A method for constructing a mouse model for studying vascular aging

Abstract

The application discloses a method for constructing a mouse model for studying blood vessel aging, and belongs to the technical field of genetic engineering. The 6030442E23Rik gene is modified by flox by using the principle of homologous recombination and the method of homologous recombination of fertilized eggs. 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 an In-Fusion cloning method, the vector comprising a 2.9kb 5' homologous arm, a 2.2kb flox region and a 2.6kb 3' homologous arm. The Cas9 mRNA, gRNA and donor vector are microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice, and the positive F0 generation mice are identified by PCR amplification and sequencing.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a method for constructing a mouse model for studying vascular aging. Background Technology

[0002] The increasingly aging global society is one of the major public health challenges worldwide. Aging is the most important cardiovascular risk factor, and vascular aging is a structural abnormality and functional impairment in the process of individual aging, characterized by accelerated vascular cell senescence, vascular dysfunction, and vascular remodeling. Therefore, further understanding of the specific molecular mechanisms of age-induced vascular dysfunction may provide new treatment options for delaying vascular aging and reducing the incidence and mortality of age-related cardiovascular diseases.

[0003] Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs longer than 200 nt and are an important component of the noncoding genome. Numerous studies have shown that lncRNAs participate in a variety of biological processes, including cellular senescence, DNA methylation, histone modification, post-transcriptional regulation of RNA, and protein translation regulation, and influence the regulation of various physiological and pathological processes. They are important molecules that regulate genes in cells and even individuals.

[0004] In our previous studies, using aged and young aortic tissues and lncRNA array technology, we screened for an age-related lncRNA—6030442E23Rik—and named it the "Aging-Associated Transcript" (AAT). Furthermore, we validated in mouse aortic tissue that the expression of 6030442E23Rik increases with age, and that mice with the 6030442E23Rik mutation exhibited improved vascular function, activated energy metabolism, and enhanced activity.

[0005] In summary, humans also possess the same AAT sequence as mice, and targeting this sequence holds promise as a potential therapeutic strategy for delaying vascular aging and improving vascular function. Summary of the Invention

[0006] To address the aforementioned problems, this invention employs homologous recombination of fertilized eggs to modify the 6030442E23Rik gene with flux, thereby obtaining a mouse model capable of conditionally knocking out the 6030442E23Rik gene.

[0007] The purpose of this invention is to provide a method for constructing a mouse model for studying vascular aging, comprising the following steps:

[0008] Step 1: Design a gRNA that recognizes the 6030442E23Rik gene, perform in vitro transcription, and obtain Cas9 mRNA and gRNA;

[0009] Step 2: Construct homologous recombination vectors;

[0010] Step 3: Cas9 mRNA, gRNA and homologous recombination vector were microinjected into mouse zygotes to obtain a mouse model with the 6030442E23Rik gene knocked out.

[0011] Furthermore, the primer sequences used in step 1 to prepare the gRNA include gRNA1 and gRNA2, and the nucleotide sequences of gRNA1 and gRNA2 are shown below:

[0012] gRNA1: 5'-GCCAATAAGGTTTTATCAGCTGG-3';

[0013] gRNA2: 5'-GCTGGAGACCTTATTCTATTGGG-3'.

[0014] Furthermore, in step 2, the homologous recombination vector includes a 2.9kb 5' homologous arm, a 2.2kb flux region, and a 2.6kb 3' homologous arm.

[0015] Furthermore, the target fragments of the 5' homologous arm, flux region, and 3' homologous arm were ligated to the pBR-322 vector using the in-fusion cloning method.

[0016] Furthermore, in step 3, Cas9 mRNA, gRNA, and homologous recombination vector were microinjected into mouse fertilized eggs. Mice born 20 days later were F0 generation mice. PCR identification of F0 generation mice yielded positive F0 generation mice, which are mouse models with the 6030442E23Rik gene knocked out.

[0017] Furthermore, the identification protocol for the F0 generation mice is as follows:

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

[0019] Furthermore, the PCR primer sequences for the 5' homologous arm are shown below:

[0020] Forward-GGCATGGTAAAGGATTCACATCAAA;

[0021] Reverse-TACACTCTTGATGCTTGGGTTTTC.

[0022] Furthermore, the PCR primer sequences for the 3' homologous arm are shown below:

[0023] Forward-TCCTGTGTTGACATAGTTCTTTGGA;

[0024] Reverse-TGTCATGTTTACCTCTCCAGACATT.

[0025] Furthermore, the mice were C57BL / 6J mice.

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

[0027] The 6030442E23Rik gene conditional knockout mouse model constructed in this application exhibits high reproducibility, good controllability, and is easy to breed and raise. It is also more stable and better reflects clinical characteristics, providing a better platform for research on the pathogenesis of cardiovascular and cerebrovascular diseases related to vascular aging and the exploration of intervention measures. Using this model will allow for more in-depth and scientific research on the pathogenesis of vascular aging, as well as drug treatment and drug screening. Attached Figure Description

[0028] Figure 1 A schematic diagram of the strategy for constructing a mouse model, where 5' homology arm, 3' homology arm, Wild type allele, Targeting vector, Targeted allele, Knockout allele, Cre recombination, Exon1, Exon2, Coding region, and Uncoding region are all shown.

[0029] Figure 2 Map of homologous recombination plasmids;

[0030] Figure 3 Electrophoresis diagram for homologous recombination vector restriction enzyme digestion identification: 1: EcoRI restriction enzyme digestion identification results, theoretical band sizes are 7594bp, 5614bp, and 466bp; M: 1 kb DNA ladder.

[0031] Figure 4This diagram illustrates the identification strategy for F0 generation mice, where: homology arm, wild type allele, targeted allele, exon1, exon2, coding region, uncoding region, primer location, PCR product, and PCR sequenced region.

[0032] Figure 5 Electrophoresis image of F0 generation mice positive for homologous recombination, numbers: F0 generation mouse number; M is a 1kb DNA marker;

[0033] Figure 6 Electrophoresis images of PCR identification of the 5' and 3' homologous arms of F1 generation mice; where A represents the 5' homologous arm; B represents the 3' homologous arm; numbers represent F1 generation mouse numbers; M represents a 1kb DNA ladder.

[0034] Figure 7 This is a schematic diagram illustrating the sequencing verification of PCR products from F1 generation mice.

[0035] Figure 8 A schematic diagram of F1 generation positive mice;

[0036] Figure 9 This is a schematic diagram of the 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.

[0037] Figure 10 A schematic diagram showing the primer positions for PCR identification in flox mice;

[0038] Figure 11 Electrophoresis image for genotype identification of flox mice by enzyme digestion;

[0039] Figure 12The sequence alignment results are for the 5' homologous arm 1# of F1 generation mice; Query is the target sequence (E23Rikrecombined genomic DNA sequence), Subject is the sequencing result, and the red underlined part is the 5' homologous arm sequence;

[0040] Figure 13 The sequence alignment results are for the 5' homologous arm 2# of F1 generation mice; Query is the target sequence (E23Rikrecombined genomic DNA sequence), Subject is the sequencing result, the bases in the red box are the Loxp sequence, and the red underline is the 5' homologous arm sequence;

[0041] Figure 14 The sequence alignment results are for the 3' homologous arm 1# of F1 generation mice; Query is the target sequence (E23Rikrecombined genomic DNA sequence), Subject is the sequencing result, the bases in the red box are the Loxp sequence, and the red underline is the 3arm homologous arm sequence;

[0042] Figure 15 The sequence alignment results are for the 3' homologous arm 2# of F1 generation mice; Query is the target sequence (E23Rikrecombined genomic DNA sequence), Subject is the sequencing result, and the red underlined part is the 3' homologous arm sequence. Detailed Implementation

[0043] The invention will be further illustrated below with specific examples.

[0044] This application provides a method for constructing a mouse model to study vascular aging, such as... Figure 1 As shown, the 6030442E23Rik gene was modified with flux using homologous recombination in fertilized eggs. The simplified process is as follows: Cas9 mRNA and gRNA were obtained through in vitro transcription; a homologous recombination vector (donor vector) containing a 2.9kb 5' homologous arm, a 2.2kb flux region, and a 2.6kb 3' homologous arm was constructed using in-fusion cloning. The Cas9 mRNA, gRNA, and donor vector were microinjected into fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. PCR amplification and sequencing confirmed the positive F0 generation mice.

[0045] The target gene name (Ensembl number) mentioned above is: 6030442E23Rik (ENSMUSG00000100664).

[0046] Target gene Ensembl website link:

[0047] http: / / asia.ensembl.org / Mus_musculus / Gene / Summary?db=core;g=ENSMUSG00000100664;r=7: 70866797-70874157;t=ENSMUST00000186618

[0048] The transcript targeted by the protocol (Ensembl number): 6030442E23Rik-201 (ENSMUST00000186618.2)

[0049] Flox targets the following exon: exon 1

[0050] 1. Design of Guide RNA (gRNA):

[0051] gRNA design is primarily conducted using http: / / crispr.mit.edu / and http: / / www.rgenome.net / cas-designer / , involving processes such as selecting the editing type, choosing the species, and inputting the target gene sequence.

[0052] The gRNA sequence information is shown in the table below:

[0053]

[0054] 2. Construction of homologous recombination plasmids

[0055] 2.1 Map of homologous recombination plasmids

[0056] Methods for constructing homologous recombination plasmids:

[0057] 1. Based on the restriction enzyme sites of the target fragment, select the corresponding restriction enzyme, and cut the circular vector plasmid into a linearized vector by double enzyme digestion. The enzyme digestion products are then recovered by agarose gel electrophoresis.

[0058] 2. Amplify the sequence of the target fragment using PCR and recover the amplification product using agarose gel electrophoresis;

[0059] 3. Ligate the linearized vector and the amplified fragment in proportion, at 37 ℃ for 1-2 h. Transform the ligated recombinant plasmid (10 μl) into cloning competent cells DH5α, gently pipette to mix, incubate on ice for 30 min, then incubate in a 42 ℃ water bath for 90 s, and immediately place on ice to cool for 2-3 min.

[0060] 4. Add 500 μl of LB liquid medium without antibiotics, and incubate at 37 ℃ for 1 h with shaking at 250 rpm.

[0061] 5. Centrifuge the bacterial culture from the previous step at 5000 rpm for 5 min and discard 300 μl of supernatant. Resuspend the bacterial culture in the remaining culture medium, preferably spreading it in different gradients to prevent the single clones from being too dense or too sparse. Spread it evenly on a plate containing plasmid resistance using a sterile spreader and incubate it in a 37 ℃ incubator for 10-12 h.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Homologous recombination plasmid map, such as Figure 2 As shown.

[0067] 2.2 Identification by Enzyme Digestion of Homologous Recombinant Plasmids

[0068] After PCR, the product was subjected to agarose gel electrophoresis to observe the band size, such as... Figure 3 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.

[0069] 2.3 Genotyping of F0 generation mice

[0070] 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.

[0071] 2.3.1 Genotyping Strategy for F0 Generation Mice

[0072] like Figure 4 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.

[0073] PCR identification protocol for homologous recombination positive mice:

[0074] A 5' arm homologous recombination-positive genome should amplify a 5.4 kb fragment, and a negative genome should amplify an 8.3 kb fragment; a 3' arm homologous recombination-positive genome should amplify a 5.7 kb fragment, and a negative genome should amplify a 9.4 kb fragment.

[0075] 2.3.2 PCR identification results of homologous recombination positive F0 generation mice

[0076] The F0 generation mice that were positive for homologous recombination in both arms were mice 1, 9, and 14. The electrophoresis results of long-fragment PCR identification are as follows: Figure 5 As shown, in the 5' and 3' homologous arms, positive mice had obvious 5.4kb and 5.7kb bands, respectively, while WT mice had obvious 8.3kb and 9.4kb bands.

[0077] 2.3.3 PCR Identification Method for 5' Homologous Arm Recombinant Positive F0 Generation Mice

[0078] Primer information

[0079]

[0080] Reaction system:

[0081]

[0082] KOD-Multi&Epi-(TOYOBO, Code No: KME-101)

[0083] Reaction conditions:

[0084]

[0085] 2.3.4 PCR Identification Method for 3' Homologous Arm Recombinant Positive F0 Generation Mice

[0086] Primer information

[0087]

[0088] Reaction system:

[0089]

[0090] KOD-Multi&Epi-(TOYOBO, Code No: KME-101)

[0091] Reaction conditions:

[0092]

[0093] 2.4 F1 generation mouse acquisition and genotyping

[0094] Because the early cleavage rate of fertilized eggs is very rapid, the resulting F0 generation mice are chimeras and may not necessarily possess stable heritability. Therefore, they need to be passaged to obtain the stably heritable F1 generation mice. F0 positive mice were mated with wild-type C57BL / 6J mice to obtain F1 generation mice, which were then genotyped using PCR and sequencing.

[0095] 2.4.1 PCR identification of 5' and 3' homologous arms in F1 generation mice

[0096] The PCR identification strategy and method are the same as in section 2.3, F0 generation mouse identification. The electrophoresis results of PCR identification of the 5' and 3' homologous arms of F1 generation mice are shown in Figures 6A and 6B. The mice that were positive for PCR identification were: 1, 6, 8, 10, 11, 12, 13, 14, 15, 20, 22, and 23; all were confirmed to be positive by sequencing.

[0097] 2.4.2 PCR identification and sequencing comparison results of F1 generation mice

[0098] Sequencing of PCR products from F1 generation positive mice involved four sequencing reactions. The regions corresponding to the sequencing reactions are shown in Figure 7. Specifically, the 5' homologous arm identification involved two sequencing reactions, labeled 1 and 2; the 3' homologous arm identification involved two sequencing reactions, labeled 3 and 4.

[0099] 2.4.3 F1 generation positive mouse numbering and basic information

[0100] The F1 generation positive mice's identification numbers and basic information are shown in the table below;

[0101] Basic information of F1 generation positive mice

[0102]

[0103] 3. Follow-up work after mouse delivery

[0104] 3.1 Mouse Validation

[0105] Upon receiving the mice from our company, please first verify the quantity and serial numbers of the delivered mice against the information on the delivery note. Our mouse numbering system is as follows: Figure 8 As shown.

[0106] 3.2 Subsequent mouse breeding protocol

[0107] Unless otherwise specified, all delivered flox mice are flox heterozygous mice (geneflox / +).

[0108] 3.2.1 Brief Procedure and Recommendations for Subsequent Experimental Mouse Reproduction

[0109] 3.2.1.1 The simplified procedure for subsequent mouse breeding is as follows: Figure 9 As shown:

[0110] (1) The obtained flox heterozygous mice (gene flox / + Divided into two parts:

[0111] A subset of flox mice were mated with Cre mice to obtain flox-positive and Cre-positive mice (these mice are abbreviated as gene mice). flox / + Cre+ mice and flux-positive and Cre-negative mice (gene) flox / + A subset of flux mice were self-crossed to obtain flux homozygous mice (these mice are abbreviated as: gene). flox / flox ) and flox heterozygous mice (gene flox / + ).

[0112] (2) To obtain flux homozygous and Cre positive mice, two breeding methods can be selected:

[0113] One approach is to obtain flox and Cre double-positive heterozygous mice (gene flox / + Cre+) and flox heterozygous mice (gene flox / + Mice were mated to obtain a group of experimental mice that were homozygous for flux and positive for Cre (these mice are abbreviated as gene mice).flox / flox Cre+ mice (representing 1 / 8 of the offspring) and a control group of flux-homozygous, Cre-negative mice (gene) flox / flox The proportion of this mouse in the offspring is 1 / 8.

[0114] Another method is to obtain flox and Cre double-positive heterozygous mice (gene flox / + Cre+ mice and flux homozygous mice (gene flox / flox Mice were mated to obtain experimental group mice that were homozygous for flux and positive for Cre (gene). flox / flox Cre+ mice (accounting for 1 / 4 of the offspring) and flux-homozygous, Cre-negative control mice (gene) flox / flox The proportion of this mouse in the offspring is 1 / 4.

[0115] Note: The above breeding protocol for laboratory mice is only a reference protocol. During the breeding process, the number and situation of mice of various genotypes will vary. Please adjust it according to the actual situation.

[0116] 3.2 Subsequent mouse genotyping protocol

[0117] After the delivered flux mice are verified to be problem-free, their genotypes can be identified using short-fragment PCR during subsequent mouse breeding. The primers and conditions required for genotype identification in each step of the subsequent mouse breeding process are shown below. There are multiple methods for mouse genotype identification; this report only provides one method. Clients can also design their own primers based on the design principles we provide. No special software requirements are specified for primer design; standard DNASTAR software such as PrimerSelect is sufficient (this software can be downloaded from the internet).

[0118] 3.2.1 genotyping of flox mice (used to identify flox homozygous, heterozygous, and wild-type mice)

[0119] The principle of Cas9-based genotyping of flux mice is to differentiate genotypes by utilizing the difference in PCR product fragment size between wild-type and mutant mice after loxp site insertion. This report only provides primer position diagrams for two schemes (P1, P2 primer pairs and P3, P4 primer pairs). The primer sequence information and specific PCR identification method for one scheme are provided for reference; for the other scheme, please design and verify your own primer sequence information and specific PCR identification method. Users can perform identification according to the identification schemes and methods we provide, or design their own.

[0120] 3.2.1.1 Schematic diagram of primer positions for PCR identification

[0121] like Figure 10 As shown, WT mice have only a 291 bp band, heterozygous mice have two bands, 291 kb and 350 kb, and homozygous mice have only a 350 bp band.

[0122] 3.2.1.2 PCR Identification

[0123]

[0124] 4. Sequencing Results Analysis

[0125] 4.1 Sequencing and alignment results of F1 generation mouse PCR identification products with target sequences

[0126] The sequencing and alignment results are based on mouse number 1, which is a positive mouse.

[0127] 4.1.1 PCR identification and sequencing alignment results of the 5' homologous arm of F1 generation mice

[0128] The alignment results of sequencing reaction #1 are as follows: Figure 12 As shown, Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, and the red underlined part is the 5-arm homologous arm sequence.

[0129] The alignment results of sequencing reaction #2 are as follows: Figure 13 As shown, Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, the bases in the red box are the Loxp sequence, and the red underline is the 5arm homologous arm sequence.

[0130] 4.1.2 PCR identification and sequencing alignment results of the 3' homologous arm of F1 generation mice

[0131] The alignment results of sequencing reaction #3 are as follows: Figure 14 As shown, Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, the bases in the red box are the Loxp sequence, and the red underline is the 3arm homologous arm sequence.

[0132] The alignment results of sequencing reaction #4 are as follows: Figure 15 As shown, Query is the target sequence (E23Rik recombinated genomic DNA sequence), Subject is the sequencing result, and the red underlined part is the 3arm homologous arm sequence.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions created by the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions created by the present invention without departing from the essence and scope of the technical solutions created by the present invention.

Claims

1. A method for constructing a mouse model for studying vascular aging, characterized in that, The construction method includes: Step 1: Design a gRNA that recognizes the 6030442E23Rik gene, perform in vitro transcription, and obtain Cas9 mRNA and gRNA; Step 2: Construct a homologous recombination vector, which includes a 2.9kb 5' homologous arm, a 2.2kb flux region, and a 2.6kb 3' homologous arm; Step 3: Microinject Cas9 mRNA, gRNA and homologous recombination vector into mouse zygotes to obtain F0 generation mice; Step 4: PCR amplification and sequencing identification. Positive F0 generation mice were mated with C57BL / 6J mice to obtain positive F1 generation mice; flox targets the following exon: exon 1.

2. The construction method of claim 1, wherein, The primer sequences used in step 1 to prepare gRNA include gRNA1 and gRNA2, and the nucleotide sequences of gRNA1 and gRNA2 are shown below: gRNA1: 5'-GCCAATAAGGTTTTATCAGCTGG-3'; gRNA2: 5'-GCTGGAGACCTTATTCTATTGGG-3'.

3. The construction method of claim 1, wherein, The method for constructing the homologous recombination vector includes: using the in-fusion cloning method to ligate the target fragment of the 5' homologous arm, the flux region, and the 3' homologous arm to the pBR-322 vector.

4. The construction method according to claim 3, characterized in that, The identification protocol for F0 generation mice is as follows: A 5' arm homologous recombination-positive genome should amplify a 5.4 kb fragment, and a negative genome should amplify an 8.3 kb fragment; a 3' arm homologous recombination-positive genome should amplify a 5.7 kb fragment, and a negative genome should amplify a 9.4 kb fragment.

5. The construction method according to claim 4, characterized in that, The PCR primer sequences for the 5' homologous arm are shown below: Forward-GGCATGGTAAAGGATTCACATCAAA; Reverse-TACACTCTTGATGCTTGGGTTTTC.

6. The construction method of claim 5, wherein, The PCR primer sequences for the 3' homologous arm are shown below: Forward-TCCTGTGTTGACATAGTTCTTTGGA; Reverse-TGTCATGTTTACCTCTCCAGACATT.

7. The construction method of claim 6, wherein, The mice were C57BL / 6J mice.

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