CN121160801A - Construction method of keshan disease rat model - Google Patents

Abstract

This invention provides a method for constructing a Keshan disease rat model, belonging to the field of animal model construction. The method involves constructing rats with a G deletion at base 874 of the SEPHS2 gene; the SEPHS2 gene transcript number is ENSRNOTT00000074114.3. This invention has found that the c.874delG mutation in the SEPHS2 gene is significantly associated with Keshan disease in cardiomyopathy. Deletion of G at base 874 of the SEPHS2 gene in SD rats resulted in dilated cardiomyopathy-like changes accompanied by significant systolic dysfunction, typical of Keshan disease-related myocardial damage. Therefore, by inducing the c.874delG mutation in the SEPHS2 gene in SD rats, a cardiomyopathy model, particularly a Keshan disease model, can be prepared, providing a model basis for cardiomyopathy research, especially Keshan disease research, and showing promising application prospects.

Description

Construction method of keshan disease rat model

Technical Field

The invention belongs to the field of animal model construction, and particularly relates to a construction method of a keshan disease rat model.

Background

Keshan disease (KESHAN DISEASE, KD) is an endemic cardiomyopathy which is characterized by myocardial degenerative necrosis as the major pathological change, clinically manifested as acute or chronic congestive heart failure and various arrhythmias, severe ones can lead to cardiogenic shock and even death. The etiology of keshan disease is not completely elucidated, keshan disease and dilated cardiomyopathy (dilated heart disease) are clinically difficult to identify, and keshan disease has poorer clinical prognosis, which causes burden to families and society. Thus elucidating the pathogenesis of keshan disease and proposing effective preventive and interventional measures becomes critical for improving prognosis.

Regarding the etiology hypothesis of keshan disease, one hypothesis is nutritional bio-geochemical etiology hypothesis, the selenium deficiency is the main causative factor causing keshan disease, the other is biological etiology theory, and the theory of virus infection such as coxsackievirus is mainly included, but the pathogenesis and clinical characteristics of keshan disease cannot be satisfactorily explained. In keshan disease research, there is a significant problem of the lack of keshan disease models that can be used in keshan disease research.

The method for constructing the keshan disease model is found, and has important significance for researches on pathogenesis of keshan disease, therapeutic drugs and the like.

Disclosure of Invention

The invention aims to provide a construction method of a keshan disease rat model.

The invention provides a construction method of a myocardial disease rat model, which comprises the steps of constructing a rat with a SEPHS gene 874 th base G deletion, wherein the number of a SEPHS gene transcript is ENSRNOTT00000074114.3, and the construction method comprises the following steps:

(1) Constructing a rat fertilized egg with a SEPHS gene 874 th base G deletion by using a CRISPR/Cas9 technology, and culturing to obtain a positive F0 generation rat, wherein the sequence of sgRNA adopted by the CRISPR/Cas9 technology is shown as SEQ ID NO. 1;

(2) And hybridizing the positive F0 generation rats with wild rats to obtain positive F1 generation rats, namely a rat model.

Further, in the step (1), the sequence of the targeting vector adopted by the CRISPR/Cas9 technology is shown as SEQ ID NO. 2.

Further, step (1) and step (2) include further identification of the resulting rat genotype.

The identification method further comprises the steps of taking rat tail genes, amplifying target sequences through PCR, and determining gene mutation conditions of PCR amplified products through sequencing, wherein the forward primer sequences used for identification are shown as SEQ ID NO. 3, and the reverse primer sequences are shown as SEQ ID NO. 4.

Further, the rat is an SD rat.

Further, the cardiomyopathy rat model is a keshan disease rat model.

The invention also provides application of the cardiomyopathy rat model obtained by the construction method in research of cardiomyopathy.

Further, the cardiomyopathy is keshan disease.

Compared with the prior art, the invention has the beneficial effects that:

The study of the invention finds that the c.874delG mutation of SEPHS gene (namely 874 th base G deletion of coding region in SEPHS gene) is obviously related to keshan disease in cardiomyopathy. After deletion of 874 th base G in coding region of SEPHS gene of SD rat, the obtained SD rat has dilated cardiomyopathy-like change with obvious contraction failure, which is typical of keshan disease myocardial damage manifestation. Therefore, the myocardial disease model, particularly the keshan disease model, can be prepared by making the SD rat SEPHS gene generate c.874delG mutation, can provide a model foundation for myocardial disease research, particularly keshan disease research, and has good application prospect.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention.

Detailed Description

The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.

Example 1 construction of Crohn's disease rat model

SD rats are used as study subjects in the invention.

SEPHS2 Gene (murine) on chromosome 1 opposite strand, full length about 2.3kb,NCBI ID:308993. The SEPHS gene (murine) transcript number referred to in the present invention is ENSRNOTT00000074114.3.

1. Design scheme

1. Targeting strategy

The delegated baioci plot company prepares point mutant gene knock-in mode rats based on the EGE system developed by CRISPR/Cas 9. sgRNA was designed in the 3' UTR, and the actual sizes of the 5' -and 3' -homology arms of the targeting vector for homologous recombination were 1.4kb and 1.0kb, respectively.

2. Southern blotting screening strategy

In order to screen the gene targeting rats with correct recombination, the invention adopts PCR and Southern blotting to verify that F1 generation positive rats are verified by using a 3' probe and an LR probe. The specific design of the Southern blot screen is shown in Table 1.

TABLE 1 specific design of southern blot screening

BglII and ScaI were used as Southern blotting cleavage sites. The 3' probe is used to detect if proper recombination has occurred. If correct recombination occurs, both wild-type and mutant bands will occur. LR probes were used to detect if random insertions were included. Without random insertion, both wild-type and mutant bands will occur.

2. Model preparation

1. Sequencing validation of target sequences and design of sgrnas

The target gene DNA sequences of different strains may have differences, in order to ensure the correctness of the designed Cas9/sgRNA, the SD rat tail target gene DNA sequence is firstly subjected to PCR amplification and sequencing verification, and the verification result shows that the sgRNA target sequence is completely consistent with the standard sequence given by a Genebank database.

Based on the design principle of the sgRNA, 1 sgRNA is designed in the target site region, and the corresponding targeting sequence (SEQ ID NO: 1) is shown as follows:

GCTGCCGGCATAGTGCCTCTAGG(5’-3’)。

2. Construction of targeting vectors

Constructing a targeting vector, identifying and sequencing by enzyme digestion, confirming that the targeting vector is constructed correctly, and preparing the targeting vector for microinjection. The sequence of the targeting vector (SEQ ID NO: 2) is shown below:

agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat ttaggtgacg cgttagaata ctcaagctat gcatcaagct tatccgacgc tttaggagtg aataccttta agaaggagat atacatgaat tcccagagct tccttctata agtaacttat tcttcggtaa agcgattcca tgactttggt agaggtgact tccccccagc caactcaaag accaggttca cagtggaatg gggggaaggg aacacgacac gcacaggtaa cgtggctccc acctgtaaca ccagcactta ggaatctgag acagacagat ggatgcgagt tcgagttcat cctgatctat acagagaact gcgttccagg ccagttacaa gggattcacg tgacgacctc gcggggaccg gtggtgcaag cagacggtta atttgttata cacctttttg aataaaggaa gcccgcctct cggtacgaga atgatcattg gatggagcga ctgtcaatca tgctgtatat aaatacaggc cgctcggcgc tcggacaggc agcgtggctt gagtcttccg atcggtttct cttggtagat catctccagg cctaaccttt acccaccgtg gcaattagaa tcgcctgcag gtaccagggc cttctggttc gcacggcctc ccgggcgggc ggtgcgatgg cggaagcggc ggcaggcgcc agcggagaag ccatggcggc actagtggcc gcggaaggtt ccttgggccc ggcgggctgg tctgctggcc ggagtttctc caactaccgg ccgttcgagc cccagacact gggcttcagc ccgagctggc ggctgacgag cttctccggc atgaaaggct gaggctgcaa ggtcccccag gagaccctgc tcaaactcct ggagggactg acgcggcccg cgctgcagcc cccgcttacc tcgggtctgg tcgggggcca ggaagagacg gtgcaggaag ggggcctgac caccaggccc ggcccgggct cagccttccc ctcgctgagc attgggatgg actcctgcgt catccccctg aggcacggag gcctgtcgct ggtgcagacc accgacttct tttacccctt ggtggaagat ccctatatga tggggcgcat agcttgtgcc aatgtgctca gtgacctcta tgccatgggc attaccgagt gtgacaacat gttgatgtta ctcagtgtga gccagagcat gagtgaaaag gaacgagaga aggtaacgcc gctcatgatc aaaggctttc gtgacgctgc ggaagaggga ggaactgcag tgactggtgg acagacagtg gtcaaccctt ggattatcat cggtggggtt gccactgtgg tgtgtcagca aaatgaattc ataatgcctg acagcgctgt ggtaggagat gtgctggtat taaccaagcc tttaggaacc caggttgctg ccaatgccca ccaatggctg gataatcctg agaaatggaa taaaatcaag atggtggttt ccagagagga agtagagcta gcctatcaga agctatgttc aacatggcta ctctgaacag gactgctgct ggcttgatgc acacttttaa tgctcacgca gccacggata tcacaggctt tggcatatta ggacactccc agaacctcgc aaaacagcaa aaaaatgaag tgtcctttgt cattcacaat ctgccaatca ttgccaagat ggctgcgatc agcaaagcca gtgggcgctt tggcctcctc caaggaacat cagctgaaac ctctggggga ttactgatct gtctgccaag agaacaggcg gcccgctttt gttcggaaat caaatcttcc aagtacggag agggtcacca agcttggatt gttggcatcg tggagaaggg aaaccggaca gcccggatca ttgacaagcc tcgcgttatt gaagttctac ctcggggagc ctctgcttct gctgctgctg ctcctgacaa ttccaatgca gcctctgagc ctagttcttg aagatctatg catagtactg agctcaatgg aatagccctt gttgggaact cggagccatt ctacacgctc acagactgtt ggccagggtt gattttaaga cctttccaaa ggctgccggc atagtgcctc tagctacttt tgctagttcc atcaaaagct gcctgtgtac atccagaccc agaagtgcag gggatgtgtg ctcatctgtt gagagaatga ggagtaaaaa aaccttttcc caaagcaaga tgaggctatt ccagttttag ggattttttg cactgagttg attcatttct gcacagggag taagattatt aagattacat acagaaaaaa gtaaactgca acatgaaaaa aattatctgg accaacatat tgataagtct aaattgttag gagaactctt actgatttat tgtcaaattt gtcattaatt atttttctga gcaactgcct cttttcctgt tctgagcaag agttgagcaa cttgtccagc caggaaagga aggccatagc cacctgactt ggtctctgat aatgatgttt ctccctctaa ctcctaataa ggactgggag aggctgatca aacctcagag ccaggtgttg gcggccgtta agatactaaa tcttacactg aaaatttcca gagatttaat taataaaaaa aatttcttaa cagctaacta aataaaaggg actttgttgg aaaactcttg tggctaatgc ttggttctgt gggtcacttt atcttgtttc agacaggttc ttgggtcctg gagaatggcc ttgaaccctt gatgttgctt ctgcctccaa tctgctgcca ggaccagctg gaactctgac ttcttatccc ttgacattgc acccagagcc tgcagaatag aaagtagatg ttgcccttgc ccctgagcca agggatttgg gtttgcactg tcccccgaga tgacaaaaga ggactcgaat ggatataaat tctggagatt ttagggggaa aaaaacttaa aagatggctt agcagcatag aagaggccat ctgaagatgg ggacgataac caaaagctct agaaggattc agtgaggtga aggcaaggag ttggcatgag aagagacggt ggctaaaagc ttgtaataca gggggatcct gagatccggc tgctaacaac cgacgcttta ggagtgaata cctctagagg gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct atacgtacgg cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc ggggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gcatgagatt atcaaaaagg atcttcacct agatcctttt cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaatta ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag

3. microinjection

CRISPR/Cas9 related reagents such as sgRNA and targeting vectors are microinjected into fertilized eggs (222) of rats, and F0 generation rats are born after injection.

Genotyping of F0 Generation rats

The invention uses sgRNA to inject fertilized eggs to prepare gene point mutation knock-in rats. Since embryo early cleavage is fast, the resulting F0 rats are chimeras. Therefore, the F0 genotype identified by the rat tail of F0 is only used as a reference, and cannot represent that the rat tail is necessarily a genetic mutant type. The heritable genotype is determined after rat tail detection in F1.

4.1 Identification primer design

Primer information is shown in table 2:

TABLE 2 primer information

PCR conditions were as shown in Table 3:

Enzyme KOD-FX

TABLE 3 PCR conditions

4.2 Rat tail genotype identification of F0 generation

The obtained F0 generation rats were subjected to rat tail genotype identification according to the PCR conditions in 4.1. 3 positive F0 rats were obtained by PCR amplification and sequencing of the products.

5. Genotyping of F1-generation rats

The F0-generation rats identified as positive were mated with wild-type SD rats, respectively, to obtain F1-generation rats having stable genotypes. The rat tail genotype of the F1 generation rat is identified by adopting the same identification method as that of the F0 generation rat. 8 positive F1 rats were obtained.

5.1 Southern blot detection of F1 generation PCR positive rats

And extracting the rat tail DNA of the F1 generation positive to the PCR primary screening for Southern blot detection. Through Southern blot detection, rats with correct recombination and no random insertion in the positive F1 generation rats are screened. These rats are SD rats in which the SEPHS gene had a c.874delG mutation.

3. Relation of SEPHS Gene to the occurrence of c.874delG mutation to keshan disease

SD rats (8 positive F1 generation rats) and wild type rats (2) with SEPHS gene subjected to c.874delG mutation are detected by heart ultrasound, and whether the keshan disease model is constructed successfully is observed. LVIDd (left ventricular end-diastole inner diameter) and EF (ejection fraction) are important ultrasound markers of success for the keshan disease myocardial injury model. Cardiac ultrasound was performed on 2 wild-type SD rats and 8 positive F1-generation SD rats with SEPHS c.874delG mutations in the gene, and the results are shown in tables 4 to 13.

TABLE 4 ultrasonic detection results of wild SD rats (M224)

TABLE 5 ultrasonic detection results of wild SD rats (M229)

TABLE 6 ultrasonic test results for positive F1 rats (B204)

TABLE 7 ultrasonic test results of positive F1 rats (B203)

TABLE 8 ultrasonic test results for positive F1 rats (B206)

TABLE 9 ultrasonic test results of positive F1 rats (B205)

TABLE 10 ultrasonic test results of positive F1 rats (B202)

TABLE 11 ultrasonic test results for positive F1 rats (B201)

TABLE 12 ultrasonic test results of positive F1 rats (B207)

TABLE 13 ultrasonic test results of positive F1 rats (B208)

As is clear from the results of the above-mentioned SD rat heart ultrasound, LVIDd (left ventricular end-diastole inner diameter) of SD rat having SEPHS d gene c.874delG mutation was significantly increased compared with wild-type rat, EF (ejection fraction) was significantly decreased, indicating that SD rat had dilated cardiomyopathy-like changes with obvious systolic function failure. This is a typical manifestation of keshan disease myocardial damage. And the SD rat keshan disease model is successfully constructed.

In conclusion, the present invention found that the c.874delG mutation of SEPHS gene (i.e. 874 th base G deletion in coding region of SEPHS gene) was significantly related to keshan disease in cardiomyopathy. After deletion of 874 th base G in coding region of SEPHS gene of SD rat, the obtained SD rat has dilated cardiomyopathy-like change with obvious contraction failure, which is typical of keshan disease myocardial damage manifestation. Therefore, the myocardial disease model, particularly the keshan disease model, can be prepared by making the SD rat SEPHS gene generate c.874delG mutation, can provide a model foundation for myocardial disease research, particularly keshan disease research, and has good application prospect.

Claims (6)

1. A method for constructing a rat model of cardiomyopathy, characterized in that: the method involves constructing rats with a G deletion at base 874 of the SEPHS2 gene; the SEPHS2 gene transcript number is ENSRNOTT00000074114.3; the construction method is as follows: (1) Rat zygotes with G deletion at position 874 of the SEPHS2 gene were constructed using CRISPR/Cas9 technology and positive F0 generation rats were obtained by culturing; the sequence of the sgRNA used in the CRISPR/Cas9 technology is shown in SEQ ID NO:1; (2) Cross positive F0 generation rats with wild-type rats to obtain positive F1 generation rats, which are the rat models. 2. The construction method according to claim 1, characterized in that: in step (1), the sequence of the targeting vector used in the CRISPR/Cas9 technology is as shown in SEQ ID NO:2. 3. The construction method according to claim 1, characterized in that: steps (1) and (2) include further identification of the obtained rat genotype. 4. The construction method according to claim 3, characterized in that: the identification method includes: taking a rat tail gene, amplifying the target sequence by PCR, and determining the gene mutation status by sequencing the PCR amplification product; the forward primer sequence used for identification is shown in SEQ ID NO:3; the reverse primer sequence is shown in SEQ ID NO:4. 5. The construction method according to any one of claims 1 to 4, wherein the rat is an SD rat. 6. The construction method according to any one of claims 1 to 4, wherein the cardiomyopathy rat model is a Keshan disease rat model.