Mouse model with conditional knockout of vitamin d-binding protein gene, and use thereof

Abstract

A mouse model with conditional knockout of a vitamin D-binding protein gene, and the use thereof. A method comprises the following steps: S1, performing flox modification on the mouse vitamin D-binding protein gene to obtain F0-generation positive mice having undergone the flox modification; S2, mating said F0-generation positive mice with wild type mice to obtain F1-generation positive mice with preserved flox modification; S3, hybridizing the F1-generation positive mice with tissue-specific Cre tool mice to obtain heterozygous mice, and mutually mating the heterozygous mice to obtain homozygous mice; and S4, selecting the obtained homozygous mice to undergo tamoxifen injection, so as to obtain mice with conditional knockout of the vitamin D-binding protein gene.

Description

A vitamin D binding protein gene conditional knockout mouse model and its application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a vitamin D binding protein gene conditional knockout mouse model and application thereof. Background Art

[0002] Depression is a complex, multifactorial disease involving multiple factors, including genes, neurotransmitters, neural development, stress response, cognitive biases, interpersonal relationships, and social support. The etiology remains unclear, and the diagnosis of depression is still primarily based on clinical manifestations and questionnaires, lacking objective indicators such as experimental diagnosis. This study found that knocking out vitamin D-binding protein in microglia can effectively mitigate depressive-like behaviors induced by chronic stress.

[0003] Summary of the Invention

[0004] The purpose of the first aspect of the present invention is to provide a method for constructing a vitamin D binding protein gene conditional knockout mouse model.

[0005] The purpose of the second aspect of the present invention is to provide the use of the vitamin D binding protein gene conditional knockout mouse constructed by the construction method described in the first aspect of the present invention in at least one of the following.

[0006] The third aspect of the present invention aims to provide an sgRNA.

[0007] The fourth aspect of the present invention aims to provide a reagent.

[0008] The fifth aspect of the present invention aims to provide the use of vitamin D binding protein gene as a drug target in screening drugs for treating depression.

[0009] The sixth aspect of the present invention aims to provide the use of a vitamin D binding protein gene inhibitor in the preparation of a drug for treating depression.

[0010] The technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a method for constructing a vitamin D binding protein gene conditional knockout mouse model, comprising the following steps:

[0012] S1: flox-modify the mouse vitamin D binding protein gene to obtain the F0 generation of flox-modified positive mice;

[0013] S2: F0 generation positive mice were mated with wild-type mice to obtain F1 generation positive mice that retained the flox modification;

[0014] S3: F1 generation positive mice were crossed with tissue-specific Cre tool mice to obtain heterozygous mice (fl / +, Cre / +), and heterozygous mice were mated with each other to obtain homozygous mice (fl / fl, Cre / +);

[0015] S4: The homozygous mice obtained were selected and injected with tamoxifen to obtain vitamin D binding protein gene conditional knockout mice.

[0016] Preferably, in step S1, Exon2-Exon4 of the mouse vitamin D binding protein gene is flox-modified.

[0017] Preferably, in step S1, Exon2-Exon4 of the mouse vitamin D binding protein gene is flox-modified using CRISPR / Cas9 technology.

[0018] Preferably, step S1 specifically includes: introducing sgRNA1, sgRNA2, Cas9, and a targeting vector into a fertilized egg to obtain a flox-modified F0 generation positive mouse; the Cas9 protein binds to the target site under the guidance of the sgRNA, and the sgRNA target site is designed based on the non-conserved region sequence downstream of Intron1 and Intron4 of the vitamin D binding protein gene.

[0019] Preferably, the sequence of the sgRNA1 is shown in any one of SEQ ID NOs. 1 to 8;

[0020] Preferably, the sequence of the sgRNA1 is shown as SEQ ID NO.1.

[0021] Preferably, the sequence of the sgRNA2 is shown in any one of SEQ ID NOs. 9 to 16;

[0022] Preferably, the sequence of the sgRNA2 is shown in SEQ ID NO.9.

[0023] Preferably, the Cre enzyme of the tissue-specific Cre tool mouse is specifically expressed in microglia.

[0024] Preferably, the tissue-specific Cre tool mouse includes B6.129P2(Cg)-Cx3cr1tm2.1(cre / ERT2)Litt / WganJ gene mouse.

[0025] Preferably, the method further comprises the step of genotyping the F0 generation positive mice, F1 generation mice, heterozygous mice, and homozygous mice.

[0026] Preferably, the method for genotyping is PCR.

[0027] Preferably, the dosage of tamoxifen is 60-90 mg / kg / day, injected for 4-8 days.

[0028] The second aspect of the present invention provides the use of a vitamin D binding protein gene conditional knockout mouse constructed by the construction method described in the first aspect of the present invention in at least one of the following:

[0029] A1) Research on the function of vitamin D binding protein gene and / or vitamin D binding protein gene-related diseases;

[0030] A2) Study the occurrence and development of depression;

[0031] A3) Research on the diagnosis and treatment of depression.

[0032] In a third aspect of the present invention, an sgRNA is provided, wherein the sequence of the sgRNA is as shown in sgRNA1 and / or sgRNA2 of the first aspect of the present invention.

[0033] A fourth aspect of the present invention provides a reagent comprising any one of the following:

[0034] B1) the sgRNA and the Cas9 protein described in the first aspect of the present invention;

[0035] B2) biological materials related to the sgRNA described in the first aspect of the present invention and the Cas9 protein;

[0036] B3) the sgRNA described in the first aspect of the present invention and biological materials related to the Cas9 protein;

[0037] B4) biological materials related to the sgRNA and the Cas9 protein described in the first aspect of the present invention;

[0038] The sgRNA-related biological material is any one of the following:

[0039] C1) a nucleic acid molecule encoding the sgRNA;

[0040] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0041] C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0042] C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3).

[0043] The Cas9 protein-related biological material is any one of the following:

[0044] D1) a nucleic acid molecule encoding the Cas9 protein;

[0045] D2) an expression cassette containing the nucleic acid molecule described in D1);

[0046] D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0047] D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3).

[0048] Preferably, the reagents further include primers.

[0049] Preferably, the sequences of the primers are shown in SEQ ID NOs. 17 to 20.

[0050] The fifth aspect of the present invention provides an application of a vitamin D binding protein gene as a drug target in screening drugs for treating depression.

[0051] Preferably, the vitamin D binding protein is vitamin D binding protein in microglia.

[0052] The sixth aspect of the present invention provides the use of a vitamin D binding protein inhibitor in the preparation of a drug for treating depression.

[0053] Preferably, the vitamin D binding protein is vitamin D binding protein in microglia.

[0054] Preferably, the vitamin D binding protein inhibitor includes a substance that inhibits vitamin D binding protein gene expression and / or protein activity.

[0055] Preferably, the vitamin D binding protein inhibitor is a nucleic acid molecule, a protein molecule or a small molecule compound;

[0056] Preferably, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotide;

[0057] Preferably, the protein molecule is a specific antibody against vitamin D binding protein;

[0058] Preferably, the compound is a small molecule or polypeptide compound that binds to vitamin D binding protein;

[0059] Preferably, the sequence of the sgRNA is as shown in the second aspect of the present invention.

[0060] The beneficial effects of the present invention are:

[0061] The present invention constructs a knockout mouse that specifically knocks out vitamin D binding protein in microglia and finds that it can resist chronic stress-induced depressive-like behavior. This indicates that vitamin D binding protein in microglia is a key factor in the occurrence and development of depressive-like behavior. By knocking out vitamin D binding protein in microglia, a significant effect can be achieved in resisting depressive-like behavior. Vitamin D binding protein in microglia can be used as a drug target to screen drugs for treating depression. Microglia vitamin D binding protein inhibitors can be used to prepare drugs for treating depression. This opens up a new direction for in-depth research on depression and provides more options for the clinical treatment of depression, which has very important theoretical and practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 is a schematic diagram of a targeting vector.

[0063] Figure 2 is a schematic diagram of the Southern blot screening strategy.

[0064] Figure 3 is a map of the precut pCS vector.

[0065] Figure 4 shows the results of sgRNA activity detection.

[0066] FIG5 shows the detection results of the targeting vector.

[0067] Figure 6 is a map of the targeting vector.

[0068] Figure 7 shows the primer design for flox mouse genotype detection.

[0069] FIG8 shows the results of genotype identification of the F0 generation.

[0070] FIG9 shows the genotype identification results of F1 generation mice.

[0071] FIG10 shows the Southern blot results of F1 generation PCR-positive mice.

[0072] FIG11 is a mating scheme for obtaining conditional gene knockout mice.

[0073] FIG12 is a mating scheme for obtaining floxed heterozygous mice from conditional gene knockout mice.

[0074] FIG13 shows a mating scheme for obtaining floxed homozygous mice from conditional gene knockout mice.

[0075] FIG14 shows different types of neural cells screened by flow cytometry.

[0076] FIG15 shows that RNA was extracted from cells screened by flow cytometry and then RT-qPCR was performed to verify the expression of VDBP gene, proving that the knockout was successful.

[0077] FIG16 shows the results of the tail suspension test (TST) and the forced swim test (FST) for detecting the depressive-like behavior of VDBP knockout mice. DETAILED DESCRIPTION

[0078] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0079] Example 1. Design of lox model mice

[0080] 1. Information about the EGE-YMX-011-A gene (vitamin D binding protein (VDBP) gene)

[0081] The EGE-YMX-011-A gene is located on the reverse strand of chromosome 5 and is 40.4 kb in length. Gene ID: 14473.

[0082] 2. Target shooting strategy

[0083] The structure of the EGE-YMX-011-A gene was analyzed to confirm the targeting strategy. Exons 2-4 of the EGE-YMX-011-A gene were floxed. sgRNAs were designed into the non-conserved regions downstream of intron 1 and intron 4 of the EGE-YMX-011-A gene. The 5' and 3' homology arms were 1.7 kb and 0.9 kb, respectively. The EGE mouse model was generated using the CRISPR / Cas9-based EGE system developed by Biocytogen. A schematic diagram of the targeting vector is shown in Figure 1.

[0084] 3. Southern blot screening strategy

[0085] To screen for gene-targeted mice that had correctly recombined, PCR and Southern blot were used for verification. Positive F1 mice were identified using 3' and LR probes. A schematic diagram of the Southern blot screening strategy is shown in Figure 2.

[0086] The specific design is as follows:

[0087] Table 1

[0088] NcoI and AseI were used as restriction sites for Southern blot analysis. The 3' probe was used to detect correct recombination; if correct recombination occurred, two bands, wild-type and mutant, would appear. The LR probe was used to detect random insertions; if not, two bands, wild-type and mutant, would appear.

[0089] Example 2 Preparation of EGE-YMX-011-Aflox Model Mice

[0090] 1. CRISPR / sgRNA design and construction

[0091] 1.1 sgRNA design

[0092] Based on the design principles of sgRNA, 8 sgRNAs were designed in the 5' target site and 3' target site regions, and their corresponding targeting sequences are shown in the following table;

[0093] Table 2

[0094] 1.2 Construction of Cas9 / sgRNA plasmid

[0095] Oligos were synthesized according to the designed sgRNA sequence and ligated into the pCS-4G vector via the Gibson method. After transformation, the ligation product was sent for sequencing to verify its correctness.

[0096] 1.3 Vector Map

[0097] The map of the precut pCS vector is shown in Figure 3;

[0098] 2. sgRNA activity detection

[0099] A self-developed CRISPR / Cas9 activity detection method, the UCATM method, was used; it has the advantages of no species restriction, high throughput, wide adaptability, high sensitivity, and simplicity. The test results are shown in Figure 4. As shown in the figure, considering the comprehensive consideration of activity, neither high activity can easily cause toxic effects nor too low activity can lead to knockdown failure. Therefore, two sequences with activities of 50-100 were selected. Therefore, EGE-YMX-011-A-sgRNA1 and EGE-YMX-011-A-sgRNA9 were comprehensively selected for the next experiment.

[0100] 3. Construction of targeting vector

[0101] According to the targeting scheme, primers were designed to construct the targeting vector, which was then identified by enzyme digestion and sequencing, as shown in Table 3 and Figure 5 ;

[0102] Table 3

[0103] Confirm that the construction of the targeting vector is complete; the map of the targeting vector is shown in Figure 6.

[0104] 4. Microinjection of fertilized eggs

[0105] The Cas9 / sgRNA-targeting vector was microinjected into mouse fertilized eggs. The birth status of F0 generation mice after injection is shown in the following table;

[0106] Table 4

[0107] 5. Genotype identification of F0 generation flox mice

[0108] Flox mice are generated by injecting Cas9 / sgRNA into fertilized eggs. Because F0 mice obtained through fertilized egg injection may be mosaic, heterozygous, or homozygous, the genotype of F0 mice obtained by tail genotyping is for reference only and does not guarantee a heritable genetic mutation. Heritable genotypes must be confirmed after genotyping of F1 mice.

[0109] 1) Primer design for flox mouse genotype detection

[0110] The primer design principles are shown in the figure below (used to detect whether the recombination is correct) as shown in Figure 7; the primer information is shown in the following table:

[0111] Table 5

[0112] PCR reaction conditions (Touchdown) are as shown in Table 6; Enzyme: KOD-FX;

[0113] Table 6

[0114] 2. Genotype identification of F0 generation

[0115] The results are shown in Figure 8. PCR amplification and product sequencing indicated that E7X11-0027 and E7X11-0033 were F0 generation positive flox mice. E7X3-0010, E7X11-0025, and E7X11-0105 were suspected F0 generation PCR positive flox mice.

[0116] 3. Genotype and Southern blot identification of F1 generation mice

[0117] 1) Select some of the F0 generation positive mice and mate them with wild-type mice to obtain the F1 generation. The mating results are shown in the following table.

[0118] Table 7

[0119] 2) Genotype identification of F1 generation mice

[0120] The principles of primer design are the same as those for F0 generation genotype identification, and the identification results are shown in Figure 9 below;

[0121] The results showed that PCR identification showed that 1E7X11-0001, 1E7X11-0007, 1E7X11-0009, 1E7X11-0012 and 1E7X11-0033 were PCR-positive F1 generation flox mice.

[0122] 3) Southern blot detection of F1 generation PCR-positive mice

[0123] Southern blot analysis and sequencing were performed on the tail DNA of some F1 mice that were positive for PCR. The results are shown in Figure 10 . The results showed that 1E7X11-0001, 1E7X11-0007, 1E7X11-0009, 1E7X11-0012, and 1E7X11-0033 were all correctly recombined, with no random insertions.

[0124] In summary, 1E7X11-0001, 1E7X11-0007, 1E7X11-0009, 1E7X11-0012, and 1E7X11-0033 were all positive by PCR assay, and Southern blot assay showed correct recombination without random insertions. Sequencing was also correct, indicating that they were positive F1 generation flox mice.

[0125] Example 3 Mating plan for flox model mice

[0126] 1. Obtaining conditional knockout mice

[0127] The floxed heterozygous mice were mated with tissue-specific Cre mice (B6.129P2(Cg)-Cx3cr1tm2.1(cre / ER T2)Litt / WganJ) to achieve tissue-specific knockout of the target gene.

[0128] The mating scheme is shown in Figure 11;

[0129] Step 1: Mating with tissue-specific Cre mice to obtain floxed heterozygous mice (Figure 12);

[0130] The genotype detection strategy is shown in Table 8 ;

[0131] Table 8

[0132] This step can only obtain heterozygous mice (fl / +, Cre / +), and homozygous mice (experimental mice) need to be further mated to obtain; mice with genotypes of + / +, Cre / +, fl / +, + / +, + / +, and + / + can all be used as controls.

[0133] Step 2: Obtain floxed homozygous mice

[0134] The obtained heterozygous mice (fl / +, Cre / +) were mated with each other to obtain homozygous mice (fl / fl, Cre / +) ( FIG13 ).

[0135] The genotype detection strategy is shown in Table 9 ;

[0136] Table 9

[0137] Mice with the genotype fl / fl, Cre / + are the experimental group, and mice with other genotypes are the control group. The best control group is + / +, Cre / + mice.

[0138] Example 4

[0139] Mouse brain tissue was prepared into a single-cell suspension, and microglia were screened using the BD FACSLyric flow cytometry system. First, the clumps of cells were removed by gating and the single-cell population was selected. The live cell population was obtained by DAPI signal, and microglia, neurons, astrocytes, oligodendrocytes, etc. were screened (Figure 14). Then, RNA was extracted from the cells screened by flow cytometry, and RT-qPCR experiments were performed to verify the expression of the vitamin D binding protein gene. The results showed that the mice achieved specific knockdown of the vitamin D binding protein gene in microglia, and the knockdown efficiency reached more than 80% (Figure 15).

[0140] Example 5

[0141] Cre+ / -—VDBP+ / + transgenic mice were generated by crossing B6.129P2(Cg)-Cx3cr1tm2.1(cre / ERT2)Litt / WganJ transgenic mice with self-constructed VDBP loxp / loxp transgenic mice. After tamoxifen injection at 5 weeks of age, 75 mg / kg was injected for 5 days. The cre enzyme is expressed in microglia and can specifically cleave the Exon2-Exon4 fragment of the vitamin D binding protein gene, achieving gene knockdown. The mice were then subjected to 5 weeks of chronic, unpredictable mild stress, and depressive-like behavior of the mice was assessed using the classic sugar water preference test, tail suspension test, and forced swim test (Figure 16).

[0142] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. A method for constructing a vitamin D binding protein gene conditional knockout mouse model, comprising the following steps: S1: flox-modify the mouse vitamin D binding protein gene to obtain the F0 generation of flox-modified positive mice; S2: F0 generation positive mice were mated with wild-type mice to obtain F1 generation positive mice that retained the flox modification; S3: F1 generation positive mice were crossed with tissue-specific Cre tool mice to obtain heterozygous mice, and heterozygous mice were mated with each other to obtain homozygous mice; S4: The homozygous mice obtained were selected and injected with tamoxifen to obtain vitamin D binding protein gene conditional knockout mice.

2. The construction method according to claim 1, characterized in that In step S1, Exon2-Exon4 of the mouse vitamin D binding protein gene are floxed.

3. The construction method according to claim 1, characterized in that In step S1, Exon2-Exon4 of the mouse vitamin D binding protein gene is flox-modified by CRISPR / Cas9 technology; preferably, step S1 specifically includes: introducing sgRNA1, sgRNA2, and Cas9 into fertilized eggs to obtain F0 generation positive mice that have completed flox modification.

4. The construction method according to claim 3, characterized in that The sequence of the sgRNA1 is shown as any one of SEQ ID NOs. 1 to 8; preferably, the sequence of the sgRNA1 is shown as SEQ ID NO. 1; preferably, the sequence of the sgRNA2 is shown as any one of SEQ ID NOs. 9 to 16, preferably, the sequence of the sgRNA2 is shown as SEQ ID NO. 9; preferably, the Cre enzyme of the tissue-specific Cre tool mouse is specifically expressed in microglia.

5. Use of the vitamin D binding protein gene conditional knockout mouse constructed by the construction method according to any one of claims 1 to 4 in at least one of the following: A1) Research on the function of vitamin D binding protein gene and / or vitamin D binding protein gene-related diseases; A2) Study the occurrence and development of depression; A3) Research on the diagnosis and treatment of depression.

6. An sgRNA, the sequence of which is as shown in sgRNA1 and / or sgRNA2 according to claim 4.

7. A reagent comprising any one of the following: B1) the sgRNA and the Cas9 protein of claim 6; B2) biological materials related to the sgRNA of claim 6 and the Cas9 protein; B3) the sgRNA of claim 6 and biological materials associated with the Cas9 protein; B4) biological materials related to the sgRNA of claim 6 and biological materials related to the Cas9 protein; The sgRNA-related biological material is any one of the following: C1) a nucleic acid molecule encoding the sgRNA; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) a recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) a recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); The Cas9 protein-related biological material is any one of the following: D1) a nucleic acid molecule encoding the Cas9 protein; D2) an expression cassette containing the nucleic acid molecule described in D1); D3) a recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3).

8. Use of the vitamin D binding protein gene as a drug target in screening drugs for treating depression; preferably, the vitamin D binding protein is the vitamin D binding protein in microglia.

9. Use of a vitamin D binding protein inhibitor in the preparation of a drug for treating depression; preferably, the vitamin D binding protein is the vitamin D binding protein in microglia.

10. The use according to claim 9, characterized in that The vitamin D binding protein inhibitor includes a substance that inhibits vitamin D binding protein gene expression and / or protein activity; preferably, the vitamin D binding protein inhibitor is a nucleic acid molecule, a protein molecule or a small molecule compound; Preferably, the nucleic acid molecule is microRNA, siRNA, shRNA, dsRNA, sgRNA and / or antisense oligonucleotide; Preferably, the protein molecule is a specific antibody against vitamin D binding protein; Preferably, the compound is a small molecule or polypeptide compound that binds to vitamin D binding protein; Preferably, the sequence of the sgRNA is as described in claim 6.

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