GRK4R65L humanized salt-sensitive hypertension animal model as well as construction method and application thereof

Abstract

The invention relates to a GRK4R65L humanized salt-sensitive hypertension animal model as well as a construction method and application thereof. According to the model, a CRISPR / Cas9 technology is combined with homologous recombination, the third exon and the fourth exon of a rat endogenous GRK4 gene are replaced with a human GRK4 cDNA sequence which is driven by a CAG promoter and carries R65L point mutation, expression is driven by the CAG promoter, the genetic background of the animal model is clear, and gene pollution and phenotype instability under the multi-gene background are avoided; experiments prove that the construction method is accurate in targeting, the obtained homozygous rats can be stably inherited, the blood pressure rise characteristic is obvious under the stimulation of high-salt diet, an ideal carrier is provided for researching the molecular mechanism of the disease, and meanwhile, as an accurate preclinical verification platform, the construction method has good application prospects. The model can be effectively used for screening treatment drugs aiming at GRK4 targets, and development of personalized diagnosis and treatment schemes is promoted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a GRK4 R65L humanized salt-sensitive hypertensive animal model and a construction method and application thereof. BACKGROUND

[0002] Salt-sensitive hypertension (SS-HT) refers to a high blood pressure that is very sensitive to salt intake. When high salt is ingested, blood pressure will increase significantly, and when salt intake is reduced, blood pressure will decrease, which will significantly increase the risk of cardiovascular and renal complications. Despite extensive research over the past few decades, it has not been fully elucidated why some people exhibit salt-sensitive blood pressure responses while others do not, and in-depth study of the pathogenesis is particularly important. The study of salt-sensitive hypertension requires the use of a salt-sensitive hypertensive animal model, and the most commonly used model is the salt-sensitive hypertensive rat (Salt-Sensitive Hypertensive Rat) model. The commonly used salt-sensitive hypertensive animal models currently include: (1) Based on Sprague-Dawley rats, individuals with a sharp increase in blood pressure (salt-sensitive, SS) and no significant change in blood pressure (salt-resistant, SR) are screened out by high-salt diet, and are respectively inbred, and finally purebred Dahl SS or SS inbred lines are bred. The rat carries multiple genetic defects, leading to hyperfunction of renal tubular sodium reabsorption and / or impaired sodium excretion. When ingesting normal or low-salt diet, blood pressure can remain normal or mildly elevated; once switched to high-salt diet (containing 4%-8% NaCl), sodium ions are retained in the body, blood volume increases, and a series of neuroendocrine and vascular responses are triggered, ultimately leading to rapid elevation of blood pressure. (2) Using normal rats (such as Wistar or SD rats), the compensatory ability is reduced by surgical removal of one kidney, then a continuous subcutaneous injection of salt corticosteroid (such as deoxycorticosterone acetate, DOCA) is performed for several weeks, and at the same time, 1% salt water is given as drinking water. DOCA is a potent salt corticosteroid that mimics the effects of aldosterone, strongly promoting the reabsorption of sodium ions in the distal tubule and collecting duct of the kidney. A high-salt diet provides a large amount of sodium load. Unilateral nephrectomy amplifies the effect of sodium retention. The combination of the three leads to severe sodium and water retention and blood volume expansion, thereby inducing hypertension.

[0003] However, the existing salt-sensitive hypertensive rat has obvious limitations: research evidence shows that the first type of SS rat selected by genetic screening has been contaminated by genes, and the phenomenon of blood pressure not rising frequently occurs during experimental research. Since this strain is the result of multiple gene interactions, without whole genome sequencing, it is impossible to find all contaminated DNA regardless of how many markers are used.

[0004] The second type of salt-sensitive hypertensive rat model is artificially created by surgery, exogenous hormones and extreme diet and other strong intervention means, and the mechanism of hypertension (extreme blood volume expansion, low renin) is quite different from the physiological and pathological process of most human primary hypertension (usually normal or high renin level), and the clinical relevance is weak. Therefore, it is urgent to establish a salt-sensitive hypertensive rat model that is closer to the human pathological and physiological process. SUMMARY

[0005] In view of the deficiencies in the prior art, the present application provides a GRK4 R65L humanized salt-sensitive hypertensive animal model, a construction method thereof and an application thereof.

[0006] The technical scheme of the present application is as follows: A construction method of a GRK4 R65L humanized salt-sensitive hypertensive animal model, comprising the following steps: the animal is a rat, and the third and fourth exons of the endogenous GRK4 gene of the rat are replaced by a human GRK4 cDNA sequence carrying a R65L point mutation driven by a CAG promoter through a CRISPR-Cas9 technology.

[0007] Further, the specific steps include: S1. Construct a donor vector: construct a donor vector containing a homologous recombination element, the donor vector comprising: a 5' homologous arm homologous to the upstream rat genomic sequence of the GRK4 target site, a CAG promoter, a Kozak sequence, a full-length human GRK4 cDNA coding sequence carrying a R65L point mutation, a rabbit beta-globulin polyadenylic acid signal, and a 3' homologous arm homologous to the downstream genomic sequence; S2. Fertilized egg injection and transplantation: design sgRNA targeting the target region of the rat GRK4 gene, mix Cas9 mRNA, sgRNA and the donor vector constructed in step S1, and microinject into rat fertilized eggs, and transplant the surviving fertilized eggs into pseudopregnant female mice to obtain F0 generation rats; S3. Breeding and identification: cross the positive F0 generation rats with wild type rats to obtain F1 generation, and cross the F1 generation heterozygotes to obtain homozygous rats, which are the GRK4 R65L humanized salt-sensitive hypertensive animal model.

[0008] Further, the sgRNA in step S2 includes gRNA1 targeting the reverse strand upstream of the target region and gRNA2 targeting the forward strand downstream of the target region; wherein the nucleotide sequence of gRNA1 is shown in SEQ ID NO. 1, and the nucleotide sequence of gRNA2 is shown in SEQ ID NO. 2.

[0009] Further, the primers used for typing after the F1 generation hybrids are crossed in step S3 include: primer group A composed of F1 and R3 for amplifying mutant alleles, and primer group B composed of F5 and R3 for amplifying wild-type alleles, wherein the nucleotide sequence of F1 is shown in SEQ ID NO. 4, the nucleotide sequence of R3 is shown in SEQ ID NO. 5, and the nucleotide sequence of F5 is shown in SEQ ID NO. 6.

[0010] The GRK4 R65L humanized salt-sensitive hypertension animal model is constructed according to the construction method.

[0011] The animal model is applied to screening of drugs for treating salt-sensitive hypertension.

[0012] Further, the method for screening drugs comprises diet stimulation of at least 4% sodium chloride to the rats.

[0013] The animal model is applied to research on the pathological mechanism of hypertension caused by GRK4 gene mutation.

[0014] Further, the application comprises diet stimulation of at least 4% sodium chloride to the rats.

[0015] Compared with the prior art, the present application has at least the following advantages: The present application relates to a GRK4 R65L humanized salt-sensitive hypertension animal model and a construction method and application thereof; the model successfully simulates the real pathophysiological process of a human individual carrying a risk gene after high-salt intake by introducing a human GRK4 R65L mutation, has a clear genetic background, avoids gene pollution and unstable phenotypes in a multi-gene background, and its pathogenesis does not depend on extreme blood volume expansion and other strong artificial intervention, is closer to human primary hypertension, and significantly improves the clinical relevance of the model; in terms of the construction method, the present application uses CRISPR / Cas9 technology combined with homologous recombination to precisely replace a specific exon of a rat GRK4 gene with a human sequence containing a R65L mutation, and uses a CAG promoter to drive expression; this method is targeted and precise, has a high success rate, and the homozygous rats obtained can be stably inherited and exhibit significant blood pressure elevation characteristics under high-salt stimulation; in terms of application, the model not only determines the causal relationship between the GRK4 R65L mutation and the occurrence of salt-sensitive hypertension, but also provides an ideal carrier for researching the molecular mechanism of the disease; at the same time, as a precise preclinical verification platform, the model can be effectively used for screening of therapeutic drugs targeting the GRK4 target, and helps to promote the development of personalized diagnosis and treatment programs. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the specific embodiments of the present application, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced as follows.

[0017] Figure 1 Schematic diagram of construction strategy of replacing the third and fourth exons of GRK4 of a rat in Example 1 of the present application with human GRK4 R65L gene; Figure 2 Southern Blot identification of F1 generation GRK4 R65L rats in Example 1 of the present application; Figure 3 Sequencing results of F1 generation GRK4 R65L rats in Example 1 of the present application; Figure 4 Identification results of GRK4 R65L and GRK4 WT rats in Example 1 of the present application; Figure 5 GRK4 protein expression results of GRK4 R65L and GRK4 WT rats in Example 2 of the present application; Figure 6 Changes of systolic blood pressure of Control, GRK4 WT and GRK4 R65L groups under high-salt and low-salt diets in Example 3 of the present application; Figure 7 Urine sodium excretion of Control, GRK4 WT and GRK4 R65L groups under high-salt and low-salt diets in Example 3 of the present application. DETAILED DESCRIPTION

[0018] Further detailed description of the present application will be given as follows, it is necessary to point out that the following specific embodiments are only used to further illustrate the present application, and cannot be understood as limiting the protection scope of the present application, all other embodiments obtained by the ordinary skilled in the art without making creative efforts belong to the protection scope of the present application.

[0019] The materials used in the experiment and the test method are generally and / or specifically described in the present application. The test method or test method involved, if no special description, is a conventional method; the reagent or instrument used, if no manufacturer is indicated, is a conventional product on the market, which is prepared or used by a conventional method, and no special description is given, and the raw materials used in parallel experiments are the same batch of raw materials.

[0020] Example 1 Construction of GRK4 R65L humanized salt-sensitive hypertensive animal model The present application entrusts Cyagen to carry out gene editing and preparation of animal models, and the main experimental steps are as follows: S1. Design stage: Take rat GRK4 gene as a molecular target, the principle of construction is to use CRISPR-Cas9 technology to replace the third and fourth exons of rat endogenous GRK4 gene with a segment of human GRK4 cDNA sequence containing R65L (CGT to CTT) point mutation, in order to ensure that the human gene after replacement can be expressed efficiently and stably in rats, a powerful universal promoter CAG is used to drive its expression; in molecular design, two guide RNAs are designed targeting the intron sequences on both sides of the target region, gRNA1 (the sequence is shown as SEQ ID NO. 1) targets the reverse strand and is located upstream of the target region; gRNA2 (the sequence is shown as SEQ ID NO. 2) targets the forward strand and is located downstream, the purpose of designing two gRNAs is to completely remove the target exon, so as to improve the efficiency of subsequent homologous recombination; the construction of donor vector, the donor vector is a plasmid containing complete homologous recombination elements, its structure is in turn, 5' homologous arm with a length of about 500-1500 bp, homologous to the rat genomic sequence upstream of the GRK4 target site; strong CAG promoter; Kozak sequence (the sequence is shown as SEQ ID NO. 3) for optimizing translation initiation; full-length human GRK4 cDNA coding sequence carrying R65L pathogenic mutation; and rabbit beta-globulin polyadenylation signal (rBG pA) to ensure mRNA stability and 3' homologous arm homologous to the downstream genomic sequence Figure 1 ).

[0021] S2. Microinjection and embryo transfer: After mixing the in vitro transcribed Cas9 mRNA, the above two gRNAs and the constructed donor vector DNA, they are microinjected into the zygotes of SD rats; the surviving zygotes after injection are transplanted into the oviducts of pseudopregnant female mice, and after full-term pregnancy, the first generation of gene edited rats, called F0 generation (Founder generation), are obtained.

[0022] S3. F0 generation gene screening and identification, the screening and identification of F0 generation rats is the basis for subsequent work: at the time of weaning of rats, tail tissue samples are collected to extract genomic DNA; two pairs of specific primers spanning the integration site are used for PCR amplification to preliminarily screen positive individuals, wherein primer pair 1 (F2 / R2, SEQ ID NO. 7 and SEQ ID NO. 8) is composed of a human gene internal primer, and a 2.6 kb band is expected to be amplified; primer pair 2 (F1 / R1, SEQ ID NO. 9 and SEQ ID NO. 10) is composed of a human gene internal primer and a 3' homologous arm primer, and a 2.2 kb band is expected. For samples with positive PCR results, the product also needs to be further subjected to Sanger sequencing to finally confirm the accurate existence of R65L point mutation (CGT→CTT) and verify the correctness of the entire integrated sequence.

[0023] S4. F1 generation acquisition and stable line: To obtain a stably inherited line, the verified F0 generation positive rats were mated with wild type SD rats, and the offspring were the F1 generation. The F1 generation rats were subjected to long fragment PCR again to confirm the genetic stability of gene integration. Southern Blot technology, which is considered a gold standard, was also used for strict verification: after digestion of genomic DNA with specific restriction enzymes (such as BstE II and Avr II), hybridization was performed using a probe designed against the human GRK4 sequence. The expected specific band completely consistent with the design should appear, thereby firmly proving the correct integration of the human gene at the molecular level. The results, as shown in Figures 2-3 , indicate successful integration. Thereafter, the heterozygous rats in the F1 generation were interbred, and according to Mendelian inheritance law, homozygous, heterozygous and wild type individuals can be obtained in the offspring. To quickly and routinely genotype a large number of offspring, we established a short fragment PCR identification method: two specific primer sets were used, of which primer set A (F1 / R3, SEQ ID NO. 4 and SEQ ID NO. 5) can specifically amplify the mutant allele to produce an 838 bp product; primer set B (F5 / R3, SEQ ID NO. 6 and SEQ ID NO. 5) specifically amplifies the wild type allele to produce a 694 bp product, and the reaction program is shown in Table 1: Table 1 PCR reaction program The PCR reaction system is shown in Table 2: Table 2 PCR reaction system The PCR products were analyzed by 1.5-2% agarose gel electrophoresis: after electrophoresis at 120-150V for 15-20 minutes, the bands were observed under the gel imaging system. The result interpretation standard is: homozygotes (Hom) only appear 838 bp band; heterozygotes (Het) appear 838 bp and 694 bp bands; wild type (WT) only appears 694 bp band, as shown in Figure 4 .

[0024] Example 2 PCR verification of human GRK4 (R65L) protein expression The steps of this example are shown below.

[0025] Sample collection: human GRK4 R65L homozygous rat, GRK4 R65L WT rat and SD rat kidney cortex tissue were taken respectively.

[0026] Protein extraction: Weigh about 50-100 mg of tissue, and grind into powder after quick-freezing in liquid nitrogen. Transfer the powder into a pre-cooled centrifuge tube, and add 500ul-1 mL pre-cooled RIPA lysis buffer (containing 1x protease inhibitor cocktail and 1x phosphatase inhibitor). Lysate on ice for 30-60 minutes. Centrifuge at 4°C, 12000-14000 rpm for 15 minutes. Carefully pipette the supernatant (i.e., total protein lysate) into a new pre-cooled centrifuge tube.

[0027] Protein concentration determination (BCA method): Use BCA protein concentration determination kit, and follow the instructions. Draw a standard curve, and determine the protein concentration of all samples. Adjust all samples to a uniform concentration with lysis buffer.

[0028] Protein denaturation: Take an appropriate amount of protein solution, and add 6x protein loading buffer. Heat in a metal bath at 95-100°C for 5-10 minutes to fully denature the protein. After a brief centrifugation, the sample can be immediately loaded or stored at -20°C / -80°C.

[0029] Gel preparation and loading: According to the size of the target protein (66 kDa), prepare 10% separating gel and 5% stacking gel. Add protein molecular weight marker (Marker) and denatured protein sample to the loading well in turn.

[0030] Electrophoresis: Add 1x SDS-PAGE electrophoresis buffer to the electrophoresis tank. The initial voltage is 80 V (stacking gel stage), and the voltage is adjusted to 120 V after the sample enters the separating gel. Stop the electrophoresis when the bromophenol blue indicator reaches the bottom of the gel.

[0031] Membrane transfer: Place the "sandwich" in the membrane transfer tank, and fill the tank with pre-cooled 1x membrane transfer buffer. Perform membrane transfer in an ice bath or refrigerator at a constant current of 400 mA for 25-30 minutes.

[0032] Blocking: After membrane transfer, take out the PVDF membrane, and place it in 5% skimmed milk (prepared with TBST). Block at room temperature on a shaker for 1-2 hours.

[0033] Primary antibody incubation: Dilute the human GRK4 specific antibody (OriGene; TA323501) with blocking solution at a ratio of 1:500 according to the antibody instructions. Place the membrane in the primary antibody dilution, and incubate overnight on a shaker at 4°C. After incubation, wash the membrane with TBST on a shaker for 3 times, each for 5-10 minutes.

[0034] Secondary antibody incubation: Dilute the corresponding anti-rabbit IgG secondary antibody (1:10,000 dilution) with TBST. Place the membrane in the secondary antibody dilution and incubate on a rocking platform at room temperature for 1-2 hours. After incubation, wash the membrane thoroughly with TBST 3 times for 5-10 minutes each to remove unbound secondary antibody.

[0035] The results are shown in Figure 5 From the figure, it can be seen that the expression of GRK4 protein in GRK4 R65L and GRK4 WT rats is higher than that in normal SD rats.

[0036] Example Three: Blood pressure and urine sodium excretion determination This example sets up three groups of normal SD rats (Control), experimental control group (GRK4 WT) and model group (GRK4 R65L), each group is divided into low salt diet and 4% high salt diet, and the systolic blood pressure of each group of rats is measured after modeling, and the results are shown in Figure 6 Figure 6 a The systolic blood pressure of the three groups of rats under low salt diet shows no significant difference, Figure 6 b High salt diet shows that the systolic blood pressure of the GRK4 R65L group is significantly higher than that of the Control group and the GRK4 WT group.

[0037] The urine sodium excretion of each group of mice within 24 hours is shown in Figure 7 Figure 7 a The urine sodium excretion under low salt diet shows no significant difference between the three groups, Figure 7 b The urine sodium excretion under 4% high salt diet is significantly lower in the GRK4 R65L group than in the other two groups.

[0038] Under low salt diet conditions, the blood pressure and urine sodium excretion of the three groups of rats show no significant difference, indicating that the GRK4 R65L gene mutation itself does not affect the basic physiological state, while under high salt diet, the GRK4 R65L rats show significantly elevated blood pressure and significantly decreased urine sodium excretion, which have clear pathological relevance and high clinical relevance, and can be directly translated to the exploration and treatment of clinical relevant human diseases, providing a clear model for the study of salt-sensitive hypertension in human populations.

[0039] The sequence according to the present application is: SEQ ID NO. 1: TAACCCCAGGGGGGATTTCAGGG.

[0040] SEQ ID NO. 2: AGACAAAATTCTTACCACGTGCGG.

[0041] SEQ ID NO. 3:​​ GCCACC.

[0042] SEQ ID NO. 4: 5'-GCATCTGACTTCTGGCTAATAAAG-3'.

[0043] SEQ ID NO. 5: 5'-CTCATCCCTCTTTTAACACTGAAAGC-3'.

[0044] SEQ ID NO. 6: 5'-TATAGTGCCCATTCGCCACAAAG-3'.

[0045] SEQ ID NO. 7: 5'-CTTTGACCTGACCTTCTAACCTTG-3'.

[0046] SEQ ID NO. 8: 5'-AGATGTACTGCCAAGTAGGAAAGTC-3'.

[0047] SEQ ID NO. 9: 5'-GCATCTGACTTCTGGCTAATAAAG-3'.

[0048] SEQ ID NO. 10: 5'-CTAGGGTAAGCAATTCCATACAGC-3'.

[0049] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

Claims

1. A method for constructing a GRK4 R65L humanized salt-sensitive hypertension animal model, characterized in that, Includes the following steps: The animal in question was a rat. The third and fourth exons of the rat endogenous GRK4 gene were replaced with a human GRK4 cDNA sequence carrying the R65L point mutation, driven by the CAG promoter, using CRISPR-Cas9 technology.

2. The construction method according to claim 1, characterized in that, The specific steps include: S1. Constructing a donor vector: Constructing a donor vector containing homologous recombination elements, wherein the donor vector comprises: a 5' homologous arm homologous to the upstream rat genome sequence of the GRK4 target site, a CAG promoter, a Kozak sequence, a full-length human GRK4 cDNA coding sequence carrying the R65L point mutation, a rabbit β-globulin polyadenylate signal, and a 3' homologous arm homologous to the downstream genome sequence; S2. Injection and transplantation of fertilized eggs: sgRNA targeting the target region of the rat GRK4 gene was designed. Cas9 mRNA, sgRNA and the donor vector constructed in step S1 were mixed and microinjected into rat fertilized eggs. The surviving fertilized eggs were then transplanted into pseudopregnant female rats to obtain F0 generation rats. S3. Breeding and identification: Positive F0 generation rats were mated with wild-type rats to obtain F1 generation. The F1 generation heterozygotes were crossbred to obtain homozygous rats, which are the GRK4 R65L humanized salt-sensitive hypertension animal models.

3. The construction method according to claim 2, characterized in that, The sgRNA mentioned in step S2 includes gRNA1, which targets the upstream reverse strand of the target region, and gRNA2, which targets the downstream forward strand of the target region; wherein the nucleotide sequence of gRNA1 is shown in SEQ ID NO.1, and the nucleotide sequence of gRNA2 is shown in SEQ ID NO.

2.

4. The GRK4 R65L humanized salt-sensitive hypertension animal model constructed according to any one of claims 1-3.

5. The use of the animal model described in claim 4 in screening drugs for treating salt-sensitive hypertension.

6. The application according to claim 5, characterized in that, The method for screening drugs includes administering a dietary stimulus to rats containing at least 4% sodium chloride.

7. The application of the animal model described in claim 4 in studying the pathological mechanism of hypertension caused by GRK4 gene mutation.

8. The application according to claim 7, characterized in that, The application includes providing rats with a dietary stimulus containing at least 4% sodium chloride.

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