Method for constructing mammalian leucopenia model based on Crispr gene editing technology

Abstract

The invention discloses a method for constructing a mammalian leucopenia model based on a Crispr gene editing technology. The invention belongs to the technical field of biology, and particularly relates to a method for constructing a mammalian leucopenia model based on a Crispr gene editing technology. The construction method of the leucopenia mouse model comprises the following steps: specifically mutating an H4K31 site in a mouse fertilized egg cell, so that the site is changed into arginine from lysine, and the leucopenia mouse model is obtained. A corresponding gene sequence is mutated at an H4K31 site in a fertilized egg cell of the specifically mutated mouse by means of cutting of a Crispr gene editing system and a fixed-point knock-in technology of homologous recombination. By detecting the immune function regulation and control mechanism of a homozygous mouse with the H4K31 site after the homozygous mouse with the H4K31 site is prepared, the mouse shows a natural leucopenia phenotype after lysine at the H4K31 site is mutated into arginine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for constructing a mammalian leukopenia model based on Crispr gene editing technology. BACKGROUND

[0002] Leukopenia refers to a disease caused by unknown reasons and secondary to other diseases, and is divided into primary and secondary types. Leukopenia weakens the anti-infection ability of animals and human bodies, and is prone to be infected by pathogenic microorganisms such as bacteria and viruses.

[0003] The cause of primary leukopenia has not been completely clarified, secondary leukopenia can be secondary to systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis disease, and can also be secondary to leukopenia caused by chemotherapy, antibiotics, immunosuppressants and other drugs, and is often secondary to leukopenia caused by mycotoxins and other foodborne toxins. Leukopenia has a high incidence in humans, is a common blood disease, reduces the body's immunity, causes immune suppression of the body, and seriously affects human health and life.

[0004] The development of drugs, health foods and food or feed additives for the disease requires the use of a leukopenia model. At present, the common leukopenia modeling mainly uses chemotherapy drugs such as cyclophosphamide to construct by intraperitoneal injection or gavage to model animals, and can also be constructed by irradiating mice. Such methods are complicated, have large additional damage, are high in cost, and the effect cannot be maintained for a long time. Therefore, while the pathogenesis of leukopenia is studied, it is an urgent scientific problem to construct an effective animal model to seek new effective prevention, treatment means or treatment targets. At present, there is still a lack of a natural leukopenia animal model without drug treatment.

[0005] Mouse H4 histone is produced by H4C1 gene on chromosome 13, and H4K31 is the 32nd amino acid on histone H4, which is lysine. Long-term research has not revealed the specific switch gene that controls the number of leukocytes in the body. SUMMARY

[0006] The main problem to be solved by the present application is how to construct a leukopenia mouse model.

[0007] In order to solve the above problems, the present application provides a method for constructing a leukopenia mouse model.

[0008] The present application provides a method for constructing a leukopenia mouse model, comprising: specifically mutating H4K31 site in mouse zygote cells, so that the site is mutated from lysine to arginine, to obtain a leukopenia mouse model.

[0009] In the above method, the specific mutation of the H4K31 site in the mouse zygote cell is achieved by means of the cutting and homologous recombination of the Crispr gene editing system.

[0010] The Crispr gene editing system can be a Crispr Cas9, Crispr Cas12, or Crispr Cas13 editing system.

[0011] Further, the specific mutation of the H4K31 site in the mouse zygote cell includes the specific cutting of the sequence near the corresponding gene site of H4K31 in the mouse zygote cell by using a Cas9 protein.

[0012] Further, the sequence used for the specific knock-in can be gRNA-A1 and gRNA-A2, the nucleotide sequence of gRNA-A1 is SEQ ID No: 1, and the nucleotide sequence of gRNA-A2 is SEQ ID No: 2.

[0013] Further, the gRNA sequence used for the knock-in is synthesized by a solid-phase phosphoramidite method, and the Cas9 protein used is purchased from NEB (catalog number: M0646M).

[0014] In the above method, the Crispr gene editing system further comprises a Donor DNA sequence, and the nucleotide sequence of the vector of the Donor DNA is SEQ ID No: 3.

[0015] In a specific embodiment, the above method comprises the following steps: Step one, synthesis of the above-mentioned gRNA; Step two, construction of a Donor containing a homologous arm of the H4K31 site, and the nucleotide sequence of the Donor DNA vector is SEQ ID No: 3; Step three, microinjection of the gRNA and Cas9 protein of step 1 and the Donor DNA vector of step 2 into mammalian zygote cells or embryonic stem cells at the same time, and transplantation of the injected zygote or embryonic stem cells into the uterus of a surrogate mother to obtain F0 generation; Step four, gene sequencing analysis of the mutation region of the F0 offspring of step three by toe clipping or tail clipping to obtain heterozygous mutant male and female offspring; Step five, mating of the heterozygous mutant male and female obtained in step four, and obtaining homozygous mutant F1 generation animals by sequencing technology analysis.

[0016] In step one, the nucleotide sequence of the gRNA can be SEQ ID No: 1 and SEQ ID No: 2.

[0017] In step two, the knock-in sequence in the vector of the Donor DNA is located at positions 2502-4141 of SEQ ID No: 3, the upstream homologous arm sequence is located at positions 690-2479 of SEQ ID No: 3, the downstream homologous arm sequence is located at positions 4163-5845 of SEQ ID No: 3, the AGG mutation part in the knock-in sequence is located at positions 2940-2942 of SEQ ID No: 3, and the inserted kanamycin resistance gene sequence is located at positions 5970-7101 of SEQ ID No: 3.

[0018] In step three, the concentration of the gRNA in the microinjection buffer is 1 pmol / μL, the concentration of the Cas9 protein in the microinjection buffer is 15 pmol / μL, the concentration of the Donor DNA vector in the microinjection buffer is 15 ng / μL, and the injection dose is about 1.5 μL to the nucleus region with obvious swelling.

[0019] The application further provides the use of the method described above in the preparation of a mouse model of leukopenia.

[0020] The application further provides the use of the mouse model in the development or screening of a drug for preventing and / or treating leukopenia or in the research of a leukopenia condition.

[0021] The lysine at the H4K31 site can be modified by crotonylation and acetylation, which significantly affects the immune function of the body. By preparing homozygous mice at the H4K31 site and detecting the mechanism of regulating immune function, it is found that when the lysine at the H4K31 site is mutated to arginine, the mouse presents a natural leukopenia phenotype, indicating that the H4K31 site is an immune switch of the body, and the mouse with the H4K31 site mutated to arginine is a natural leukopenia model. After obtaining the mouse once, it can be continuously and massively bred for scientific research, drug development, functional testing of additives, etc., avoiding the use of immunosuppressive drugs, without additional harm, and the subsequent operation is simple, convenient, low in cost, and the effect can be permanently maintained, stable and reliable. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The results of determining the wild type, heterozygous type and homozygous mutant type of H4K31 in the gene sequencing of this embodiment 1 are shown in the figure. In the figure, A is the sequencing result of the wild type mouse, B is the sequencing result of the heterozygous mouse, and C is the sequencing result of the homozygous mutant mouse.

[0023] Figure 2The results of absolute value comparison of peripheral blood cells of H4K31 wild type and homozygous mutant male mice in Example 1 are shown in the following figures. In the figures, A shows the comparison results of red blood cell number of mice, B shows the comparison results of platelet number of mice, C shows the comparison results of absolute value of nucleated red blood cells of mice, D shows the comparison results of white blood cell number of mice, E shows the comparison results of absolute value of lymphocytes of mice, F shows the comparison results of absolute value of monocytes of mice, G shows the comparison results of absolute value of neutrophils of mice, H shows the comparison results of absolute value of eosinophils of mice, and I shows the comparison results of absolute value of basophils of mice.

[0024] Figure 3 The results of percentage comparison of peripheral blood cells of H4K31 wild type and homozygous mutant male mice in Example 1 are shown in the following figures. In the figures, A shows the comparison results of percentage of nucleated red blood cells of mice, B shows the comparison results of percentage of lymphocytes of mice, C shows the comparison results of percentage of monocytes of mice, D shows the comparison results of percentage of neutrophils of mice, E shows the comparison results of percentage of eosinophils of mice, and F shows the comparison results of percentage of basophils of mice. DETAILED DESCRIPTION

[0025] The application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application.

[0026] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0027] In the following examples, unless otherwise specified, the quantitative tests are set up in triplicate, and the results are averaged.

[0028] In the following examples, the mice are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the professional name is C57BL / 6JNifdc Mice, and the item number is 219.

[0029] The following examples use GraphPad Prism 9.0.0 statistical software to process data, and the experimental results are expressed as mean ± standard deviation, Student's t test is used for inspection, P<0.05 (*) indicates significant difference, P<0.01 (**) indicates extremely significant difference, P<0.001 (*** ) indicates extremely significant difference.

[0030] Example 1, construction of a mammalian leukopenia model based on Crispr gene editing technology The genomic sequence near the H4K31 DNA site of the wild-type mouse is SEQ ID No: 4 in the sequence listing.

[0031] 1. Two cutting sites near H4K31 were designed, and two primers were designed as follows: gRNA-A1 (for matching the reverse strand of the cutting gene): 5'-TCATCCTGTCGCTATTAGCTAGG-3' (SEQ ID No: 1, located at positions 3378-3400 of SEQ ID No: 4); gRNA-A2 (for matching the forward strand of the cutting gene): 5'-AAGACGCAAAAGCCCGCCGGCGG-3' (SEQ ID No: 2, located at positions 1742-1764 of SEQ ID No: 4); 2. Synthesize gRNA1 and gRNA2 to construct a Donor DNA vector gRNA1 and gRNA2 were synthesized by solid-phase phosphoramidite method, respectively.

[0032] The sequence of the Donor DNA vector is SEQ ID No: 3. In SEQ ID No: 3, the knock-in sequence is located at positions 2502-4141 of SEQ ID No: 3, the upstream homologous arm sequence is located at positions 690-2479 of SEQ ID No: 3, the downstream homologous arm sequence is located at positions 4163-5845 of SEQ ID No: 3, the AGG mutant part in the knock-in sequence is located at positions 2940-2942 of SEQ ID No: 3, and the inserted kanamycin resistance gene sequence is located at positions 5970-7101 of SEQ ID No: 3.

[0033] The structure of the recombinant vector pUC19-H4K31 is described as follows: the DNA fragment of sequence 5 from 1 to 5185 is inserted between the 5'-tgggtacggcgcgccatgcttattcaggctgcgcaactgttg-3' and 5'-gacaatcggctgctctgatgcgcatccgcggccgc-3' fragments of the starting vector pUC19 (P0368, Moli Plasmid Platform), the DNA fragment of sequence 3 from 5970 to 7101 is inserted between the 5'-atcgaattccgctagcttggctgcaggtcgtcgaaatct-3' and 5'-cttctgaggcggaaagaaccagctggggctcgactagag-3' fragments, and the other sequences of the starting vector pUC19 remain unchanged to obtain the recombinant vector.

[0034] After amplifying the mouse genomic fragment with the H4K31 gene site homologous arm using a high-fidelity DNA polymerase (R040A, Takara), the fragment (from 1 to 5185 of SEQ ID No: 5) and the kanamycin resistance gene (from 5970 to 7101 of SEQ ID No: 3) were inserted into the pUC19 vector, respectively, and transformed into DH5a E. coli. Positive colonies were obtained by screening with the kanamycin resistance marker. The positive colonies were amplified in LB medium with kanamycin, and the recombinant vector pUC19-H4K31 plasmid was extracted using a plasmid extraction kit (CW2106S, Kangwei Century).

[0035] The plasmid mutation was performed at the H4K31 site of the recombinant vector pUC19-H4K31 using a Beijing Polymerase Biological Point Mutation Kit (MF296-01), and the positive colonies were again transformed into DH5a E. coli and amplified in LB medium with kanamycin to obtain the Donor DNA vector for microinjection.

[0036] The difference between the recombinant vector pUC19-H4K31 and the Donor DNA vector is that the nucleotide at position 2941 of the recombinant vector pUC19-H4K31 is A, and the nucleotide at position 2941 of the Donor DNA vector SEQ ID No: 3 is mutated to "G".

[0037] 3. Transfection of mouse zygote cells gRNA-A1, gRNA-A2, Cas9 protein, and Donor DNA vector were injected into mouse zygotes via microinjection. The concentration of gRNA in the microinjection buffer was 1 pmol / μL, the concentration of Cas9 protein in the microinjection buffer was 15 pmol / μL, and the concentration of Donor DNA vector in the microinjection buffer was 15 ng / μL. The injection dose was approximately 1.5 μL until significant swelling was observed in the nucleus region.

[0038] 4. When multiple fertilized oocytes injected via microinjection are cultured to the morula (8-16 cells) or blastocyst (32-200 cells) stage (approximately 1-2 days), the embryos are transferred to the oviduct dilatation of a synchronized estrus female mouse (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) using glass microtubes.

[0039] 5. After the mother mice give birth, the toes or tails of the obtained F0 generation mice are clipped, and DNA is crudely extracted. PCR amplification is performed using identification primers F: 5'-GTCTTATTGGCGGGGTTTATGAC-3' and R: 5'-TAGCCGCCGAATCCGTAGAG-3' to identify the region containing H4K31, thus obtaining wild-type and heterozygous mutant mice.

[0040] Sequencing results as follows Figure 1 As shown, Figure 1 Type A is wild-type. Figure 1 B is a heterozygous type.

[0041] Compared to the wild type, the heterozygous mutant mice had the following mutations at the H4K31 DNA site on their chromosomes: Two homologous chromosomes near the H4K31 DNA site, “5'-CCACCGTAAGGTTCTCCGCGATAACATCCAGGGCATCACCAAGCCCGCCATCCGCCGCCTGGCCCGGCGCGGGGAGTGA-3'” (corresponding to positions 2146-2225 of SEQ ID No. 4), were mutated into two heterozygous chromosomes, “5'-CCACCGTAAGGTTCTCCGCGATAACATCCAGGGCATCACCAAGCCCGCCATCCGCCGCCTGGCCCGGCGCGGGGAGTGA-3'” and “5'-CCACCGTAAGGTTCTCCGCGATAACATCCAGGGCATCACCAGGCCCGCCATCCGCCGCCTGGCCCGGCGCGGGGAGTGA-3'”.

[0042] 6. After reaching sexual maturity, the heterozygous male and female mice from step 5 were mated, and the selected mice were screened according to the identification method in step 5. Figure 1 Homozygous mutant mice of the C-type.

[0043] Compared with the wild type, the homozygous mutant mice had the following mutations in the DNA site corresponding to H4K31 on their chromosomes: the two homologous chromosomes near the H4K31 DNA site “5'-CCACCGTAAGGTTCTCCGCGATAACATCCAGGGCATCACCAAGCCCGCCATCCGCCGCCTGGCCCGGCGCGGGGAGTGA-3' (corresponding to positions 2146-2225 of SEQ ID No. 4) were both mutated to “5'-CCACCGTAAGGTTCTCCGCGATAACATCCAGGGCATCACCAGGCCCGCCATCCGCCGCCTGGCCCGGCGCGGGGAGTGA-3'.

[0044] The homozygous mutant mouse was named the H4K31 mutant. The H4K31 mutant was propagated for 10 generations.

[0045] 7. Perform five-cell differential hematologic analysis on wild-type mice and homozygous mutant male mice.

[0046] Five-cell differential hematology analysis was performed using a Sysmex XN-1000V fully automated animal blood analyzer, following the instructions in the manual.

[0047] The results are as follows Figure 2 As shown: the dark-colored group represents the wild-type group, and the light-colored group represents the homozygous H4K31 mutant group. According to... Figure 2 As shown in the AI, the number of white blood cells, lymphocytes and eosinophils in homozygous mutant male mice was generally reduced, proving that the mice exhibited a leukopenia phenotype.

[0048] The results are as follows Figure 3 As shown: the dark-colored group represents the wild-type group, and the light-colored group represents the homozygous H4K31 mutant group. According to... Figure 3 According to the AF, the overall reduction in the number of white blood cells in homozygous mutant male mice did not affect the proportion of most white blood cells, while the proportion of eosinophils in homozygous mutant mice was significantly reduced, proving that the overall number of white blood cells was reduced, but the decrease in eosinophils was greater.

[0049] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A method for constructing a mouse model of leukopenia, characterized in that, include: A mouse model of leukopenia was obtained by specifically mutating the H4K31 site in mouse zygote cells, changing the lysine residue to arginine.

2. The method according to claim 1, characterized in that, The H4K31 site in the fertilized egg cells of the specifically mutated mice was mutated using the CRISPR gene editing system's cutting and homologous recombination site-directed knock-in technology to mutate the corresponding gene sequence.

3. The method according to claim 1 or 2, characterized in that, The H4K31 site in the specifically mutant mouse zygote includes a sequence near the gene site corresponding to H4K31 in the mouse zygote, specifically cleaved using the Cas9 protein.

4. The method according to claim 1 or 2, characterized in that, The gRNA sequences used for site knock-in are gRNA-A1 and gRNA-A2, wherein the nucleotide sequence of gRNA-A1 is SEQ ID No:1; and the nucleotide sequence of gRNA-A2 is SEQ ID No:

2.

5. The method according to claims 2-4, characterized in that, The gRNA sequence used for the site-directed mutagenesis was synthesized using the solid-phase phosphoramide method.

6. The method according to claims 3-5, characterized in that, The CRISPR gene editing system also includes a Donor DNA vector, the nucleotide sequence of which is SEQ ID No:

3.

7. The method according to claims 1-6, characterized in that, Includes the following steps: Step 1: Chemically synthesize the gRNA as described in claim 4; Step 2: Construct a Donor DNA vector containing the homologous arm of the H4K31 gene locus; Step 3: Simultaneously microinject the gRNA and Cas9 protein described in Step 1 and the Donor DNA vector from Step 2 into mammalian fertilized egg cells or embryonic stem cells, and then transplant the injected fertilized egg or embryonic stem cells into the uterus of a surrogate mother to obtain F0 generation. Step 4: Obtain heterozygous mutant male and female offspring by performing gene sequencing analysis on the mutant regions of the F0 offspring described in Step 3 through toe clipping or tail clipping. Step 5: Mating the heterozygous mutant males and females obtained in Step 4, and using sequencing technology to analyze and obtain homozygous mutant F1 generation animals.

8. The method according to claim 7, characterized in that, In step three, the concentration of the gRNA in the microinjection buffer is 1 pmol / μL, the concentration of the Cas9 protein in the microinjection buffer is 15 pmol / μL, the concentration of the Donor DNA vector in the microinjection buffer is 15 ng / μL, and the injection dose is about 1.5 μL until the nuclear region shows obvious expansion.

9. The application of the method according to claims 1-8 in the preparation of a mouse model of leukopenia.

10. The use of the mouse model described in claims 1-8 in the development or screening of drugs for the prevention and / or treatment of leukopenia or in research on leukopenia.

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