CN112522312B - WKH rat model construction method - Google Patents

Abstract

The invention relates to the technical field of a method for constructing a hereditary hypertension rat model, in particular to a method for constructing a WKH rat model, which comprises the following steps: adopting gene sequencing technology to detect coding regions and flanking regions of 43 pathogenic genes related to single-gene hypertension of clinical teenager hypertension patients with familial aggregation, predicting the influence of newly-discovered mutant genes on protein structure and function through a plurality of software, and determining the pathogenic genes through pathogenicity judgment; then, the WKH rat model is constructed by utilizing a gene engineering CRISPR/Cas9 technology, the postnatal blood pressure of the mutant gene animal is continuously increased along with the age of the animal, and the systolic pressure of most offspring tail animals is more than 140mmHg. Provides a research tool for the function of the gene in the development of hypertension, and provides a research model for continuously exploring the pathogenesis related to hypertension and exploring a new target point of the antihypertensive effect.

Description

WKH rat model construction method

Technical Field

The invention relates to the technical field of a method for constructing a hereditary hypertension rat model, in particular to a method for constructing a WKH rat model, namely a method for constructing a humanized hereditary hypertension rat model.

Background

With the improvement of living standard of people, the life of people is continuously increased, the prevalence rate of hypertension in China is also continuously increased, and the guideline of the prevention and treatment of hypertension in China in 2019 shows that the total number of people suffering from hypertension in China is about 2 hundred million, namely, 2 hypertension patients exist in 10 adults, as the cardiovascular and cerebrovascular diseases are the leading cause of death and disability in China, the hypertension is also the first risk factor of cardiovascular and cerebrovascular diseases, therefore, the control of the blood pressure level of people is very important, but the current situation is that the treatment control rate of the hypertension patients in China is only 16%, one of the causes is related to the complication and complication of the pathogenesis of hypertension, and a plurality of problems of genetic factors need to be solved urgently.

For the research of human hypertension pathogenesis and prevention and treatment scheme, a genetic hypertension model can be constructed by a genetic method, such as a selective recent propagation hypertension model and a genetic engineering hypertension model. The former model is to perform close breeding of the screened congenital hypertension animal for a plurality of generations (more than 20 generations) to obtain stable hypertension inheritance. Hypertensive rabbits, rats and mice have been developed. Rats are commonly used, and more mature varieties include: spontaneous Hypertensive Rats (SHR), dahl salt-sensitive rats (DS), milan hypertensive rats (MHS), and hereditary hypertensive rats (GH) are also called New Zealand hypertensive rats, israel hypertensive rats (SBH), and Lyon hypertensive rats (LH). Although these models are very similar to human hypertension, the following deficiencies still exist: (1) it is obtained by selective reproduction and is different from human diseases. (2) Abnormalities in thyroid and immune function exist.

Genetically engineered hypertension models can be divided into two categories: one is that a transgenic technology is adopted to randomly integrate exogenous genes into an animal genome and cause hypertension through over expression, and the transgenic animal is called hypertension; the other is that the endogenous gene of the animal is directionally knocked out by adopting a gene targeting technology to cause hypertension, and the animal is called a hypertension gene knockout animal. Because the technology and laboratory requirements for preparing the gene engineering hypertension model are higher, the reports of successfully preparing the gene engineering hypertension model in China are less. With the development of CRISPR-Cas9 technology, a gene manipulation tool, it has recently been widely applied to various experimental models such as cell lines, laboratory animals, plants, and even human clinical trials. The CRISPR-Cas9 system includes a guide Cas9 nuclease, guided by small RNA molecules, creating site-directed double-stranded DNA breaks. A process that allows permanent modification of a genomic target sequence can repair DNA damage. The application of the system is proposed to provide a new approach for treating hypertension and cardiovascular diseases. Therefore, the animal model aims to screen pathogenic genes from hypertension families, a specific genetic variation genetic hypertension rat model (WKH rat model) is constructed by using a CRISPR-Cas9 gene editing technology, and a new tool is provided for screening antihypertensive drugs with specific action targets.

The Cav1.3L-type calcium channel alpha 1 subunit coding gene CACNA1D is related to hypertension, the CACNA1D gene is reported to be a newly found blood pressure susceptible region in literature, chinese scholars find that rs9810888 polymorphism is obviously related to diastolic pressure and average arterial pressure, poor living mode and rs9810888 GG genotype synergy can influence the blood pressure level of Chinese teenagers, and the change of the CACNA1D gene function can influence the blood pressure reduction effect of a calcium channel blocker.

Disclosure of Invention

The invention aims to provide a WKH rat model construction method, which overcomes the defects of the prior art, provides a research tool for the action in the development of hypertension, and provides a research model for continuously exploring the pathogenesis related to hypertension and exploring a new target point of the antihypertensive action.

One of the technical schemes of the invention is realized by the following measures: a WKH rat model construction method comprises the following steps: adopting gene sequencing technology to detect coding regions and flanking regions of 43 pathogenic genes related to single-gene hypertension of clinical teenager hypertension patients with familial aggregation, predicting the influence of newly-discovered mutant genes on protein structure and function through a plurality of software, and determining the pathogenic genes through pathogenicity judgment; then, the WKH rat model is constructed by utilizing a genetic engineering CRISPR/Cas9 technology, the postnatal blood pressure of the mutant gene animal is continuously increased along with the age of the animal, and the systolic pressure of most offspring tail animals is more than 140mmHg.

The following is a further optimization or/and improvement of one of the above-mentioned technical solutions of the invention:

the mutant gene is the gene detection result of a special type of hypertension patient.

The experimental animal is selected from a mouse, a rat or a rabbit.

The specific gene point mutation F0 mouse is constructed by a CRISPR/Cas9 technology.

The specific gene mutation mouse is a descendant bred by the constructed F0 mouse.

The breeding condition of the mutant mice is an SPF animal breeding room, the room temperature is kept at 22 +/-2 ℃, the day and night period is 12 hours, and the mutant mice can be fed with food and water freely.

The invention provides a construction method of a human Cav1.3 hereditary hypertension rat model (Wang-Kang-Hui, WKH rat model). Firstly, discovering new sick Cav1.3 gene mutation sites of hypertension clinically, determining research target genes through literature retrieval and software prediction, constructing and screening a specific Cav1.3 gene mutation (WKH) rat model by utilizing a gene engineering CRISPR/Cas9 technology, comparing the model with a wild type WKH rat, and increasing the blood pressure of the screened specific single-gene mutation rat from 6 months of age, increasing the volume of glomeruli at 8 months of age, generating contraction, swelling the renal bursa, expanding the renal tubules, unclean lumen structures, thickening the walls of the renal arteriolar vessels and increasing fibrous tissues; cardiomyocyte enlargement; thickening secondary artery of mesentery, abnormal function of endothelium contraction and relaxation, etc.; the progress of these pathologies can be prevented or alleviated by therapeutic measures such as hypotensive drugs; when the Cav1.3 gene is further hybridized with WKH rats with two site variations, hypertension is completely developed in three months. The similarity protein structure search based on NCBI blasts shows that these Cav1.3 gene variation sites can be homologously modeled. In a word, the WKH rat model has specific humanized mutation, can provide a research tool for the effect of the gene in the development of hypertension, and provides a research model for continuously exploring pathogenesis related to hypertension and exploring a new target point of the antihypertensive effect.

Drawings

FIG. 1 shows the comparison of the heterozygous variation and homozygous variation of WKH gene in WKH wild type SD rats with the 7-32 week systolic blood pressure (SBP, 6 in each group): ^* P<0.05 and ^** P<0.01: comparison of heterozygous mice with wild type mice， ^# P<0.05: homozygous mice were compared to heterozygous mice. A, a CACNA1D p.307 locus male mouse SBP time-varying trend chart; b: trend graph of time-dependent change of female mouse SBP of CACNA1D p.307 locus; c: trend graph of male mouse SBP of CACNA1D p.936 locus with time; d: trend plot of female mouse SBP at CACNA1D p.936 locus with time; e: a trend graph of the change of the SBP of the male mouse at the CACNA1D p.1516 locus along with time; f: trend graph of time-dependent change of SBP of female mouse with CACNA1D p.1516 locus; g: a trend graph of the change of the single gene heterozygous site variation of the CACNA1D p.307 site and the CACNA1D p.936 site and the change of the SBP of the male mouse combined variation of the two sites along with time; h: and (3) a time-dependent trend chart of the male mouse SBP of the single-site heterozygous variation of the CACNA1D p.307 site and the CACNA1D p.1516 site and the combined variation of the two sites.

FIG. 2 shows the results of the assay of 6 month WKY male rats (6 individuals in each group) plasma vasoconstrictor endothelin-1 with different genotypes of CACNA1D p.307: * : compared with wild mouseP<0.05。

FIG. 3 shows the change of kidney histomorphology of different genotypes of 8-month CACNA1D p.307 WKH rats.

FIG. 4 is a graph showing the change in kidney histomorphology for different genotypes of 8 month CACNA1D p.307 WKH rats.

FIG. 5 is a graph showing the change in kidney histomorphology for different genotypes of 8 month CACNA1D p.307 WKH rats.

FIG. 6 shows EH staining comparisons of mesenteric secondary arterioles of WKH rats of different genotypes (AA, AG and GG genotypes from left to right).

FIG. 7 shows the morphology and statistics of heart tissues of different genotypes of 8-month-old WKH rats.

FIG. 8 shows the three-dimensional structure analysis of protein after mutation of exon sites of CACNA1D gene and the action sites of different drugs on L-type calcium channel, in which A is the position of CACNA1D p.307 site, CACNA1D p.936 site and CACNA1D p.1516 site in L-type calcium channel cav1.3, B: action position of dihydropyridine calcium channel antagonist nifedipine in L-type calcium channel cav1.3, C: non-dihydropyridine nifedipine calcium channel antagonists act at the site of the L-type calcium channel cav 1.3.

Detailed Description

1. Animal preparation

The clean wild SD rat and CACNA1D gene point mutation rat are 24 (half of male and female), and the age of the rat is 4 weeks. Free diet, drinking water and life rhythm according to the circadian rhythm.

2. Animal grouping and model construction

The rat CACNA1D gene (GenBank accession number: NM-017298.1, ensembl. Contains 51 exons, the ATG initiation codon is located in exon 1, and the TAG termination codon is located in exon 51. And selecting the exon where the site screened in the early stage of the subject group is as a target site. Design gRNA targeting vector and donor oligonucleotide (with targeting sequence flanked by 130 bp homologous sequence): the site of mutation in the donor oligonucleotide will be introduced into this exon by homology directed repair. Silent mutations (from GAG to GAA or from GAC to GAT) will also be introduced to prevent homology directed repair of gRNA binding and re-cleavage of sequences. Cas9mRNA, gRNA produced by in vitro transcription, and donor oligonucleotide were co-injected into fertilized eggs to breed knock-in rats. These pups will be subjected to sequence analysis.

The model rat is characterized in that: to pairCacna1dP, D307G and p, R1516Q site mutation is carried out on the p.D307 and p.R1516 site of the No. 7 exon and the No. 35 exon respectively, and thenCacna1d ^G307G Homozygous mutant rat andCacna1d ^Q1516Q the homozygous mutant rat is hybridized to obtain DNA with two strandsCacna1d ^{D307G/ R1516Q} Two-site heterozygous mutant WKH rat model.

Grnas of the rat CACNA1D gene, donor oligonucleotides containing mutation sites and silent mutation (GAG to GAA) sites, and Cas9mRNA were co-injected into fertilized rat eggs to generate targeted knock-in offspring. F0 rats were subjected to sequence analysis and subsequently mated with wild type SD rats to produce F1-generation rats.

Co-injection of gRNA, donor oligonucleotide and Cas9mRNA of the rat CACNA1D gene into fertilized largeIn murine eggs to produce targeted knock-in offspring; identifying the F0 initial mouse by PCR, then carrying out sequencing identification, and finally hybridizing the F0 initial mouse to a wild rat to generate an F1 generation mouse; f1 generation mice are hybridized to respectively obtainCacna1d ^G307G Homozygous mutation andCacna1d ^Q1516Q homozygous mutant F2 rats, and hybridization of single-site homozygous mutant rats to obtain DNA fragments on both strandsCacna1d ^{D307G/ R1516Q} Two-site heterozygous mutant rat model.

The WKH rat model construction method comprises the following steps:

(1) RatCacna1dGene, geneBank accession No.: NM _017298.1, combination: ENSRNOG00000013147, located on rat chromosome 16;

(2) Fifty one exons have been identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 51; p.d307 and p.r1516 are located at exons 7 and 35, thus exons 7 and 35 are selected as target sites, respectively;

(3) gRNA targeting vectors and donor oligonucleotides were designed;

(4) The p.d307g, c.920atog and p.r1516q, c.4475 AGGtoCAG mutation sites in the donor oligonucleotides will be introduced into the corresponding exons by homology-directed repair; introduction of silent mutations (from GAG to GAA or from GAC to GAT) was required for the p.d307g (c.920atog) mutation to prevent gRNA binding and re-cleavage of the sequence following homology-directed repair;

(5) Mixing and injecting a cas9 sample mixing system into fertilized eggs, and transplanting to obtain an F0 generation rat;

(6) The positive F0 generation rats and the common SD rats are bred to obtain the compoundCacna1dGene knock-in positive F1-generation heterozygous SD rats;

(7) Hybridizing the F1 generation rat to obtain a single-site mutation homozygote F2 generation rat;

(8) Two single-site F2 generation homozygous rats were crossed to obtainCacna1d ^{D307G/ R1516Q} Two-site mutant rats.

Mating the F1 generations to obtain F2 generations, and then dividing the generations into a wild type group, a heterozygous subgroup and a homozygous subgroup according to different genotypes, wherein the specific grouping and modeling method comprises the following steps:

	wild type group	Hybrid group	Group of homozygotes
				Treatment of	Keeping room temperature at 22 + -2 deg.C for 12h day and night, and freely collecting and drinking water	Keeping room temperature at 22 + -2 deg.C for 12h day and night, and freely collecting and drinking water	Keeping room temperature at 22 + -2 deg.C for 12h day and night, and freely collecting and drinking water
Time length (moon)	8	8	8

And (3) measuring physiological and biochemical indexes: blood pressure was measured weekly from 50 days old, after 8 months of feeding, venous blood, content of endothelin-1 in serum was taken from each group of rats after fasting for 12 hours, heart and aorta were collected after sacrifice, and morphology of heart, kidney and aorta was examined.

The model is verified that SBP is larger than or equal to 140mmHg and is used as a standard for successful modeling of the hypertensive WKH rat.

Claims (6)

1. A WKH rat model construction method is characterized in that firstly, CRISPR/Cas9 technology is utilized to constructCacna1dA point mutation rat model, and then hybridizing two point mutation rats to obtain a double point mutation WKH rat model;

the rat model is characterized in that: to pairCacna1dP, D307G and p, R1516Q site mutation is carried out on the p.D307 and p.R1516 site of the No. 7 exon and the No. 35 exon respectively, and thenCacna1d ^G307G Homozygous mutant rat andCacna1d ^Q1516Q the homozygous mutant rat is hybridized to obtain DNA with two strandsCacna1d ^{D307G/ R1516Q} Two-site heterozygous mutant WKH rat model.

2. The method for constructing a WKH rat model according to claim 1, wherein: the rat model is selected from SD rats.

3. The method for constructing a WKH rat model according to claim 1 or 2, wherein: the method comprises the following two major steps, wherein the first step is particularly critical, and the specific steps are as follows:

(1) For ratsCacna1dGene, design of gene knock-in strategy, selection of knock-in region: p.D307 and p.R1516 are located in exons 7 and 35, respectively, and therefore exons 7 and 35 are selected as target sites, respectively;

(2) Construction by CRISPR/Cas9 techniqueCACNA1DGene single site mutant F0 rats;

(3) Hybridizing the F0 initial mouse and the wild type SD rat to respectively obtain the compoundCacna1d ^D307G AndCacna1d ^R1516Q site-mutated F1-generation mice;

(4) Hybridizing the F1 generation rat to obtain an F2 generation homozygote rat;

(5) Will be provided withCacna1d ^G307G Homozygous mutant rats andCacna1d ^Q1516Q homozygous mutant rat F2 mice were crossed to obtainCACNA1D ^D307G/R1516Q Double-site processAnd (5) changing into rats.

4. The WKH rat model construction method according to claim 3, characterized in that gRNA of rat CACNA1D gene, donor oligonucleotide and Cas9mRNA are co-injected into fertilized rat eggs to generate targeted knock-in offspring; identifying the F0 initial mouse by PCR, then carrying out sequencing identification, and finally hybridizing the F0 initial mouse to a wild rat to generate an F1 generation mouse; f1 generation mice are hybridized to respectively obtainCacna1d ^G307G Homozygous mutation andCacna1d ^Q1516Q homozygous mutant F2 rats, and hybridization of single-site homozygous mutant rats to obtain DNA fragments on both strandsCacna1d ^{D307G/ R1516Q} Two-site heterozygous mutant rat model.

5. The WKH rat model construction method as claimed in claim 4, comprising the steps of:

(1) Rat modelCacna1dGene, geneBank accession No.: NM-017298.1, ensembl: combining: ENSRNOG00000013147, located on rat chromosome 16;

(2) Fifty exons have been identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 51; p.D307 and p.R1516 are located at exons 7 and 35, respectively, thus exons 7 and 35 are selected as target sites, respectively;

(3) gRNA targeting vectors and donor oligonucleotides were designed;

(4) The p.d307g, c.920atog and p.r1516q, c.4475 AGGtoCAG mutation sites in the donor oligonucleotides will be introduced into the corresponding exons by homology-directed repair; for p.d307g, the c.920atog mutation requires introduction of silent mutations, from GAG to GAA or from GAC to GAT, to prevent gRNA binding and re-cleavage of the sequence after homology-directed repair;

(5) Mixing and injecting the cas9 mixed sample system into fertilized eggs, and transplanting to obtain F0 generation rats;

(6) The positive F0 generation rats and the common SD rats are bred to obtain the compoundCacna1dPositive F1 heterozygous for knock-insub-SD rats;

(7) Hybridizing the F1 generation rat to obtain a single-site mutation homozygote F2 generation rat;

(8) Two single-site F2 generation homozygous rats were crossed to obtainCacna1d ^{D307G/ R1516Q} Two-site mutant rats.

6. The WKH rat model construction method of claim 5, wherein the cas9 mixed sample system comprises sgRNA and cas9 mRNA.

Images