 TATAAGCACCCTAACTTGGTTTCTGG (SEQ ID NO: 9) Table 6 PCR reaction composition Volume (µL) <![CDATA[ddH2O]]> 30 PCR Buffer 10 2.5 mM dNTP 4 Primer 1 (20 pmol / µL) 1 Primer 2 (20 pmol / µL) 1 DNA Polymerase 2 genomic DNA 2 total 50 The PCR reaction program for primer pair P3 / P4 is shown in Table 7: Table 7 step Temperature (℃) time Remark 1 94 2 min 2 94 30 seconds 3 60 30 seconds 4 72 1 min Repeat steps 2-4 for a total of 34 cycles 5 72 5 min 6 10 Hold After identification of two F0 generation rats, one contained a 328 bp knockout and the other contained a 251 bp knockout and a 6-base mutation ( Figure 3At A in the middle). The latter were mated with wild-type SD rats after adulthood to obtain F1 generation rats. After birth, the F1 generation rats were tail-cut and genomic extraction was performed. PCR identification was performed using the same conditions as for the identification of the F0 generation rats, targeting primers specific for both ends of the second exon (primers P3 and P4). F1 generation Cd47 knockout heterozygous rats were obtained. F1 generation heterozygous rats were then hybridized to obtain F2 generation rats, and PCR identification was performed using primers P3 and P4. Representative identification results are shown in Figure 2. Figure 3 As shown in B (No. 3 and 7 are knockout homozygotes; No. 1, 2, 8, 9, 10 are heterozygotes; No. 4, 5, 6 are wild-type homozygotes). Figure 3 As shown in Figure C, Western blot revealed that CD47 protein was not expressed in Cd47 knockout rats. The inventors detected CD47 protein expression in peripheral blood cells by flow cytometry. Figure 3 As shown in center D, CD47 protein expression was almost undetectable in peripheral blood cells of Cd47 knockout rats (-0.13 ± 0.08, N=3).
[0026] Example 4 Growth and development of important immune organs in Cd47 knockout rats The growth and development of important immune organs of Cd47 knockout rats were tested. Figure 4 As shown in Figure A, from the 4th to 32nd week after birth, the knockout rats weighed less than the heterozygous controls, but there was no statistically significant difference. Cd47 knockout rats did not show reproductive disorders. Figure 4 As shown in center B, Cd47 knockout rats had enlarged spleen (0.0030 ± 0.000086 vs 0.0022 ± 0.00016, N=3) and reduced thymus (0.0015 ± 0.00021 vs 0.0018 ± 0.00023, N=3) at postnatal week 8 compared with heterozygous controls.
[0027] Example 5 Phenotypic Identification of Red Blood Cells and T Cells in Cd47 Knockout Rats In order to verify the phagocytic effect of macrophages on erythrocytes mediated by CD47, the inventors identified the number of erythrocytes in the peripheral blood of Cd47 knockout rats by routine blood tests at 8 weeks after birth. Figure 5 As shown in Figure A, the number of peripheral blood red blood cells in Cd47 knockout rats was not significantly different from that in heterozygous controls (7.46 ± 0.64 vs 7.71 ± 0.77). However, the proportion of peripheral blood T cells was detected by flow cytometry. Figure 5As shown in center B, the proportion of T cells in the peripheral blood of Cd47 knockout rats was significantly reduced (37.89 ± 0.62 vs 49.39 ± 6.48). This phenotype indicates that Cd47 knockout rats have abnormal T cell differentiation and development, and have a certain immune deficiency phenotype.
[0028] In summary, the inventors provide a method for constructing a Cd47 knockout rat model. Based on the common characteristics of Cd47 gene-encoded isoforms, a pair of highly active gRNA target sites were screened at both ends of the second exon, and a Cd47 knockout rat model was constructed based on the CRISPR / Cas9 system. The inventors characterized the gene knockout and protein expression, growth, immune organ growth and development, and red blood cell and T cell phenotypes of the model. This model can be applied to the fields of CD47 protein research in neurology, immunity, and tumor-related fields.
Claims
1. A method for constructing a Cd47 knockout rat model, characterized in that: The steps include: (1) For all isoforms of the rat Cd47 gene, a pair of target site recognition gRNA1 and gRNA2 were designed and synthesized at both ends of the second exon; the nucleotide sequence of the gRNA1 is shown in SEQ ID NO: 1, and the nucleotide sequence of the gRNA2 is shown in SEQ ID NO: 2; (2) The Cd47 gene of rats was knocked out using CRISPR gene editing technology to construct a Cd47 knockout rat model.
2. The method for constructing a Cd47 knockout rat model according to claim 1, wherein: In step (2), Cas9 mRNA and gRNA are mixed and microinjected into fertilized eggs. The injected fertilized eggs are briefly cultured in an incubator and then transplanted into the oviduct of the recipient mother mouse to obtain the F0 generation of gene-modified rats, which are then subcultured to obtain recombinant positive rats.
3. The method for constructing a Cd47 knockout rat model according to claim 2, wherein: The method also includes an identification step: using a primer pair P3 and P4 specific to both ends of the insertion site to identify whether knockout has occurred in the genomes of the obtained F0 generation of gene-modified rats and recombinant-positive mice; The nucleotide sequences of primers P3 and P4 are shown in SEQ ID NOs: 8 and 9, respectively.
4. A Cd47 knockout rat model constructed according to the method according to any one of claims 1 to 3.
5. Use of the Cd47 knockout rat model according to claim 4 in the fields of neurology, immunity, and tumor research.
6. The use according to claim 5, characterized in that The Cd47 knockout rat model is used to study the functions of Cd47 in various tissues / organs in individual animals.
7. The use according to claim 5, characterized in that The applications described include: a) Development of products involving immune processes in human cells, and applications in the manufacture or screening of human antibodies; b) use as a model system in medical research in pharmacology, immunology, microbiology and oncology; c) Production and use of animal experimental disease models involving human cells for the study of immune processes and their application in etiology research; d) In vivo screening, efficacy testing, efficacy assessment, validation or evaluation of modulators of human CD47-related signaling pathways; or e) Research on CD47 gene function, human CD47 antibodies, drugs targeting human CD47 target sites and their efficacy, and the use of drugs for immune-related diseases as well as anti-tumor or inflammatory drugs.