CN114868705A - Retinitis pigmentosa mouse model and construction method thereof - Google Patents
Retinitis pigmentosa mouse model and construction method thereof Download PDFInfo
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Abstract
The invention relates to a retinitis pigmentosa mouse disease model, which is characterized in that based on a RHO gene of a retinitis pigmentosa patient, a 75nt DNA fragment of the patient is knocked into a corresponding site in a mouse genome by using CRISPR/Cas9, and the fragment contains pathogenic mutation, so that the T17M gene knock-in mouse disease model is constructed, wherein the pathogenic mutation is named as c.C50T at a nucleic acid level, and the pathogenic mutation is named as p.T17M at a protein level. The retinitis pigmentosa mouse disease model provided by the invention has patient-specific mutant fragments, and provides powerful support for research and development of corresponding medicines and gene therapy methods.
Description
Technical Field
The invention relates to the field of genetic engineering, and relates to a retinitis pigmentosa mouse model and a construction method thereof.
Background
Retinitis Pigmentosa (RP) is an inherited ocular disease in which progressive apoptosis of retinal photoreceptor cells is caused by genetic mutations. The disease is mainly manifested by progressive decline of vision, visual field constriction, night blindness, fundus pigmentation and Electroretinogram (ERG) abnormalities. Statistically, the population incidence of retinitis pigmentosa worldwide is about 1/4000, and has become a major blinding disease. The genetic patterns are divided into three categories, namely Autosomal Dominant RP (ADRP), Autosomal Recessive RP (ARRP) and sex-linked recessive RP (X-linked RP, XLRP). In autosomal dominant inheritance (ADRP), the mutation rate of Rhodopsin (RHO) gene is high, accounting for about 25% -30% of the incidence rate of ADRP. The RHO gene is located at 3q22.1 and consists of 5 exons, encoding 348 amino acids. The functional abnormality of rhodopsin protein caused by RHO gene mutation finally causes the apoptosis of photoreceptor cells. Therefore, these mutant sites also become important targets in gene therapy. The construction of ADRP disease models caused by RHO gene mutation is of great significance.
Because of the difference between the mouse genome and the human genome, the genes of the retinal pigment degeneration mouse model reported at present are greatly different from those of patients, so that the existing gene editing method developed based on the retinal pigment degeneration mouse model cannot be directly used for clinical treatment of patients, and the gene editing method is seriously influenced for treating the retinal pigment degeneration diseases.
Disclosure of Invention
In view of the above, the present invention aims to develop a retinitis pigmentosa mouse model, which carries patient-specific mutant fragments at corresponding genomic sites and provides a strong support for the development of corresponding drugs and gene therapy methods.
In order to achieve the aim, the invention develops a retinitis pigmentosa mouse disease model, based on the RHO gene of a retinitis pigmentosa patient, uses CRISPR/Cas9 to knock a 75nt DNA fragment of the patient into a corresponding site in a mouse genome, wherein the fragment contains a pathogenic mutation, so as to construct a T17M gene knock-in mouse disease model, wherein the pathogenic mutation is named as c.C50T at a nucleic acid level, and the pathogenic mutation is named as p.T17M at a protein level.
Preferably, the protein sequence corresponding to the mutant Rho gene is shown in SEQ ID NO. 1.
Preferably, the T17M gene knock-in mouse Rho partial gene sequence is shown in SEQ ID NO. 3.
Preferably, the method for constructing the retinitis pigmentosa mouse model by using CRISPR/Cas9 comprises the following steps,
(1) designing two specific sgRNA sequences;
(2) introducing missense mutation RHO and p.T17M on exon 1 of mouse Rho gene, designing a repair template for knocking in gene, wherein the sequence of the repair template is shown as SEQ ID NO. 2;
(3) cas9 mRNA transcribed in vitro, sgRNA and a repair template are injected into fertilized eggs of a C57BL/6J mouse in a microinjection mode, a CRISPR/Cas9 gene editing system cuts genes in the fertilized eggs of the mouse, homologous recombination repair is induced, and the fertilized eggs develop into embryos;
(4) the embryo after gene editing is immediately transferred into the uterus of a pseudopregnant female mouse, and a knock-in mouse is obtained after production.
Preferably, the two specific sgRNA sequences include sgRNA1 and sgRNA2, which are respectively targeted to specific genomic sites, the drawn sequences are PAM, and the sequences of the sgRNA1 and the sgRNA2 are as follows:
sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；
sgRNA2:CTTCTCCAACGTCACAGGCGTGG。
preferably, the CRISPR/Cas9 gene editing system binds to a target site of a genome, Cas9 exerts cleavage activity to generate DNA double strand breaks, thereby inducing DNA damage repair, cells repair DNA through homologous recombination, and repair templates are knocked into the mouse Rho gene at a fixed point.
Preferably, a 195nt repair template is designed comprising a 75nt patient DNA sequence and the causative mutation RHO, p.T17M, which comprises BstXI enzyme cleavage sites 5 '-CCANNNNNNTGG-3' but not present in the wild type mouse RHO gene sequence, by comparison with the wild type mouse RHO gene sequence, and thus this enzyme cleavage site can be used for genotyping.
Preferably, the repair template sequence is shown in SEQ ID NO. 2;
the partial gene sequence of the T17M knock-in mouse Rho is shown in SEQ ID NO. 3.
Preferably, the step of mouse identification comprises:
obtaining an F0 generation mouse after the pseudopregnant female mouse farrow;
taking the tail and the toe of an F0-generation mouse, extracting a whole genome, carrying out PCR amplification and sequencing, hybridizing an F0-generation positive mouse with a wild-type mouse to obtain an F1-generation mouse, taking the tail and the toe of the mouse, extracting the whole genome, carrying out enzyme digestion verification by BstXI or carrying out gene sequencing identification after PCR amplification.
In the invention, the structural and functional abnormalities of the retina of the mouse with the knock-in T17M gene are obvious, and the characteristics are the same as those of a clinical patient. Therefore, the invention provides powerful support for the research and development of clinical therapeutic drugs and gene therapy methods for retinitis pigmentosa.
Drawings
FIG. 1 is a diagram showing the construction of a mouse model of retinitis pigmentosa;
FIG. 2 is a graph showing the results of identifying mice into which T17M gene was knocked;
FIG. 3 shows the results of the identification of the retinal structure (OCT and immunohistochemistry) of mice into which the T17M gene was knocked;
FIG. 4 shows the result of identifying the function of the retina (ERG) of a mouse in which the T17M gene is knocked in;
FIG. 5 is a photograph showing the fundus oculi and the blood vessel of the mouse knocked in with the T17M gene.
Detailed Description
To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, structures, features and effects according to the present invention will be made with reference to the accompanying drawings and preferred embodiments.
Referring to fig. 1 to 5, the retinitis pigmentosa transgenic mouse model disclosed by the invention is based on the RHO gene of a retinitis pigmentosa patient, and a 75DNA fragment of the patient is knocked into the genome of the mouse by using CRISPR/Cas9, wherein the fragment contains RHO and p.t17m pathogenic mutation, so that a T17M gene knock-in mouse disease model is constructed. Wherein T17M is a mutation site on the RHO gene.
The protein sequence corresponding to the T17M knock-in mouse model Rho gene is shown in SEQ ID NO. 1:
MNGTEGPNFYVPFSNAMGVVRSPFEQPQYYLAEPWQFSMLAAYMFLLIVLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVVFTWIMALACAAPPLVGWSRYIPEGMQCSCGIDYYTLKPEVNNESFVIYMFVVHFTIPMIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIFFLICWLPYASVAFYIFTHQGSNFGPIFMTLPAFFAKSSSIYNPVIYIMLNKQFRNCMLTTLCCGKNPLGDDDASATASKTETSQVAPA the mutated amino acids are underlined.
The method for constructing the retinitis pigmentosa transgenic mouse model comprises the following steps:
(1) two specific sgRNA sequences were designed.
Two specific sgRNA sequences including sgRNA1 and sgRNA2, targeting specific sites in the genome, respectively, the sequences of sgRNA1 and sgRNA2 of CRISPR/Cas9 are as follows:
sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；
sgRNA2:CTTCTCCAACGTCACAGGCGTGG。
(2) missense mutation (RHO, p.T17M) is introduced into exon 1 of the mouse Rho gene, a repair template sequence of gene knock-in is designed,
the repair template sequence is shown in SEQ ID NO. 2:
5'-GGGAGCCGTCAGTGGCTGAGCTCGCCAAGCAGCCTTGGTCTCTGTCTACGAAGAGCCCGTGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGATGGGCGTGGTGCGGAGCCCCTTCGAGCAGCCGCAGTACTACCTGGCGGAACCATGGCAGTTC-3', introducing missense mutation (RHO, p.T17M) in exon 1 of mouse Rho gene, the mutation site is underlined, and the comparison analysis with wild mouse Rho gene sequence shows that the repair template contains BstXI restriction site 5 '-CCANNNNNNTGG-3', but the wild mouse Rho gene sequence does not exist, so the restriction site can be used for genotype identification.
(3) The T7 promoter sequence was inserted before the coding region of Cas9 (SpCas 9 used, coding sequence from https:// www.addgene.org/42230/) and the sgRNA sequence by PCR amplification and used as an in vitro transcription template. Cas9 mRNA, sgRNA and a repair template which are transcribed in vitro are injected into C57BL/6J fertilized eggs in a microinjection mode, and a CRISPR/Cas9 system is injected into mouse fertilized eggs in a microinjection mode to carry out gene cutting, so that homologous recombination repair is induced.
The CRISPR/Cas9 gene editing system is combined with a target site of a genome, Cas9 exerts cleavage activity to generate DNA double-strand break so as to induce DNA damage repair, cells repair DNA through homologous recombination, and a repair template is knocked into a mouse Rho gene at a fixed point.
(4) The fertilized ovum after gene editing develops into embryo, which is transferred into the uterus of a pseudopregnant female mouse, and a gene knock-in mouse is obtained after production.
The partial sequence of the T17M knock-in mouse Rho gene is shown in SEQ ID NO. 3:
gcgttagtatgatatctcgcggatgctgaatcagcctctggcttagggagagaaggtcactttataagggtctggggggggtcagtgcctggagttgcgctgtgggagccgtcagtggctgagctcgccaagcagccttggtctctgtctacgaagagcccgtGGGTCAGCCACAAGGGCCACAGCCATGAATGGCACAGAAGGCCCTAACTTCTACGTGCCCTTCTCCAATGCGATgggcgtggtgcggagccccttcgagcagccgcagtactacctggcggaaccatggcagttctccatgctggcagcgtacatgttcctgctcatcgtgctgggcttccccatcaacttcctcacgctctacgtcaccgtacagcacaagaagctgcgcacacccctcaactacatcctgctcaacttggccgtggctgacctcttcatggtcttcggaggattc, the DNA sequence (upper case) is the 75ntDNA fragment from human, and the mutation site (RHO, p.T17M) is underlined.
In the present invention, the steps of mouse breeding and progeny identification include:
after the pseudopregnant female mouse farrow, the F0 mouse is obtained; and taking the tail and the toe of the mouse, carrying out PCR amplification, and then carrying out enzyme digestion verification by BstXI or carrying out gene sequencing identification. Hybridizing an F0 generation positive mouse with a wild mouse to obtain an F1 generation mouse, taking the tail and the toe of the F1 generation mouse, extracting a whole genome, performing PCR amplification, BstXI enzyme digestion verification or performing gene sequencing identification. Breeding was continued in the F1 mouse generation to obtain more diseased mice.
Referring to fig. 2, sgRNA1 and sgRNA2 targeting target sequences and a 195nt repair template are designed, respectively, wherein sgRNA1 and sgRNA2 targeting target sequences, respectively, are shown in fig. 2A. The repair template comprises a 75nt human DNA sequence and a pathogenic mutation (RHO, p.t 17m). Mouse zygotes were edited and developed into embryos. The gene-edited embryos were then transferred into the uterus of pseudopregnant females to produce knock-in mice (see FIG. 1). In designing a repair template, the repair template was found to contain a BstXI cleavage site (5 '-CCANNNNNNTGG-3') that was included in the knockin mouse Rho gene sequence, but not in the wild-type (WT) mouse Rho gene sequence, since it contained a 75nt human DNA sequence, compared to the wild-type mouse Rho gene sequence. After amplification by PCR and BstXI cleavage, two bands were visualized by agarose gel electrophoresis, indicating that knock-in mice, only one band representing wild-type mice, were identified (FIG. 2B). Genotyping was also performed by Sanger sequencing (fig. 2C).
As shown in FIG. 3 of the present invention, structural changes in the retinas of the knock-in mice were further observed. OCT (Optical Coherence Tomography) showed clear retinal structures in wild-type (WT) mice, while the photoreceptor layer, especially the outer nuclear layer, was significantly thinned in knock-in mice (fig. 3A, B). Further hematoxylin-eosin staining was performed as shown in fig. 3C, and the knock-in mouse was abnormal in retinal structure, most notably the thinning of the outer nuclear layer to almost disappear, compared to the WT mouse.
As is clear from the above, referring to FIG. 4 of the present invention, it is clear that the structural change of the mouse retina upon knock-in of the T17M gene is significant, and the electrophysiological function of the mouse retina is evaluated. Electroretinograms are the total electrophysiological responses of the retina recorded from the corresponding sites when the retina is light-stimulated. When dark-adapted electroretinograms are used to detect rod responses (A, E), b-wave amplitude is significantly reduced compared to WT (n is 10, P < 0.0001); in the dark adaptation mixed reaction, normal wild-type mice have a negative phase compared with a small a wave and a larger positive phase b wave in sequence, and the amplitude (B, D, E) of the a wave and the b wave of a mouse knocked in the T17M gene is obviously reduced and remarkably different (n is 10, and P is less than 0.0001); when cone function was examined (C, D, E), there was no significant difference in a-wave (n 10, P >0.05) and a significant decrease in b-wave amplitude (n 10, P <0.01) compared to WT, suggesting that there was a significant amount of rod apoptosis in mice that knocked the T17M gene into the retina, but cone cells also retained partial function. The significant reduction in function of the T17M knock-in mouse retina was consistent with OCT-detected thinning of the outer nuclear layer of the retina.
As shown by the present invention with reference to FIG. 5, it was observed by fundus retinal examination and angiography (as indicated by the arrow in FIG. 5) that significant retinitis pigmentosa, retinal artery tapering, and beaded changes occurred in the fundus of mice into which the T17M gene was knocked.
In conclusion, the structural and functional abnormalities of the retina of the T17M knock-in mouse are obvious, which is the same as the characteristics of clinical patients, and the results show that a T17M knock-in mouse disease model is successfully constructed.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (9)
1. A mouse disease model of retinitis pigmentosa is characterized in that: based on the RHO gene of a retinitis pigmentosa patient, 75ntDNA (deoxyribonucleic acid) fragments of the patient are knocked into the corresponding sites in the mouse genome by using CRISPR/Cas9, and the fragments contain pathogenic mutations, so that a T17M knock-in mouse disease model is constructed, wherein the pathogenic mutations are named as c.C50T at the nucleic acid level, and the pathogenic mutations are named as p.T17M at the protein level.
2. The mouse model of retinitis pigmentosa as set forth in claim 1, wherein: the protein sequence corresponding to the mutant Rho gene is shown in SEQ ID NO. 1.
3. The mouse model of retinitis pigmentosa as claimed in claim 1 or 2, wherein: the partial gene sequence of the T17M knock-in mouse Rho is shown in SEQ ID NO. 3.
4. A method for constructing a retinitis pigmentosa mouse model is characterized by comprising the following steps: construction of a knock-in mouse with CRISPR/Cas9 includes the following steps,
(1) designing two specific sgRNA sequences;
(2) introducing missense mutation RHO and p.T17M on exon 1 of mouse Rho gene, designing a repair template for knocking in gene, wherein the sequence of the repair template is shown as SEQ ID NO. 2;
(3) cas9 mRNA, sgRNA and a repair template which are transcribed in vitro are injected into fertilized eggs of a C57BL/6J mouse in a microinjection mode, a CRISPR/Cas9 gene editing system cuts genes in the fertilized eggs of the mouse to induce homologous recombination and repair, and the fertilized eggs develop into embryos;
(4) the embryo after gene editing is immediately transferred into the uterus of a pseudopregnant female mouse, and a knock-in mouse is obtained after production.
5. The method of claim 4, wherein the mouse model of retinitis pigmentosa is prepared by: two specific sgRNA sequences include sgRNA1 and sgRNA2, which target forward and reverse sequences, respectively, at specific sites in the genome, the sequences of sgRNA1 and sgRNA2 are as follows:
sgRNA1:CGGCTCTCGAGGCTGCCCCACGG；
sgRNA2:CTTCTCCAACGTCACAGGCGTGG。
6. the method of claim 4, wherein the mouse model of retinitis pigmentosa is prepared by:
the CRISPR/Cas9 gene editing system is combined with a target site of a genome, Cas9 exerts cleavage activity to generate DNA double-strand break so as to induce DNA damage repair, cells repair DNA through homologous recombination, and a repair template is knocked into a mouse Rho gene at a fixed point.
7. The method for constructing a mouse model of retinitis pigmentosa as set forth in claim 4, 5 or 6, wherein: 195nt repair templates were designed containing 75nt of the patient's DNA sequence and the causative mutation, RHO, p.T17M, and by comparison with the wild-type mouse RHO gene sequence, the repair templates contained BstXI restriction sites 5 ' -CCANNNNNNTGG-3 ' that were absent from the wild-type mouse RHO gene sequence, and thus this restriction site was used for genotyping.
8. The method of claim 7, wherein the mouse model of retinitis pigmentosa is prepared by:
the repair template sequence is shown as SEQ ID NO. 2;
the T17M gene knock-in mouse Rho part gene sequence is shown in SEQ ID NO. 3.
9. The method of claim 4, wherein the step of identifying the mouse comprises,
after the pseudopregnant female mouse farrow, the F0 mouse is obtained;
taking the tail and the toe of an F0-generation mouse, extracting a whole genome, carrying out PCR amplification and sequencing, hybridizing an F0-generation positive mouse with a wild-type mouse to obtain an F1-generation mouse, taking the tail and the toe of the mouse, carrying out enzyme digestion verification by BstXI after PCR amplification or carrying out gene sequencing identification.
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