Method for constructing Ddx58-R109C point mutation mouse model and application of Ddx58-R109C point mutation mouse model

Abstract

The invention discloses a method for constructing a Ddx58-R109C point mutation mouse model by using a CRISPR / Cas9 gene editing technology and an application of the Ddx58-R109C point mutation mouse model. The method comprises the following steps: (1) target construction: searching a CDS region of a Ddx58 gene, determining an exon part, and determining a gene mutation site; (2) obtaining a fertilized egg, and injecting Cas9mRNA, gRNA and donor oligo into the fertilized egg of the C57BL / 6J mouse together in vitro through a microinjection technology to obtain an F0-generation hybrid mouse; (3) mating the F0-generation hybrid mouse with the wild type mouse to obtain an F1-generation mouse; (4) the F1-generation heterozygous mice which are identified to be positive are subjected to mating, and homozygous Ddx58-R109C mutant type and wild type mice can be obtained by breeding obtained offspring; (5) phenotypic analysis shows that Ddx58-R109C point mutation mice can have pathological changes of systemic lupus erythematosus and lupus nephritis; the invention can provide an effective research approach and method for researchers to deeply explore the action mechanism of Ddx58-R109C mutation in diseases, and has important significance for explaining the pathogenesis of systemic lupus erythematosus and researching and developing targeted therapeutic drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology research, and specifically relates to a method for constructing a Ddx58-R109C point mutation mouse model and application thereof. Background Art

[0002] The DDX58 gene encodes RIG-I protein, an important cytoplasmic pattern recognition receptor that senses viral invasion and activates downstream type I interferon and nuclear factor κB (NF-κB) signaling pathways by recognizing viral double-stranded RNA (dsRNA) in the cytoplasm.

[0003] Genetic variation is the root cause of the occurrence and development of diseases. Studies have confirmed that mutations in the human DDX58 gene can lead to Singleton-Merten syndrome, which is characterized by aortic and valvular calcification, abnormal tooth development, skeletal abnormalities, glaucoma, and psoriasis-like rashes. The human DDX58-R109C mutation was first discovered in patients with systemic lupus erythematosus and can lead to lupus nephritis, psoriasis and other diseases. In vitro cell models and patient peripheral blood sample tests found that the mutation spontaneously relieved RIG-I autoinhibition and continuously activated the type I interferon signaling pathway. However, the effects and molecular mechanisms of this mutation on immune cell function are still unclear, and there are no personalized therapeutic drugs developed targeting this site. Therefore, a mouse model with edited sites is urgently needed to further study these issues. Summary of the invention

[0004] To solve the above problems, the present invention discloses a method and application for constructing a mouse model with a Ddx58-R109C point mutation. The method uses CRISPR / Cas9 gene editing technology to perform site-directed mutation at the R109C position of the mouse Ddx58 gene, successfully constructs a Ddx58-R109C point mutation mouse model, and further analyzes the mouse serum autoantibody level and renal tissue pathological structure. The present invention can provide researchers with an effective research approach and method for in-depth exploration of the mechanism of action of the Ddx58-R109C mutation in the disease, which is of great significance for explaining the pathogenesis of systemic lupus erythematosus and developing targeted therapeutic drugs.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] The first object of the present invention is to provide a method for constructing a Ddx58-R109C point mutation mouse model, comprising the following steps:

[0007] (1) Determine the site to be mutated: This site is located in the third exon of the mouse Ddx58 gene

[0008] The wild-type allele sequence information is as follows:

[0009] TTTTCAGGTTACTGTGGACTTTGTGAAGCCATCGAAAGTTGGGACTTTCAAAAAATTGAAAAGTTAGAGGAACACAGATTACTTTTAAGA CGT TTAGAACCAGAATTTAAGGCCACAGTTGATCCAAATGATATCCTTTCTGAACTATCCGAATGTTTGATTAATCAGGAATGTGAAGAAATCAGACAGGTAAACCAATGCCAGGTAC, the underlined bases are mutation sites, as shown in SEQ ID NO.2.

[0010] (2) Designing a gRNA that can specifically recognize the Ddx58 gene, wherein the DNA sequence of the gRNA is: gRNA: 5'-TGACTCACTTCTCCCAGAGA-3', as shown in SEQ ID NO.1;

[0011] (3) Using In-Fusion cloning technology to construct a vector donor oligo, the DNA sequence of the donor oligo is:

[0012] GGGT ACAGTT CTCAGT TGCTCC TTCTGT ATGAAC TCAATG TGCTGC CAGTGGTGACGCCAGGAC TCGAAG GATGTG GTCACC AGGTCC CGCAGG GTGTGG TCACCTCTAAAG ACTGTC ACCAGCTGGTGT CCAAGC CTGCAA CCTGTC AGCCTC ATGGTCTGGCTC CCCAGA CTGTCC AGTGAC TGAGGCCATTTG CAGATG GTTTTC AGTTCCCTTGCC ACTGAT TTGAAC AGGATT CCCATG ATTTTG ACTTCAAAGCAT TTTTATGTTGGA TTTGCT TAAGAA ATCCCC ATTTCT CTTTTC TTTTTC AGGTTACTGTGGACTTTG TGAAGC CATCGA AAGTTG GGACTT TCAAAA AATTGA AAAGTT AGAGGAACACAGATTACT TTTAAG A TGTTT AGAACC AGAATT TAAGGC CACAGT TGATCCAAATGA TATCCT TTCTGAACTATC CGAATG TTTGAT TAATCA GGAATG TGAAGAAATCAG ACAGGT AAACCA ATGCCA GGTACTAAATTT GAAGAA AAATGC AGAGACATTGGA AATGCC CATTTT TCTGTC TTGTTT TAGGCC CAAGGATAATTG AAACCCATAAAA GCTCTC ATCTAG CAGATA TAATGA CTAGAA TAGAAT TTTTAAAGTGAATGGGGT AATTTT TGTGCT AGACTA TTAGAA AATTAT TTAACC TATTTG CAGTTAAAGTTGCCCCCT TACTTT AAAAAA AATAGT GGTTTA TGCATA ATGCAA ATCACACCAAAC AGTGCA ACAATTAAAAGG AAAAAT ATGTCA GGCTCT TGGGCA TAGATACATTTA TTACAG TCTCGC AGTCAC TTAACTAGTGAT GTGATG CCAG

[0013] As shown in SEQ ID NO.3.

[0014] (4) Construction of F0 generation mice: gRNA, Cas9 mRNA and donor oligo are mixed and injected into mouse fertilized eggs through microinjection. The fertilized eggs that survive the injection are then transplanted into pseudopregnant female mice. The mice born are edited F0 generation mice.

[0015] (5) Identification and breeding of F0 generation mice: The tails of F0 generation mice were cut to extract DNA for PCR reaction and sequencing identification, and they were divided into wild-type mice or positive heterozygous mice. The positive heterozygous mice were then mated with wild-type mice to obtain F1 generation mice;

[0016] (6) Identification of F1 generation mice: After identification again through PCR reaction and sequencing, the F1 generation heterozygous mice identified as positive can be stably inherited. Then, the F1 generation heterozygous mice are hybridized with each other, and the offspring obtained by breeding can obtain homozygous mutant and wild-type individuals, indicating that the model was successfully constructed.

[0017] Furthermore, the primers used in the PCR reaction are:

[0018] The forward primer sequence is 5'-AGGGTGTGGTCACCTCTAAAGACTG-3', as shown in SEQ ID NO.4; the reverse primer sequence is 5'-GTACCTGGCATTGGTTTACCTGTC-3', as shown in SEQ ID NO.5; this pair of primer sequences can be used to identify whether the obtained mouse is a wild-type mouse or a mutant mouse.

[0019] The second object of the present invention is to provide a Ddx58-R109C point mutation mouse model obtained by the method for constructing a Ddx58-R109C point mutation mouse model as described above.

[0020] The third object of the present invention is to provide the use of the Ddx58-R109C point mutation mouse model as described above in genetic research on diseases caused by the Ddx58-R109C mutation.

[0021] The fourth object of the present invention is to provide the use of the Ddx58-R109C point mutation mouse model described above in the study of the biological functions of autoimmune inflammatory diseases.

[0022] As a specific embodiment of the present invention, the autoimmune inflammatory disease includes systemic lupus erythematosus.

[0023] The fifth object of the present invention is to provide the use of the Ddx58-R109C point mutation mouse model as described above in the diagnosis and treatment of autoimmune inflammatory diseases.

[0024] As a specific embodiment of the present invention, the autoimmune inflammatory disease includes systemic lupus erythematosus.

[0025] The sixth object of the present invention is to provide a kit for constructing the Ddx58-R109C point mutation mouse model as described above, comprising a gRNA as shown in SEQ ID NO.1, primers as shown in SEQ ID NO.4 and SEQ ID NO.5, and a homologous repair template donor oligo sequence as shown in SEQID NO.3.

[0026] Furthermore, Cas9 mRNA is also included.

[0027] The seventh object of the present invention is a phenotypic verification of the Ddx58-R109C point mutation mouse model as described above, including serum autoantibodies and renal tissue pathological structure of Ddx58-R109C point mutation mice.

[0028] The beneficial effects of the present invention are:

[0029] The method of constructing a Ddx58-R109C site-directed mutation mouse model based on CRISPR / Cas9 gene editing technology provided by the present invention is simple to operate, efficient and fast, highly controllable, and has a stable effect. The model can accurately simulate the onset of patients with the R109C mutation site of the human DDX58 gene, is an ideal model for studying the pathogenesis of human DDX58-R109C mutations, provides a new approach and method for further developing targeted drugs for autoimmune inflammatory diseases, can be promoted and applied at home and abroad, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure shows the location of Ddx58-R109C in the embodiment of the present invention and the comparison of the amino acid sequences corresponding to the wild type (WT) and the mutant (R109C);

[0031] Figure 2 The figure is a diagram of the gene sequence sequencing results of mice of different genotypes in the embodiment of the present invention;

[0032] Figure 3 The serum anti-DNA and anti-RNA antibody levels of mice with different genotypes in the examples of the present invention;

[0033] Figure 4 Schematic diagram of the staining results of kidney pathological sections of mice with different genotypes in the examples of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0035] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0036] Example 1

[0037] The present invention provides a method for constructing a Ddx58-R109C point mutation mouse model, comprising the following steps:

[0038] (1) Target construction

[0039] A. Determine the mutation site: The site to be mutated, R109C, is located in the third exon of the mouse Ddx58 gene. The amino acid sequences corresponding to the wild type (WT) and the mutant (R109C) are as follows: Figure 1 shown.

[0040] The wild-type allele sequence information is shown in SEQ ID NO.2:

[0041] SEQ ID NO.2:

[0042] TTTTCAGGTTACTGTGGACTTTGTGAAGCCATCGAAAGTTGGGACTTTCAAAAAATTGAAAAGTTAGAGGAACACAGATTACTTTTAAGA CGT TTAGAACCAGAATTTAAGGCCACAGTTGATCCAAATGATATCCTTTCTGAACTATCCGAATGTTTGATTAATCAGGAATGTGAAGAAATCAGACAGGTAAACCAATGCCAGGTAC, the underlined bases are the mutation sites.

[0043] B. Design a gRNA that can specifically recognize the Ddx58 gene, the DNA sequence of the gRNA is shown in SEQ ID NO.1:

[0044] SEQ ID NO.1:

[0045] gRNA: 5'-TGACTCACTTCTCCCAGAGA-3';

[0046] C. Preparation of Cas9 mixed sample system: The Cas9 mixed sample system contains Cas9 mRNA, gRNA, and DEPC water, with concentrations of Cas9 mRNA 3μg / μL and gRNA 1μg / μL, respectively.

[0047] D. Construct the vector donor oligo using In-Fusion cloning technology. The DNA sequence of the donor oligo is shown in SEQ ID NO.3:

[0048] SEQ ID NO.3:

[0049] GGGT ACAGTT CTCAGT TGCTCC TTCTGT ATGAAC TCAATG TGCTGC CAGTGGTGACGCCAGGAC TCGAAG GATGTG GTCACC AGGTCC CGCAGG GTGTGG TCACCTCTAAAG ACTGTC ACCAGCTGGTGT CCAAGC CTGCAA CCTGTC AGCCTC ATGGTCTGGCTC CCCAGA CTGTCC AGTGAC TGAGGCCATTTG CAGATG GTTTTC AGTTCCCTTGCC ACTGAT TTGAAC AGGATT CCCATG ATTTTG ACTTCAAAGCAT TTTTATGTTGGA TTTGCT TAAGAA ATCCCC ATTTCT CTTTTC TTTTTC AGGTTACTGTGGACTTTG TGAAGC CATCGA AAGTTG GGACTT TCAAAA AATTGA AAAGTT AGAGGAACACAGATTACT TTTAAG A TGT TT AGAACC AGAATT TAAGGC CACAGT TGATCCAAATGA TATCCT TTCTGAACTATC CGAATG TTTGAT TAATCA GGAATG TGAAGA

[0050]

[0051] (2) Microinjection and fertilized egg transplantation

[0052] The method for preparing F0 generation mice comprises the following steps: superovulation of mice, injection and transplantation of fertilized eggs, and obtaining F0 generation mice; wherein the mouse strain is C57BL / 6J.

[0053] The specific method is as follows:

[0054] A. Superovulation of embryo donor mice

[0055] Donor female mice were treated with pregnant mare serum gonadotropin (PMSG), injected with human chorionic gonadotropin (hCG) 46 hours later, mated with male mice, and fertilized eggs were obtained for microinjection the next day.

[0056] B. Microinjection and fertilized egg transplantation

[0057] The Cas9 mixed system was mixed with donor oligo (concentration 1 μg / μL), and the final concentrations of Cas9 mRNA, gRNA and donor oligo were 50 ng / μL, 20 ng / μL and 5 ng / μL respectively. The mixture was microinjected into the fertilized eggs of C57BL / 6J mice using a microinjector, and the surviving fertilized eggs were transplanted into pseudopregnant mother mice, and the F0 generation mice were waited for to be born.

[0058] (3) Identification and breeding of F0 generation mice: The tails of F0 generation mice were cut to extract DNA for PCR reaction and sequencing identification, wherein the PCR primers are shown in SEQ ID NO.4 and SEQ ID NO.5:

[0059] SEQ ID NO.4:

[0060] Forward primer sequence 5′-AGGGTGTGGTCACCTCTAAAGACTG-3′;

[0061] SEQ ID NO.5:

[0062] Reverse primer sequence 5'-GTACCTGGCATTGGTTTACCTGTC-3'

[0063] The PCR system is shown in Table 1 below.

[0064] Table 1

[0065] PCR components Volume (μL) Rat tail genomic DNA X*(about 100ng~1μg) 2xTaqmasterMix 25.0 Primer-F(10mM) 1.0 Primer-R(10mM) 1.0 Nuclease-freewater To50μL

[0066] The PCR process is shown in Table 2 below.

[0067] Table 2

[0068]

[0069] The sequencing identification primer for F0 generation mice is as follows: 5'-GTACCTGGCATTGGTTTACCTGTC-3'.

[0070] (4) Preparation and identification of F1 generation mice: F0 generation mice that have been identified correctly by PCR and sequencing are mated with wild-type C57BL / 6J background mice to obtain F1 generation mice. The above identification method is then used to identify the genotype of the F1 generation mice. The obtained positive F1 generation heterozygous mice can be stably inherited. The F1 generation heterozygous mice are then hybridized, and the offspring obtained by breeding can obtain homozygous mutant and wild-type individuals. The sequencing results of mice with different genotypes are shown in Figure 2. Figure 2 As shown, base mutation occurred at the target site, which was C / C in the wild type, C / T in the heterozygous type, and T / T in the homozygous type, indicating that we successfully constructed the R109C mutant mouse model.

[0071] The embodiment of the present invention also provides a kit for constructing a Ddx58-R109C point mutation mouse model, which contains gRNA and corresponding primers and a homologous repair template donor oligo sequence.

[0072] The gRNA sequence is 5'-TGACTCACTTCTCCCAGAGA-3';

[0073] The primer sequences are: forward primer sequence 5'-AGGGTGTGGTCACCTCTAAAGACTG-3'; reverse primer sequence 5'-GTACCTGGCATTGGTTTACCTGTC-3'.

[0074] The donor oligo sequence is:

[0075]

[0076]

[0077] Furthermore, it also contains Cas9 mRNA.

[0078] Example 2

[0079] The present invention also provides phenotypic verification of the Ddx58-R109C point mutation mouse model.

[0080] The specific steps are as follows:

[0081] (1) Determination of serum anti-DNA and anti-RNA antibody levels

[0082] 1) Add 100 μL of poly-lysine (final concentration 100 μg / mL) to a 96-well plate, incubate at room temperature for 2 h, and discard the supernatant.

[0083] 2) Add 100 μL DNA (final concentration 10 μg / mL) or RNA (final concentration 10 μg / mL), incubate at room temperature for 2 h, discard the supernatant and pat dry on absorbent paper.

[0084] 3) Add 100 μL of blocking solution (PBS containing 1% FBS), let stand at room temperature for 1 hour, pour out the supernatant and pat dry.

[0085] 4) Add 100 μL mouse serum (diluted 1:5000 with PBS containing 1% FBS) and incubate at 4°C overnight.

[0086] 5) Add 100 μL of washing buffer (PBS containing 0.05% Tween 20), let stand for 1 min, pour out and pat dry, and wash 4 times.

[0087] 6) Add 100 μL goat anti-mouse IgG-HRP antibody, incubate at room temperature for 1 h, and discard the supernatant.

[0088] 7) Repeat step 5).

[0089] 8) Add 100 μL HRP substrate and incubate at room temperature in the dark for 30 min (the specific time is determined by the color depth).

[0090] 9) Add 100 μL of stop solution to terminate the reaction.

[0091] 10) Within 30 minutes, use an ELISA reader to detect the OD values ​​at 450 nm and 570 nm.

[0092] like Figure 3 As shown, at 32 weeks, a large amount of anti-DNA antibodies and anti-RNA antibodies were detected in both heterozygous and homozygous mice compared with wild-type mice, showing the autoimmune phenotype of systemic lupus erythematosus.

[0093] (2) Pathological examination of renal tissue: fresh renal tissue of mice was collected, washed with PBS, and the renal capsule was removed. 1 / 2 of the renal tissue was placed in an embedding mold containing OCT embedding agent (the largest section was placed on the base), and immediately placed in liquid nitrogen for quick freezing (the bottom of the mold was slowly immersed in a small liquid nitrogen tank, and the OCT embedding agent began to solidify gradually until it was completely solidified and then taken out), and temporarily stored at -20°C for the preparation of frozen fluorescent sections. 1 / 2 of the renal tissue was placed in a 5mL centrifuge tube containing 4% paraformaldehyde, soaked completely, and stored at room temperature for the preparation of paraffin sections, followed by HE staining and PAS staining. Take a small piece of renal cortex, about 2-3mm 3 , placed in an EP tube containing 500 μL of electron microscopy fixative, fixed in the dark, and temporarily stored at 4°C for the preparation of electron microscopy samples. The subsequent preparation, sectioning, HE staining, PAS staining, immunofluorescence staining, and electron microscopy sections of kidney tissue samples were all completed by the clinical laboratory of the National Clinical Research Center for Kidney Diseases.

[0094] The results of tissue section staining are as follows Figure 4 As shown: The glomerular basement membrane and mesangial area of ​​wild-type mice were relatively normal, with no obvious pathological changes, no IgG and C3 deposition in the glomeruli, and no electron-dense deposition under the electron microscope; while the glomeruli of homozygous and heterozygous mice with the Ddx58-R109C point mutation showed mesangial cell proliferation and increased mesangial matrix, and a large amount of fuchsin deposits were seen in the capillary loop lumen, presenting a "platinum ear"-like change; IgG and C3 were deposited in the glomerular capillary loop in granular form; and a large amount of electron-dense deposition was seen in the glomerular mesangial area under the electron microscope.

[0095] The results showed that heterozygous and homozygous mice carrying the Ddx58-R109C mutation developed pathological changes of lupus nephritis, which was consistent with the renal pathological phenotype of patients carrying the DDX58 R109C mutation.

[0096] In summary, Ddx58-R109C point mutation mice show systemic lupus erythematosus and lupus nephritis phenotypes, which can be used to study the genetic function of the R109C mutation in the Ddx58 gene in diseases involved, as well as the biological function of this mutation in systemic lupus erythematosus and lupus nephritis. It can also be used for the diagnosis of systemic lupus erythematosus and lupus nephritis and the development of targeted drugs for this mutation.

[0097] It should be noted that the above content only illustrates the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications all fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a Ddx58-R109C point mutation mouse model, comprising the following steps: (1) Determine the mutation site: the site is located in the third exon of the mouse Ddx58 gene; (2) Designing a gRNA that can specifically recognize the Ddx58 gene, wherein the DNA sequence of the gRNA is shown in SEQ ID NO.1; (3) constructing a vector donor oligo using In-Fusion cloning technology, wherein the DNA sequence of the donor oligo is shown in SEQ ID NO.3; (4) Mix gRNA, Cas9 mRNA and donor oligo, inject them into mouse fertilized eggs by microinjection, and then transplant the surviving fertilized eggs after injection into pseudopregnant female mice. The mice born are edited F0 generation mice; (5) Cutting the tails of F0 mice to extract DNA for PCR reaction and sequencing identification, and classifying them as wild-type mice or positive heterozygous mice, and then mating the positive heterozygous mice with wild-type mice to obtain F1 mice; (6) After identification again through PCR reaction and sequencing, the F1 generation heterozygous mice identified as positive were crossed and the resulting offspring were bred to obtain homozygous mutant and wild-type individuals, indicating that the model was successfully constructed.

2. The method for constructing a Ddx58-R109C point mutation mouse model according to claim 1, characterized in that: The wild-type allele sequence information corresponding to the mutation site is: TTTTCAGGTTACTGTGGACTTTGTGAAGCCATCGAAAGTTGGGACTTTCAAAAAATTGAAAAGTTAGAGGAACACAGATTACTTTTAAGA CGT TTAGAACCAGAATTTAAGGCCACAGTTGATCCAAATGATATCCTTTCTGAACTATCCGAATGTTTGATTAATCAGGAATGTGAAGAAATCAGACAGGTAAACCAATGCCAGGTAC, as shown in SEQ ID NO.2, the bases in the underlined part are mutation sites.

3. The method for constructing a Ddx58-R109C point mutation mouse model according to claim 1, characterized in that: The primers used in the PCR reactions of step (4) and step (5) are: a forward primer sequence as shown in SEQ ID NO.4; The reverse primer sequence is shown in SEQ ID NO.

5.

4. The Ddx58-R109C point mutation mouse model obtained by the method for constructing a Ddx58-R109C point mutation mouse model according to any one of claims 1 to 3.

5. Use of the Ddx58-R109C point mutation mouse model as described in claim 4 in genetic research on diseases caused by Ddx58-R109C mutation.

6. Use of the Ddx58-R109C point mutation mouse model as described in claim 4 in the study of the biological functions of autoimmune inflammatory diseases.

7. Use of the Ddx58-R109C point mutation mouse model as claimed in claim 4 in the diagnosis and treatment of autoimmune inflammatory diseases.

8. A kit for constructing the Ddx58-R109C point mutation mouse model as claimed in claim 4, characterized in that: It contains the gRNA shown in SEQ ID NO.1, the primers shown in SEQ ID NO.4 and SEQ ID NO.5, and the homologous repair template donor oligo sequence shown in SEQ ID NO.

3.

9. The kit for the Ddx58-R109C point mutation mouse model according to claim 8, characterized in that: Cas9 mRNA is also included.

10. A phenotypic verification of the Ddx58-R109C point mutation mouse model as claimed in claim 4, comprising serum autoantibodies and renal tissue pathological structure of Ddx58-R109C point mutation mice.

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