A method for constructing a mouse model for studying vascular endothelial aging

Abstract

The application discloses a kind of construction methods of mouse model for studying vascular endothelial senescence, belong to genetic engineering technical field, utilize 6030442E23Rik conditional gene knockout mouse and Tek-CreERT2 mouse (utilize CRISPR gene editing technology, Cre is inserted into mouse Tek gene stop codon, is the tool mouse of endothelial cell conditional knockout, currently, this kind of strain mouse has on market), cross, obtain 6030442E23Rik- / -Tek-CreERT2 mouse.Tamoxifen is dissolved in corn oil later, so that its final concentration is 20 mg / ml.Tamoxifen dose is 120mg / kg mouse weight later, intraperitoneal injection 5 times, once every other day, and the mouse model of endothelial cell 6030442E23Rik specificity knockout can be obtained 7 days after the end of last injection.The mouse model of endothelial cell 6030442E23Rik specificity knockout is an important mouse model for studying vascular endothelial senescence.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a construction method of a mouse model for studying vascular endothelial aging. BACKGROUND

[0002] The global aging society is one of the major public health challenges in the world. Aging is the most important cardiovascular risk factor, and vascular aging is a structural abnormality and dysfunction in the aging process of individuals, characterized by accelerated vascular cell aging, vascular dysfunction and vascular remodeling. Vascular endothelial cells are a highly dynamic monolayer of cells located between the blood vessel wall and the blood, and are considered to be the most important component for maintaining vascular homeostasis. More and more studies have shown that endothelial aging is a trigger factor for vascular aging and even individual aging. Therefore, further understanding of the specific molecular mechanisms of aging-induced vascular endothelial dysfunction may provide new therapeutic options for delaying vascular aging and reducing the incidence and mortality of age-related cardiovascular diseases.

[0003] Long non-coding RNA (lncRNA) is a kind of non-coding RNA with a length of more than 200 nt, and is an important component of non-coding genome. A large number of studies have shown that lncRNAs are involved in various biological processes, including cell aging, DNA methylation, histone modification, post-transcriptional regulation of RNA and regulation of protein translation, and affect the regulation of various physiological and pathological processes, and are important molecules for regulating genes in cells and even individuals.

[0004] In our previous study, we used aging aortic tissue and young aortic tissue in combination with lncRNA array technology to screen the age-related lncRNA 6030442E23Rik, which was named "aging associated transcript" (AAT). In addition, we have verified in mouse aortic tissue and mouse aortic endothelial cells that the expression of 6030442E23Rik will increase with age, and the vascular endothelial function of mice after 6030442E23Rik endothelial knockout is improved, energy metabolism is activated, and activity is enhanced.

[0005] In general, humans also have AAT sequences homologous to mice, and targeting the sequence is expected to become a potential therapeutic strategy for delaying vascular aging and improving vascular function. SUMMARY

[0006] In view of the above problems, the present application adopts the method of homologous recombination of fertilized eggs, and utilizes the cross of 6030442E23Rik conditional gene knockout mice and Tek-CreERT2 mice to obtain an endothelial cell 6030442E23Rik gene-specific knockout mouse model.

[0007] An object of the present application is to provide a construction method of an endothelial cell 6030442E23Rik gene-specific knockout mouse model, which comprises the following steps:

[0008] The 6030442E23Rik conditional gene knockout mice are crossed with Tek-CreERT2 mice to obtain 6030442E23Rik - / - Tek-CreERT2 mice; Tamoxifen is dissolved in corn oil to obtain a final concentration of 20 mg / ml, and the mice are injected intraperitoneally with Tamoxifen at a dose of 120 mg / kg of body weight 5 times, once every other day, and the endothelial cell 6030442E23Rik-specific knockout mouse model is obtained 7 days after the last injection.

[0009] Further, the construction process of the Tek-CreERT2 mice comprises the following steps:

[0010] The Cre is inserted into the termination codon of the mouse Tek gene by using the CRISPR gene editing technology to obtain the Tek-CreERT2 mice.

[0011] Further, the construction process of the 6030442E23Rik conditional gene knockout mice comprises the following steps:

[0012] The Cas9 mRNA and gRNA are obtained by in vitro transcription, the homologous recombination vector is constructed by In-Fusion cloning, the Cas9 mRNA, gRNA and homologous recombination vector are microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 mice, the positive F0 mice are identified by PCR amplification and sequencing, and the positive F1 mice are obtained by mating the positive F0 mice with C57BL / 6J mice.

[0013] Further, the primer sequence of the gRNA comprises gRNA1 and gRNA2, and the nucleotide sequences of the gRNA1 and gRNA2 are as follows:

[0014] gRNA1: 5'-GCCAATAAGGTTTTATCAGCTGG-3';

[0015] gRNA2: 5'-GCTGGAGACCTTATTCTATTGGG-3'.

[0016] Further, the homologous recombination vector comprises a 2.9kb 5' homologous arm, a 2.2kb flox region and a 2.6kb 3' homologous arm.

[0017] Further, the F0 generation mouse identification scheme is as follows:

[0018] The 5' arm homologous recombination positive genome should amplify a 5.4kb fragment, and the negative genome should amplify an 8.3kb fragment; the 3' arm homologous recombination positive genome should amplify a 5.7kb fragment, and the negative genome should amplify a 9.4kb fragment.

[0019] Further, the 5' homologous arm PCR primer sequence is as follows:

[0020] Forward-GGCATGGTAAAGGATTCACATCAAA;

[0021] Reverse-TACACTCTTGATGCTTTGGGTTTTC.

[0022] Further, the 3' homologous arm PCR primer sequence is as follows:

[0023] Forward-TCCTGTGTTGACATAGTTCTTTGGA;

[0024] Reverse-TGTCATGTTTACCTCTCCAGACATT.

[0025] Another object of the present application is to provide the application of the endothelial cell 6030442E23Rik gene-specific knockout mouse model in the research of vascular endothelial aging.

[0026] Compared with the prior art, the present application has the following advantages:

[0027] 1、The 6030442E23Rik gene conditional knockout mouse model constructed in the present application has high repeatability, good controllability, is easy to breed and reproduce, and is more stable and more in line with clinical characteristics.

[0028] 2、6030442E23Rik gene expression in mouse aortic blood vessels (especially aortic endothelial cells) increases with age, which can be used as a detection index for mouse vascular aging. The endothelial cell 6030442E23Rik specific knockout mouse model obtained by crossing the 6030442E23Rik conditional gene knockout mouse with the Tek-CreERT2 mouse is an important mouse model for studying vascular endothelial aging, and has higher clinical value. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Schematic diagram of the construction strategy of the endothelial cell 6030442E23Rik specific knockout mouse model.

[0030] Figure 2 Construction strategy diagram of the 6030442E23Rik conditional gene knockout mouse model, wherein, 5 'homology arm: 5' homology arm, 3 'homology arm: 3' homology arm, Wild type allele: wild type allele, Targeting vector: targeting vector, Targeted allele: targeted allele, Knockout allele: knockout allele, Cre recombination: recombinase gene recombination, Exon1: exon1, Exon2: exon2, Coding region: coding region, Uncoding region: non-coding region;

[0031] Figure 3 Homologous recombination plasmid map;

[0032] Figure 4 Electrophoresis map of homologous recombination vector enzyme digestion identification, 1: EcoR I enzyme digestion identification result, the theoretical band size is 7594bp, 5614bp, 466bp; M: 1 kb DNA ladder;

[0033] Figure 5 Schematic diagram of the identification strategy of F0 mice, wherein, homology arm: homology arm, Wild type allele: wild type allele, Targeted allele: targeted allele, Exon1: exon1, Exon2: exon2, Coding region: coding region, Uncoding region: non-coding region, Primer location: primer location, PCR product: PCR product, PCR sequenced region: PCR sequenced region;

[0034] Figure 6 PCR identification electrophoresis map of F0 generation mice for homologous recombination; Number: F0 generation mice number; M: 1 kb DNA marker;

[0035] Figure 7 PCR identification electrophoresis map of F1 generation mice for 5' homologous arm and 3' homologous arm; Wherein, A-5' homologous arm; B-3' homologous arm; Number: F1 generation mice number; M: 1 kb DNA ladder;

[0036] Figure 8 PCR product sequencing verification diagram of F1 generation mice;

[0037] Figure 9 Diagram of F1 generation positive mice;

[0038] Figure 10 Diagram of subsequent mouse breeding process; Heterozygous flox mouse: heterozygous flox mouse, Cre mouse: Cre recombinase mouse, Homozygous flox mouse: homozygous flox mouse, Heterozygous flox mouse and Cre positive: heterozygous flox / Cre positive mouse, Homozygous flox mouse and Cre positive: homozygous flox / Cre positive mouse;

[0039] Figure 11 PCR identification primer position diagram of 6030442E23Rik gene of flox mouse;

[0040] Figure 12 Genotype enzyme digestion identification electrophoresis map of 6030442E23Rik gene of flox mouse;

[0041] Figure 13 6030442E23Rik - / - PCR identification primer position diagram of Cre enzyme of Tek-CreERT2 mouse;

[0042] Figure 14 6030442E23Rik - / - Genotype enzyme digestion identification electrophoresis map of Cre enzyme of Tek-CreERT2 mouse;

[0043] Figure 15F1 generation mouse 5' homologous arm 1# sequencing reaction alignment results; Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, and the red underlined sequence is the 5arm homologous arm sequence;

[0044] Figure 16 F1 generation mouse 5' homologous arm 2# sequencing reaction alignment results; Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, the red box base is the Loxp sequence, and the red underlined sequence is the 5arm homologous arm sequence;

[0045] Figure 17 F1 generation mouse 3' homologous arm 1# sequencing reaction alignment results; Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, the red box base is the Loxp sequence, and the red underlined sequence is the 3arm homologous arm sequence;

[0046] Figure 18 F1 generation mouse 3' homologous arm 2# sequencing reaction alignment results; Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, and the red underlined sequence is the 3arm homologous arm sequence. DETAILED DESCRIPTION

[0047] The content of the present application is further illustrated below in combination with specific examples.

[0048] The present application provides a method for constructing an endothelial cell 6030442E23Rik gene-specific knockout mouse model, as shown in the following formula: Figure 1 The method includes three parts:

[0049] 1. Tek-CreERT2 mouse

[0050] To add inducible property to the Cre-lox system, we used a ligand-dependent CreER recombinase. It consists of a cre recombinase fused to the hormone-binding domain of the estrogen receptor (ER). In the absence of the estrogen analog Tamoxifen, the CreER recombinase stays in the cytoplasm and cannot function; only in the presence of estrogen can CreER enter the nucleus and drive recombination at LoxP sites.

[0051] Using CRISPR gene editing technology, Cre is inserted into the mouse Tek gene stop codon, which is a tool mouse for endothelial cell conditional knockout. This strain of mouse is currently available on the market.

[0052] 2, 6030442E23Rik conditional gene knockout mouse

[0053] As shown in Figure 2 , using the principle of homologous recombination, the 6030442E23Rik gene is modified by flox using the zygote homologous recombination method. The brief process is as follows: Cas9 mRNA and gRNA are obtained by in vitro transcription; a homologous recombination vector (donor vector) is constructed by In-Fusion cloning, which contains a 2.9kb 5' homologous arm, a 2.2kb flox region, and a 2.6kb 3' homologous arm. Cas9 mRNA, gRNA and donor vector are microinjected into zygotes of C57BL / 6J mice to obtain F0 mice, and positive F0 mice are identified by PCR amplification and sequencing.

[0054] The above-mentioned target gene name (Ensembl number): 6030442E23Rik (ENSMUSG00000100664)

[0055] The target gene Ensembl website link:

[0056] http: / / asia.ensembl.org / Mus_musculus / Gene / Summary?db=core;g=ENSMUSG00000100664;r=7: 70866797-70874157;t=ENSMUST00000186618

[0057] The transcript targeted by the scheme (Ensembl number): 6030442E23Rik-201 (ENSMUST00000186618.2)

[0058] Exon targeted by Flox: exon 1

[0059] 2.1. Design of Guide RNA (gRNA):

[0060] The design of gRNA was mainly designed by http: / / crispr.mit.edu / and http: / / www.rgenome.net / cas-designer / : by selecting the editing type, selecting the species, inputting the sequence of the target gene, etc.

[0061] The sequence information of gRNAs is shown in the following table:

[0062]

[0063] 2.2. Construction of homologous recombination plasmid

[0064] 2.2.1 Homologous recombination plasmid map

[0065] Construction method of homologous recombination plasmid:

[0066] 1. According to the enzyme cutting site of the target fragment, select the corresponding endonuclease, cut the circular carrier plasmid into linearized carrier by double enzyme cutting method, and recover the enzyme cutting product by agarose gel method;

[0067] 2. Amplify the sequence of the target fragment by PCR method, and recover the amplification product by agarose gel method;

[0068] 3. Connect the linearized carrier and the amplified fragment in proportion, 37 ℃, 1~2 h. Transfer the connected recombinant plasmid (10 μl) into the clonal competent cell DH5α, gently blow and mix, stand on ice for 30 min, then 42℃ water bath for 90 s, immediately stand on ice for cooling 2~3 min;

[0069] 4. Add 500 μl of LB liquid medium without adding antibiotic, shake the bacteria at 37℃ for 1 h, at a speed of 250 rpm;

[0070] 5. Centrifuge the bacterial solution of the above step at 5 000 rpm for 5 min, discard 300 μl of supernatant. Resuspend the bacterial body with the remaining culture medium, preferably smear at different gradients to prevent single colony bacteria from being too dense or too sparse. After evenly spreading on the plate containing plasmid resistance with a sterile spreading rod, place it in a 37℃ incubator for 10~12 h;

[0071] 6. After overnight incubation, pick up a single clone of bacteria with a 1 μl pipette tip and place it in 5 ml of LB liquid medium containing antibiotics, and continue to incubate for 10-12 h.

[0072] 7. After extracting recombinant plasmids from bacterial culture using the plasmid mini-prep kit, digest them with restriction endonuclease EcoRI, and then perform enzyme digestion identification by agarose gel electrophoresis.

[0073] 8. While performing step 7, a portion of the bacterial culture can be aspirated for bacterial PCR. The PCR product can be analyzed by agarose gel electrophoresis to observe the band size and further identify whether the recombinant plasmid is correct.

[0074] 9. Perform bidirectional sequencing on the successfully identified bacterial culture. Once the sequence results are correctly aligned, it indicates that the recombinant plasmid has been successfully constructed and downstream experiments can be carried out.

[0075] like Figure 3 As shown.

[0076] 2.2.2 Identification by Enzyme Digestion of Homologous Recombinant Plasmids

[0077] After PCR, the product was subjected to agarose gel electrophoresis to observe the band size, such as... Figure 4 As shown, based on the EcoR I restriction enzyme results, combined with... Figure 2 The plasmid pattern will show three bands on agarose gel electrophoresis: 7594bp, 5614bp, and 466bp. Band 1 in the figure matches the corresponding band, confirming that the recombinant plasmid is correct.

[0078] 2.2.3 Genotyping of F0 generation mice

[0079] The injected fertilized eggs were transferred into pseudopregnant mice, and the mice born after about 20 days were designated as F0 generation mice. Genotyping was performed on these mice using PCR amplification and sequencing.

[0080] 2.2.3.1 Genotyping Strategy for F0 Generation Mice

[0081] like Figure 5 As shown, in Wild type (WT) mice, due to the absence of gene editing, an 8.3 kb fragment can be amplified using primers I / II, and a 9.4 kb fragment can be amplified using primers III / IV. In positive mice, due to the addition of the LOXP system, a 5.4 kb fragment can be amplified using primers I / II, and a 5.7 kb fragment can be amplified using primers III / IV. The genotype of the F0 generation mice can be identified based on the differences in these bands.

[0082] PCR identification protocol for homologous recombination positive mice:

[0083] 5' arm homologous recombination positive genome should be amplified 5.4 kb fragment, negative genome should be amplified 8.3 kb fragment; 3' arm homologous recombination positive genome should be amplified 5.7 kb fragment, negative genome should be amplified 9.4 kb fragment.

[0084] 2.2.3.2 Homologous recombination positive F0 generation mouse PCR identification results

[0085] The F0 generation mouse with double-arm homologous recombination positive is No. 1, 9, 14, and the electrophoresis results of long fragment PCR identification are shown in Figure 6 In the 5' and 3' homologous arms, the positive mouse has obvious 5.4 kb and 5.7 kb bands respectively, and the WT mouse has obvious 8.3 kb and 9.4 kb bands.

[0086] 2.2.3.3 5' homologous arm recombination positive F0 generation mouse PCR identification method

[0087] Primer information

[0088]

[0089] Reaction system:

[0090]

[0091] KOD-Multi & Epi- (TOYOBO, Code No: KME-101)

[0092] Reaction conditions:

[0093]

[0094] 2.2.3.4 3' homologous arm recombination positive F0 generation mouse PCR identification method

[0095] Primer information

[0096]

[0097] Reaction system:

[0098]

[0099] KOD-Multi & Epi- (TOYOBO, Code No: KME-101)

[0100] Reaction conditions:

[0101]

[0102] 2.2.4 F1 generation mouse acquisition and genotype identification

[0103] Since the early cleavage speed of zygote is very fast, the F0 generation mice obtained are chimeras, which do not necessarily have the ability of stable inheritance, and need to be passaged to obtain the F1 generation mice which can be stably inherited. The F0 generation positive mice were mated with wild type C57BL / 6J mice to breed F1 generation mice, and their genotypes were identified by PCR method and sequencing.

[0104] 2.2.4.1 PCR identification of 5' and 3' homologous arm of F1 generation mice

[0105] The PCR identification strategy and method are the same as those in 2.3 F0 generation mouse identification part, and the electrophoresis results of PCR identification of 5' and 3' homologous arm of F1 generation mice are shown in Figures 7A and 7B. The positive mice identified by PCR are: 1, 6, 8, 10, 11, 12, 13, 14, 15, 20, 22, 23; and all of them are confirmed to be positive by sequencing.

[0106] 2.2.4.2 Sequencing alignment results of PCR identification of F1 generation mice

[0107] Sequencing of PCR identification products of F1 generation positive mice was performed, and a total of 4 sequencing reactions were performed. The regions corresponding to the sequencing reactions are shown in Figure 8. Among them, for 5' homologous arm identification, a total of 2 sequencing reactions were performed, and they are marked as: 1, 2; for 3' homologous arm identification, a total of 2 sequencing reactions were performed, and they are marked as: 3, 4. Figure 8

[0108] 2.2.4.3 F1 generation positive mouse number and basic information

[0109] The F1 generation positive mouse number and basic information are shown in the following table;

[0110]

[0111] 3, 6030442E23Rik conditional gene knockout mice

[0112] ​The 6030442E23Rik conditional knockout mice were crossed with Tek-CreERT2 mice (Cre was inserted into the stop codon of mouse Tek gene by CRISPR gene editing technology, which is a tool mouse for endothelial cell conditional knockout, and this strain of mice is currently available on the market) to obtain 6030442E23Rik- / -Tek-CreERT2 mice. Then, Tamoxifen was dissolved in corn oil to a final concentration of 20 mg / ml. Then, 120 mg / kg of Tamoxifen was injected intraperitoneally for 5 times, once every other day, and the endothelial cell 6030442E23Rik-specific knockout mouse model was obtained 7 days after the last injection.

[0113] The expression of the 6030442E23Rik gene in the mouse aorta (especially in the aortic endothelial cells) increases with age, and this gene can be used as an indicator for detecting vascular aging in mice. The endothelial cell 6030442E23Rik-specific knockout mouse model is an important mouse model for studying vascular endothelial aging.

[0114] Identification of the endothelial cell 6030442E23Rik-specific knockout mouse model:

[0115] 1. qPCR method: First, isolate mouse aortic endothelial cells. Thaw the matrix gel containing growth factors completely at 4°C overnight. Coat a pre-cooled 35 mm culture dish with 1 mL of matrix gel and place it in a 37°C incubator for 20 minutes to allow the matrix gel to solidify. Isolate the mouse thoracic aorta segment and inoculate it on the ECM matrix gel supplemented with endothelial cell growth factors, 10% FBS, and 1% penicillin / streptomycin. On the 4th day, gently remove the aorta segment from the matrix gel without interrupting the growth of the endothelial cells. After 3-4 days of proliferation, trypsinize the mouse aortic endothelial cells and reseed them on 0.1% gelatin-coated cell culture plates. Then extract the mouse aortic endothelial cells for RT-PCR to verify the expression of 6030442E23Rik.

[0116] 2. Fluorescence in situ hybridization (FISH) method: First, synthesize the 6030442E23Rik-specific probe. Then, take the mouse aorta tissue, and through the processes of fixation, embedding, deparaffinization, protease treatment, denaturation, fluorescence in situ hybridization, and fluorescence microscopy, identify the expression of 6030442E23Rik in the mouse aortic endothelial cells.

[0117] 4. After the mice are delivered, subsequent related work

[0118] 4.1 Mouse validation

[0119] The principle of mouse number is as follows Figure 9 .

[0120] 4.2 Subsequent mouse breeding scheme

[0121] The delivered flox mice are flox heterozygous mice (gene flox / + ) unless otherwise specified.

[0122] 4.2.1 Brief process and suggestions for subsequent experimental mouse breeding

[0123] 4.2.1.1 The brief process of subsequent experimental mouse breeding is as shown in Figure 10 :

[0124] (1) Divide the obtained flox heterozygous mice (gene flox / + ) into two parts:

[0125] One part of the flox mice is mated with Cre mice, and flox positive and Cre positive mice (this mouse is abbreviated as: gene flox / + : Cre+) and flox positive and Cre negative mice (gene flox / + ) are obtained; one part of the flox mice is self-crossed to obtain flox homozygous (this mouse is abbreviated as: gene flox / flox ) and flox heterozygous mice (gene flox / + ).

[0126] (2) To obtain flox homozygous and Cre positive mice, there are two breeding methods to choose from:

[0127] One method is to cross the obtained flox and Cre double positive heterozygous mice (gene flox / + : Cre+) with flox heterozygous mice (gene flox / + ), and finally obtain flox homozygous and Cre positive experimental mice (this mouse is abbreviated as: gene flox / flox : Cre+, the proportion of this mouse in offspring is: 1 / 8) and flox homozygous and Cre negative control mice (gene flox / flox , the proportion of this mouse in offspring is: 1 / 8);

[0128] Another method is to cross the obtained flox and Cre double positive heterozygous mice (gene flox / + : Cre+) with flox homozygous mice (gene flox / flox) mating, finally get flox homozygous and Cre positive experimental mice (gene flox / flox : Cre+, the proportion of offspring of this mouse is: 1 / 4) and flox homozygous and Cre negative control mice (gene flox / flox , the proportion of offspring of this mouse is: 1 / 4).

[0129] Note: the breeding scheme of the above experimental mice is only a reference scheme. During the breeding process of mice, the number of mice of various genotypes is different. Please adjust it according to the actual situation.

[0130] 4.2.2 Identification scheme of subsequent mouse genotype

[0131] After the delivery of flox mice is verified to be free of problems, the genotype of mice can be identified by short fragment PCR during the subsequent mouse mating and breeding process. The primers and conditions required for mouse genotype identification in each step of subsequent mouse breeding are as follows. There are many schemes for mouse genotype identification, and this report only gives one of them. Customers can also design primers on their own according to the design principles we provide. There is no special requirement for primer design software. The PrimerSelect program in the regular DNASTAR software can meet the requirements (related software can be downloaded through network search).

[0132] 4.2.2.1 flox mouse genotype identification (used to identify flox homozygous, heterozygous and wild type)

[0133] The principle of flox mouse genotype identification based on Cas9 strategy is to use the loxp site inserted to distinguish different genotypes by the size difference of PCR product fragments in wild type and mutant PCR identification. This report only gives the primer position diagram of two sets of schemes (P1, P2 primer pair and P3, P4 primer pair), and the primer sequence information and specific PCR identification method of one set of scheme are for reference; the primer sequence information and specific PCR identification method of the other set of scheme are designed and verified by yourself. Users can identify according to the identification scheme and method we provide, or design it yourself.

[0134] 4.2.2.2 PCR identification primer position diagram

[0135] As Figure 11 shown, primers can be set according to the position of 5' homologous arm, target gene region and 3' homologous arm. Specifically, P1 and P2 primers can be designed to cover the flox sequence at the 5' end of the target gene, and P3 and P4 primers can be designed to cover the flox sequence at the 3' end of the target gene.

[0136] 4.2.2.3 PCR identification

[0137]

[0138] 4.2.3 DNA level Cre activity verification

[0139] Usually, a small piece of Cre-expressing tissue is taken, genomic DNA is extracted, and the flox region is amplified by PCR method, and whether Cre works is qualitatively determined by the presence or absence of the flox region.

[0140] 4.2.3.1 PCR identification primer position diagram

[0141] As shown in Figure 13 , primers can be set according to the positions of the 5' homologous arm, target gene region, and 3' homologous arm. Specifically, P5 and P6 primers are designed to cover the flox sequence at the 5' and 3' ends of the target gene; this sequence has different PCR products before and after Cre action.

[0142] 4.2.3.2 PCR identification conditions for whether Cre works

[0143]

[0144] 4.3 Sequencing result analysis

[0145] 4.3.1 F1 generation mouse PCR identification product sequencing and alignment results with target sequence

[0146] The sequencing alignment result is taken as an example for the 1st positive mouse.

[0147] 4.3.2 F1 generation mouse 5' homologous arm PCR identification sequencing alignment results

[0148] 1# Sequencing reaction alignment result as shown in Figure 15 , Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, and the red underlined sequence is the 5 arm homologous arm sequence.

[0149] 2# Sequencing reaction alignment result as shown in Figure 16 , Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, and the red box base is the Loxp sequence, and the red underlined sequence is the 5 arm homologous arm sequence.

[0150] 4.3.3 F1 generation mouse 3' homology arm PCR sequencing alignment results

[0151] 3# Sequencing reaction alignment results are shown in Figure 17 , Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, and the red box base is the Loxp sequence, and the red underlined is the 3 arm homology arm sequence.

[0152] 4# Sequencing reaction alignment results are shown in Figure 18 , Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, and the red underlined is the 3 arm homology arm sequence.

[0153] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not a limitation on the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for constructing a mouse model for studying vascular endothelial aging, characterized in that, The construction method comprises: The 6030442E23Rik conditional gene knockout mouse is crossed with the Tek-CreERT2 mouse to obtain the 6030442E23Rik - / - Tek-CreERT2 mouse; Tamoxifen is dissolved in corn oil to a final concentration of 20 mg / ml, and the mouse is injected intraperitoneally 5 times at a dose of 120 mg / kg of body weight of Tamoxifen, once every other day, and the 6030442E23Rik-specific knockout mouse model of endothelial cells is obtained 7 days after the last injection. The construction process of the Tek-CreERT2 mouse comprises: The Tek-CreERT2 mouse is obtained by inserting Cre into the termination codon of the mouse Tek gene by using the CRISPR gene editing technology; The construction process of the 6030442E23Rik conditional gene knockout mouse comprises: The Cas9 mRNA and the gRNA are obtained by in vitro transcription, the homologous recombination vector is constructed by the In-Fusion cloning method, the Cas9 mRNA, the gRNA and the homologous recombination vector are microinjected into the fertilized eggs of the C57BL / 6J mouse, the F0 generation mouse is obtained, the positive F0 generation mouse is identified by PCR amplification and sequencing, the positive F0 generation mouse is mated with the C57BL / 6J mouse to obtain the positive F1 generation mouse; The homologous recombination vector comprises a 2.9kb 5' homologous arm, a 2.2kb flox region and a 2.6kb 3' homologous arm; The flox is directed to the exon: exon 1.

2. The construction method of claim 1, wherein, The primer sequence of the gRNA comprises gRNA1 and gRNA2, and the nucleotide sequences of the gRNA1 and the gRNA2 are as follows: gRNA1: 5'-GCCAATAAGGTTTTATCAGCTGG-3'; gRNA2: 5'-GCTGGAGACCTTATTCTATTGGG-3'.

3. The construction method of claim 1, wherein, The F0 generation mouse identification scheme is as follows: The 5.4kb fragment should be amplified from the positive genome of the 5' arm homologous recombination, and the 8.3kb fragment should be amplified from the negative genome; the 5.7kb fragment should be amplified from the positive genome of the 3' arm homologous recombination, and the 9.4kb fragment should be amplified from the negative genome.

4. The construction method according to claim 3, characterized in that, The PCR primer sequence of the 5' homologous arm is as follows: Forward-GGCATGGTAAAGGATTCACATCAAA; Reverse-TACACTCTTGATGCTTTGGGTTTTC.

5. The construction method according to claim 4, characterized in that, The PCR primer sequence of the 3' homologous arm is as follows: Forward-TCCTGTGTTGACATAGTTCTTTGGA; Reverse-TGTCATGTTTACCTCTCCAGACATT.

Images