Method for constructing congenital heart disease mouse model

Abstract

The invention belongs to the field of biological genetic engineering, and particularly relates to a method for constructing a congenital heart disease mouse model. The invention provides gRNA (guide Ribonucleic Acid) for targeting a mouse Tmed1 gene. Nucleotide sequences of the gRNA are as shown in SEQ ID NO: 1 and SEQ ID NO: 2. Meanwhile, the invention provides a method for knocking out the mouse Tmed1 gene based on a CRISPR / Cas9 technology to obtain the mouse with the congenital heart disease. The Tmed1 mouse model is successfully constructed, a reliable mouse model is provided for congenital heart disease and visceral ectopic syndrome patients, subsequent detection and verification of upstream and downstream target gene functions and subsequent verification of development of related target drugs are facilitated, and great convenience is brought to disease mechanism research and drug research and development verification work.

Description

Technical Field

[0001] The present invention belongs to the field of biological genetic engineering, and particularly relates to a method for constructing a mouse model of congenital heart disease. Background Art

[0002] The gene knockout technology is an important tool in modern biological research, which can study the biological functions and related disease mechanisms by deleting or inactivating specific genes. Among them, the CRISPR / Cas9 technology has become the mainstream technology in the field of gene editing due to its high efficiency, specificity, and simplicity of operation. CRISPR / Cas9 is an immune system derived from bacteria for defending against virus invasion. Scientists have developed a highly efficient gene editing technology using this system. The CRISPR / Cas9 system mainly consists of two parts: CRISPR short tandem repeats and Cas9 nuclease. By designing specific guide RNAs (gRNAs), the Cas9 nuclease can be precisely guided to the position of the target gene and cleave the DNA, causing double-strand breaks in the DNA. Subsequently, the cell will repair the DNA through the non-homologous end joining (NHEJ) or homologous recombination (HDR) pathway, thereby achieving gene knockout or knock-in. Through this technology, researchers can precisely introduce double-strand breaks in the genome and then use the cell's own DNA repair mechanism to cause gene knockout or knock-in, so as to study gene functions.

[0003] In the research of human organ development and diseases, visceral heterotaxy and congenital heart disease are two important fields with a high degree of correlation. Visceral heterotaxy refers to the abnormal occurrence of the left-right asymmetry pattern of organs during embryonic development, while congenital heart disease refers to the structural abnormalities that occur in the heart during fetal development.

[0004] The human TMED1 (Transmembrane P24 Trafficking Protein 1) gene belongs to the TMED (Transmembrane emp24 domain-containing protein) family, is located on chromosome 19p13.2, is 4284 bp in length, contains 4 exons, and has 1 GOLD domain. TMED proteins have emerged as important regulators of biomolecule secretion, acting in the trafficking of molecules between cells and influencing the composition, structure, and function of the endoplasmic reticulum and Golgi apparatus in eukaryotic cells. This protein family is associated with the progression of various human diseases, such as Alzheimer's disease and cancer, and has shown specific roles in cell signaling processes, insulin secretion, and cell growth. In vivo studies have shown that TMED1 participates in interleukin-33 (IL-33) signaling by mediating the trafficking of the ST2 receptor (a member of the Toll-like / IL-1 receptor superfamily) to the cell membrane. The ST2 receptor not only interacts with IL-33 to generate T cell-mediated immune responses but also participates in infectious diseases, asthma, and allergic reactions. In IL-33 signaling, the ST2 / TMED interaction involves the TIR domain of the ST2 receptor and the GOLD domain (tp24 or p24g1) of TMED1. There is no in-depth mechanism study of this gene in visceral heterotopia or heart structure development. Summary of the Invention

[0005] An object of the present invention is to provide a gRNA for targeting the mouse Tmed1 gene.

[0006] Another object of the present invention is to provide a method for constructing a mouse model of congenital heart disease.

[0007] Another object of the present invention is to provide a mouse with congenital heart disease.

[0008] A gRNA for targeting the mouse Tmed1 gene according to a specific embodiment of the present invention, the nucleotide sequence of the gRNA is shown as SEQ ID NO:1 and SEQ ID NO:2.

[0009] SEQ ID NO:1: TAGGCGTAGCCCTCCGAGGA-CGG;

[0010] SEQ ID NO:2: TGAAGCTAAGGCACGATTTC-AGG.

[0011] The present invention also provides the application of the gRNA for targeting the mouse Tmed1 gene in constructing a mouse model of congenital heart disease.

[0012] A method for constructing a congenital heart disease mouse model according to a specific embodiment of the present invention, the method comprising the step of using the CRISPR / Cas9 gene editing technology to perform gene editing on a mouse using the above-mentioned gRNA.

[0013] A method for constructing a congenital heart disease mouse model according to a specific embodiment of the present invention, the method comprising the following steps:

[0014] (1) Prepare a mixture containing Cas9 and a gRNA for targeting the mouse Tmed1 gene;

[0015] (2) Transfect the mixture into a mouse fertilized egg;

[0016] (3) Transplant the transfected fertilized egg into a female mouse to obtain F0 generation mice;

[0017] (4) Screen for homozygous offspring with Tmed1 gene knockout based on the F0 generation mice to obtain homozygous mice with Tmed1 gene knockout and obtain congenital heart disease mice.

[0018] Preferably, the F0 generation mice are mated with wild-type C57BL / 6J mice to screen for heterozygotes of the Tmed1 gene for subsequent detection.

[0019] A method for constructing a congenital heart disease mouse model according to a specific embodiment of the present invention, the nucleotide sequence of the gRNA is as shown in SEQ ID NO:1 and SEQ ID NO:2

[0020] The present invention also provides a congenital heart disease mouse obtained by the above method.

[0021] The term "CRISPR / Cas9" used in the present invention is an adaptive immune defense formed by bacteria and archaea during long-term evolution and can be used to combat invading viruses and foreign DNA. The CRISPR / Cas9 gene editing technology is a technology for performing specific DNA modification on a target gene.

[0022] The terms "gRNA", "guide RNA", and "CRISPR guide sequence" as used herein are used interchangeably throughout and refer to a nucleic acid comprising a sequence that determines the specificity of a Cas binding protein of a CRISPR / Cas system. The gRNA hybridizes (partially or fully complementary) to a target nucleic acid sequence in the host cell genome. The length of the gRNA or a portion thereof that hybridizes to the target nucleic acid can be between 15 and 25 nucleotides, between 18 and 22 nucleotides, or between 19 and 21 nucleotides. In some embodiments, the length of the gRNA sequence that hybridizes to the target nucleic acid can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of the gRNA sequence that hybridizes to the target nucleic acid is between 10 and 30 or between 15 and 25 nucleotides.

[0023] The term "gRNA" as used in the present invention generally refers to the single molecule guide RNA or single-stranded guide RNA in the artificial CRISPR / Cas9 system, which refers to the RNA that guides the specific binding of the Cas protein to the target DNA sequence and is an important component in the CRISPR gene knockout / knock-in system.

[0024] The term "gene knockout" or "knockout" as used in the present invention refers to editing a gene in a cell (e.g., performing modifications such as insertion, replacement, and / or deletion on the gene) such that the gene loses its original function (e.g., cannot express a functional protein). Various known molecular biology techniques can be used (e.g., using zinc finger nuclease-based gene editing techniques, TALEN gene editing techniques, and CRISPR / Cas (such as CRISPR / Cas9) gene editing techniques) to edit genes in the cell genome. Gene knockout is not limited to the complete deletion or removal of an entire gene, as long as the gene loses its original function. For example, by inserting an exogenous DNA fragment into the gene, the gene can be made unable to express a functional protein, or by inserting or deleting one or several bases in the gene, a frameshift mutation can occur in the gene to achieve knockout of the gene. For example, in the present invention, gene knockout can use the CRISPR / Cas9 gene editing technique.

[0025] The term "vector" used in the present invention refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain various elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain an origin of replication.

[0026] Advantages of the present invention:

[0027] The present invention has successfully constructed a Tmed1 gene knockout mouse model, providing a reliable mouse model for patients with congenital heart disease and visceral heterotopia syndrome, facilitating the subsequent detection and verification of the functions of upstream and downstream target genes, as well as the verification of the development of related target drugs, which will greatly facilitate the research on disease mechanisms and the verification of drug development. Brief description of the drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 Showing the gRNA design position and the knockout region.

[0030] Figure 2 Is an anatomical diagram showing the mouse heart.

[0031] Figure 3 Are anatomical diagrams of Tmed1 wild-type and HO mice. Detailed implementation manners

[0032] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementation manners obtained by those of ordinary skill in the art without creative efforts fall within the scope protected by the present invention.

[0033] Example 1: Construction of a Tmed1 gene knockout mouse model

[0034] Gene design: Design a CRISPR / Cas9 system for the Tmed1 gene, including a specific guide RNA (gRNA). The design of the guide RNA needs to ensure strong specificity, capable of accurately locating and guiding the Cas9 protein to cleave the target site of the Tmed1 gene.

[0035] The Tmed1 gene (NCBI Reference Sequence: NM_010744.4; Ensembl: ENSMUSG00000032180) is located on chromosome 9 of mice. There are a total of 4 exons, and the protein-coding region is located in exons 2-4. The region specifically knocked out in this example includes exons 2 and 3 and most of exon 4.

[0036] Obtain fertilized eggs from C57BL / 6J mice. Mix the synthesized gRNA with Cas9 protein, or mix the constructed plasmid vector with gRNA, and prepare for microinjection.

[0037] gRNA-A1 (matching reverse strand of gene):

[0038] TAGGCGTAGCCCTCCGAGGA-CGG;

[0039] gRNA-A2 (matching reverse strand of gene):

[0040] TGAAGCTAAGGCACGATTTC-AGG;

[0041] Using microinjection technology, inject the synthesized gRNA and Cas9 protein or the constructed plasmid vector into the cytoplasm of the fertilized egg.

[0042] The injected fertilized eggs will be transplanted into the body of foster mother mice and wait for them to develop and mature. After about 19-21 days of gestation, the foster mother mice after transplantation will give birth to F0 generation mice.

[0043] The F0 generation of mice with successful knockout of the Tmed1 gene was screened by PCR and sequencing techniques. These mice were genotyped to confirm the knockout status of the Tmed1 gene.

[0044] Positive F0 generation mice were mated with wild-type C57BL / 6J mice to obtain F1 generation heterozygous mice. The F1 generation mice were genotyped to screen for heterozygotes of the Tmed1 gene. The heterozygotes were retained and bred for further experimental studies.

[0045] Mouse identification information:

[0046] Primers1: (Annealing Temperature 60.0℃)

[0047] F1: 5’-TCTGAGCCATTCAAAGCCCAAAAT-3’;

[0048] R1: 5’-CATAATTTATTGACTCCAGTGCCCA-3’.

[0049] Obtained product (Product size): 735bp.

[0050] Primers2: (Annealing Temperature 60.0℃)

[0051] F2: 5’-TGGCAGGAATCCATAGAGACCAT-3’;

[0052] R1: 5’-CATAATTTATTGACTCCAGTGCCCA-3’.

[0053] Obtained product (Product size): 830bp.

[0054] Homozygotes: one band with 735bp.

[0055] Heterozygotes: two bands with 735bp and 830bp.

[0056] Wildtype allele: one band with 830bp.

[0057] Example 2 Application in the study of visceral heterotopia and congenital heart disease

[0058] 1. Phenotypic observation and anatomical analysis

[0059] Situs Inversus study: Tmed1 knockout mice were observed in vitro and dissected internally to check whether there was visceral heterotopia. The positions of the heart, liver, spleen, gastrointestinal tract, lungs and other organs were observed through dissection to determine whether there was left-right reversal or abnormal position. HO refers to homozygous Tmed1 knockout mice (1 and 2 are mouse numbers); HE refers to heterozygous Tmed1 knockout mice (1 and 2 are mouse numbers); WT refers to wild-type controls of Tmed1 knockout mice (1 and 2 are mouse numbers).

[0060] The dissection showed that the heart of Tmed1 wild-type mice was located towards the left side of the mouse body; Tmed1 HO mice were smaller in size, with their hearts located towards the midline, and their lungs were also smaller due to the space occupied by the heart.

[0061] Through observation: Tmed1 knockout mice have smaller hearts than WT mice, their left ventricles are larger than WT mice, and their right ventricles are twisted to the right rear of the left ventricle. Figure 2 shown.

[0062] 2. Echocardiography and imaging examinations

[0063] Using echocardiography technology dedicated to small animals, the cardiac function of Tmed1 knockout mice was evaluated in vivo. Ultrasound images can be used to observe parameters such as cardiac structure, valve function, and blood flow velocity in real time, helping to identify cardiac developmental abnormalities. Figure 3 shown.

[0064] The results of mouse echocardiography showed that:

[0065] (1) The long axis of the heart in Tmed1 HO mice is shorter than that in WT mice;

[0066] (2) The left ventricle of Tmed1 HO mice is enlarged, and the right ventricle twists behind the left ventricle;

[0067] (3) Parasternal long axis section: the long axis of the left ventricle of homozygous mice is shorter than that of HE mice; parasternal short axis section: the inner diameter of the left ventricle of homozygous mice is larger than that of HE mice; apical four-chamber section: the left ventricle of homozygous mice appears larger than that of HE mice in this section; parasternal right ventricular long axis section: the right ventricle of Tmed1 HO mice appears larger than that of heterozygous mice in this section; aortic arch section: the ascending aorta and descending aorta of HE mice are completely visible on one section, while the ascending aorta and descending aorta of homozygous mice are not on the same section.

[0068] 3. Molecular Biology Analysis

[0069] Gene expression analysis: Detect the changes in the expression of key developmental genes and proteins in the hearts and visceral organs of mice after complete knockout of Tmed1 by qPCR, Western Blot, or immunohistochemical analysis. This can reveal the role played by the Tmed1 gene during organ development.

[0070] Through IP-MS, we screened out the gene MYH11, which has the greatest difference from the Tmed1 gene and its mutants and is related to heart development. MYH11 can be immunoprecipitated by wild-type Tmed1, while the binding between the mutant type and MYH11 is weakened.

[0071] The results of co-IP confirmed that TMED1 can bind to MYH11 and be enriched in vitro, while the binding ability of the TMED1 mutant to MYH11 is weakened. WB showed that less MYH11 was enriched in the TMED1 mutant group than in the wild-type TMED1 group, with a significant difference.

[0072] As described above, this is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claimed rights.

Claims

1. A gRNA for targeting the mouse Tmed1 gene, characterized in that, The nucleotide sequences of the gRNA are shown in SEQ ID NO:1 and SEQ ID NO:

2.

2. Use of the gRNA for targeting the mouse Tmed1 gene described in claim 1 in constructing a mouse model of congenital heart disease.

3. A method for constructing a congenital heart disease mouse model, characterized in that, The method comprises the step of gene editing of a mouse using the gRNA described in claim 1 by means of the CRISPR / Cas9 gene editing technique.

4. The method for constructing a congenital heart disease mouse model according to claim 3, characterized in that, The method comprises the following steps: (1) Prepare a mixture containing Cas9 and the gRNA for targeting the mouse Tmed1 gene; (2) Transfect the mixture into mouse fertilized eggs; (3) Transplant the transfected fertilized eggs into a female mouse to obtain F0 generation mice; (4) Screen for homozygous offspring with knockout of the Tmed1 gene based on the F0 generation mice to obtain homozygous mice with knockout of the Tmed1 gene and obtain mice with congenital heart disease.

5. The method for constructing a congenital heart disease mouse model according to claim 3, wherein, The nucleotide sequences of the gRNA are shown in SEQ ID NO:1 and SEQ ID NO:

2.

6. A mouse with congenital heart disease obtained by the method according to any one of claims 3-5.

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