A method for constructing a mouse conditional inducible Hmox1 gene knockout model and its application

Abstract

The present invention discloses a construction method and application of a mouse conditional inducible Hmox1 gene knockout model, and belongs to the field of biotechnology. The mouse conditional inducible Hmox1 gene knockout model provided by the present invention is constructed by the following steps: constructing a homologous recombination DNA vector; obtaining Cas9mRNA and gRNA for the Hmox1 gene by in vitro transcription; injecting the homologous recombination DNA vector, Cas9mRNA and gRNA into the fertilized egg of the mouse; cultivating to obtain conditional gene knockout mice; identifying the genotype by sequencing and nucleic acid gel electrophoresis; the obtained conditional knockout mice are hybridized with other tissue-specific Cre-expressing mice to obtain corresponding tissue or cell conditional knockout mice, thereby realizing precise research on specific tissues or cell types; and proving that Hmox1 inducible conditional knockout significantly aggravates acute lung injury. The present invention can overcome the difficulties of embryonic lethality of Hmox1 systemic gene knockout mice in the prior art and the inability to construct a mouse model, and assist in the study of Hmox1 in diseases.

Description

Technical Field

[0001] The invention relates to a method for constructing a mouse conditionally induced Hmox1 gene knockout model and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Gene knockout is an important genetic engineering technology. It uses homologous recombination to replace endogenous normal homologous genes with exogenous mutated genes on chromosomes, thereby inactivating endogenous genes. Researchers infer the function of specific genes by observing the phenotype of animals after the inactivation of specific endogenous genes.

[0003] Heme oxygenase-1 (HO-1) is one of the rate-limiting enzymes in the process of heme catabolism and is involved in heme catabolism and various physiological functions. The biological characteristics of HO-1 are mainly attributed to its enzymatic activity. It catabolizes heme to produce biliverdin, CO and Fe 2+ , and its synthesis is mainly regulated at the transcriptional level. Therefore, HO-1 plays an antioxidant, anti-inflammatory and anti-apoptotic role in a variety of biological processes. In recent years, HO-1 has attracted much attention in lung-related diseases. HO-1 can reduce the damage of alveolar type II epithelial cells caused by various harmful factors, promote cell proliferation and transdifferentiation into alveolar type I epithelial cells, and play a key role in the repair of lung tissue damage. HO-1 siRNA can enhance the pro-apoptotic effect of sawdust smoke extract on primary cultured alveolar type II epithelial cells and inhibit their proliferation. However, the mechanism of action of HO-1 in alveolar type II epithelial cells is still unclear. Hmox1 systemic gene knockout mice will cause embryonic lethality, so in order to more clearly study its function, it is particularly necessary to construct a Hmox1 alveolar type II epithelial cell conditional gene knockout mouse model. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an Hmox1 inducible conditional gene knockout mouse model, which can overcome the problem of embryonic lethality of Hmox1 systemic gene knockout mice in the prior art and assist in the study of the function and mechanism of Hmox1 in lung diseases.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: an Hmox1 inducible conditional gene knockout mouse model, which is constructed by the following steps: designing the FloxP position; designing the Cas9 / gRNA target; detecting the activity of the gRNA; constructing a homologous recombination template (Donor plasmid); constructing Hmox1-FloxP mice; hybridizing Hmox1-FloxP mice with mice expressing tissue-specific Cre to obtain stable genetic offspring; and detecting lung injury in a mouse acute lung injury model.

[0006] The present invention adopts CRISPR / Cas9 system to prepare Hmox1 alveolar type II epithelial cell-specific inducible gene knockout mouse animal model. The present application designs gRNA for Hmox1 gene to guide Cas9 nuclease to modify the genome at the insertion position, thereby increasing the efficiency of homologous recombination in the gene modification region, and homologously recombine the target fragment to the target site. Specifically, after Cas9 mRNA, gRNA and homologous recombination vector are mixed, they are injected into the fertilized egg of the mouse, and the Cas9 protein cuts the DNA double strand of the Hmox1 gene under the targeting action of gRNA, thereby inducing the homologous recombination repair mechanism to repair the damaged Hmox1 gene with the homologous recombination vector as a template, and integrates the LoxP sequence into a specific site in the mouse genome.

[0007] The first object of the present invention is to provide a method for constructing an Hmox1 inducible gene knockout mouse animal model, the method comprising:

[0008] (1) Design Cas9 / gRNA and Donor DNA plasmids targeting the FloxP site;

[0009] (2) Injecting the Donor DNA plasmid and Cas9 / gRNA into the fertilized eggs of wild-type mice, and then transplanting the fertilized eggs into wild-type mice, which give birth to offspring, namely F0 generation mice;

[0010] (3) Crossbreeding F0 mice with wild-type mice to obtain F1 heterozygous mice;

[0011] (4) The F1 generation heterozygotes are mated to obtain F2 generation mice, namely Hmox1-FloxP mice;

[0012] (5) Hmox1-FloxP mice were hybridized with mice expressing Cre in a tissue-specific manner to obtain stable genetic offspring.

[0013] In one embodiment, in step (1), FloxP sites are inserted at specific locations on both sides of the second exon.

[0014] In one embodiment, the amino acid sequence of the second exon is shown as SEQ ID NO.28, and the nucleotide sequence is shown as SEQ ID NO.27.

[0015] In one embodiment, in step (1), the sequence of gRNA is shown as SEQ ID NO.13 or SEQ ID NO.24 without the PAM sequence.

[0016] In one embodiment, in step (2), the Donor DNA plasmid and Cas9 / gRNA are implanted into the fertilized eggs of wild-type mice using microinjection technology.

[0017] In one embodiment, in steps (2) and (3), the strain of wild-type mice is C57BL / 6.

[0018] The second object of the present invention is to provide the use of mice constructed according to the above method in studying diseases associated with Hmox1 gene deficiency, wherein the purpose of the use is not to diagnose or treat the disease.

[0019] The third object of the present invention is to provide the use of the mouse constructed according to the above method in screening drugs for treating or preventing diseases related to the Hmox1 gene, wherein the purpose of the use is not to diagnose or treat the disease.

[0020] The fourth object of the present invention is to provide the use of the mouse constructed according to the above method in studying the functions and action mechanisms related to the Hmox1 gene.

[0021] In one embodiment, the application is to study the mechanism of the relationship between Hmox1 gene and seawater drowning-induced lung injury.

[0022] Beneficial effects of the present invention:

[0023] 1. Hmox1 alveolar type II epithelial cell-specific inducible gene knockout mice provide an animal model for studying the pathogenesis and treatment of lung diseases.

[0024] 2. There will be no phenomenon where gene knockout causes mice to die and the model cannot be constructed.

[0025] 3. In a mouse acute lung injury model, it was demonstrated that inducible conditional gene knockout of the Hmox1 gene in type II alveolar epithelial cells would aggravate lung injury, inflammation and oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the principle of conditional knockout of Hmox1 of the present invention;

[0027] Figure 2 This is a schematic diagram of the gRNA target activity detection results;

[0028] Figure 3 This is a schematic diagram of the F0 generation mouse identification strategy of the present invention;

[0029] Figure 4This is the electrophoresis diagram of homologous recombination positive F0 generation mouse PCR identification of the present invention. The arrows in Figure A indicate that 1#, 4#, and 9# are inserted into the floxp site at the 5' end. The arrows in Figure B indicate that 1#, 4#, and 9# are inserted into the floxp site at the 5' end.

[0030] Figure 5 The gene sequencing results of the homologous recombination-positive F0 generation mice of the present invention;

[0031] Figure 6 The electrophoresis diagram of PCR identification of the 5' homology arm and 3' homology arm of the F1 generation mouse of the present invention, the arrows in Figure A indicate that 9-4#, 9-5#, 9-8#, and 9-9# are inserted into the floxp site at the 5' end, and the arrows in Figure B indicate that 9-4#, 9-5#, 9-8#, and 9-9# are inserted into the floxp site at the 3' end;

[0032] Figure 7 This is the identification result of sftpc-cre in HO-1cKO mice of the present invention;

[0033] Figure 8 The present invention provides results of detecting lung injury, inflammation and oxidative stress in the mouse seawater drowning acute lung injury model, including (A) H&E staining results of mouse lung tissue sections; (B) lung tissue injury scoring results; (C) total number of cells in mouse bronchoalveolar lavage fluid; (D) protein concentration in mouse bronchoalveolar lavage fluid; (E) LDH activity in mouse serum; (F) MPO activity in mouse serum; (G) SOD activity in mouse serum; and (H) MDA content in mouse serum. DETAILED DESCRIPTION

[0034] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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.

[0035] The C57BL / 6 wild-type (WT) mice and sftpc-cre mice used in the present invention were obtained from Beijing Weishanglide Biotechnology Co., Ltd.

[0036] The present application provides an Hmox1 gene knockout mouse model and a construction method thereof. Figure 1As shown, a 5' homology arm, a flox region and a 3' homology arm were constructed for the specific region of the Hmox1 gene. The predetermined knockout region was loxp-labeled twice using the Cas / gRNA method to obtain flox mice, which were hybridized with sftpc-cre mice and combined with Cre to obtain Hmox1 gene conditional knockout mice.

[0037] Example 1 Obtaining Hmox1 gene gRNA

[0038] 1. Based on the Hmox1 genomic structure and protein function conserved regions, Hmox1 has multiple transcription products. Figure 1 As shown, Exon2 (aa9-48) is a public exon, located in the protein function conservative region: Heme domain, covering the heme binding pocket, and the number of bases in the exon2 protein coding region is 121bp, which is not a multiple of 3. After conditional deletion, the heme domain and heme binding pocket will be destroyed, and the mRNA will be re-spliced ​​to form a new mRNA, which will cause a frameshift mutation and protein inactivation. Therefore, it was decided to insert FloxP sites on both sides of exon2 to achieve conditional knockout of exon2.

[0039] The CDS sequence of Hmox1-001 is as follows (the underlined part is the conditionally deleted exon):

[0040] ATGGAGCGTCCACAGCCCGACAG CATGCCCCAGGATTTGTCTGAGGCCTTGAAGGAGGCCACCAAGGA GGTACACATCCAAGCCGAGAATGCTGAGTTCATGAAGAACTTTCAGAAGGGTCAGGTGTCCAGAGAAGGCTTTAAGCTGGTGATGGCTTCCTTGTACCATATCTACACGGCCCTGGAAGAGGAGATAGAGCGCAACAAGCAGAACCCAGTCTATGCCCCACTCTACTTCCCTGAGGAGCTGCACCGAAGGGCTGCCCTGGAGCAGGACATGGCCTTCTGGTATGGGCCTCACTGGCAGGAAATCATCCCTTGCACGCCAGCCACACAGCACTATGTAAAGCGTCTCCACGAGGTGGGGCGCACTCACCCTGAGCTGCTGGTGGCCCACGCATATACCCGCTACCTGGGTGACCTCTCAGGGGGTCAGGTCCTGAAGAAGATTGCACAGAAGGCCATGGCCTTGCCCAGCTCTGGGGAGGGCCTGGCTTTTTTTACCTTCCCGAACATCGACAGCCCCACCAAGTTCAAACAGCTCTATCGTGCTCGAATGAACACTCTGGAGATGACACCTGAGGTCAAGCACAGGGTGACAGAAGAGGCTAAGACCGCCTTCCTGCTCAACATTGAGCTGTTTGAGGAGCTGCAGGTGATGCTGACAGAGGAACACAAAGACCAGAGTCCCTCACAGATGGCGTCACTTCGTCAGAGGCCTGCTAGCCTGGTGCAAGATACTGCCCCTGCAGAGACACCCCGAGGGAAACCCCAGATCAGCACTAGCTCATCCCAGACACCGCTCCTCCAGTGGGTCCTCACTCTCAGCTTCCTGTTGGCAACAGTGGCAGTGGGAATTTATGCCATGTAA(SEQ ID NO.27);

[0041] The Hmox1 protein sequence is as follows:

[0042] MERPQPDS MPQDLSEALKEATKEVHIQAENAEFMKNFQKGQVSREGFKLVMASLYHIYTALEEEIERNKQNPVYAPLYFPEELHRRAALEQDMAFWYGPHWQEIIPCTPATQHYVKRLHEVGRTHPELLVAHAYTRYLGDLSGGQVLKKIAQKAMALPSSGEGLAFFTFP NIDSPTKFKQLYRARMNTLEMTPEVKHRVTEEAKTAFLLNIELFEELQVMLTEEHKDQSPSQMASLRQRPASLVQDTAPAETPRGKPQISTSSSQTPLLQWVLTLSFLLATVAVGIYAM(SEQ IDNO.28);

[0043] 2. Design gDNA and homologous recombination template (Donor plasmid) using CRISPR / Cas9 method for FloxP site. Design gRNA target sites at both ends of the exon to be inserted into FloxP and shear genomic DNA.

[0044] The gRNA target site is designed as follows (the underlined part is the PAM sequence):

[0045] Hmox1-L1:CTGTTTGCATGGTTAAGA CGG (SEQ ID NO.1) ；

[0046] Hmox1-L2:GACTGACATCTGTTTGCA TGG (SEQ ID NO.2) ；

[0047] Hmox1-L3:GATTCTGCCTGTGGTTTT GGG (SEQ ID NO.3) ；

[0048] Hmox1-L4:GAATAGGGATTCTGCCTG TGG (SEQ ID NO.4) ；

[0049] Hmox1-R1:AAGGTGACTTCCTTGGAC AGG (SEQ ID NO.5) ；

[0050] Reverse complement: CCTGTCCAAGGAAGTCACCTT (SEQ ID NO. 6);

[0051] Hmox1-R2:GTAATAAGGTGACTTCCT TGG (SEQ ID NO.7) ；

[0052] Publication:CCAAGGAAGTCACCTTATTAC(SEQ ID NO.8)

[0053] Hmox1-R3:CACAGTCAATGTAATA AGG (SEQ ID NO.9) ；

[0054] Publication: CCTTATTACATTGATACTGTG(SEQ ID NO.10)

[0055] Hmox1-R4:TTAACCATCCTGCTGCAC TGG (SEQ ID NO.11) ；

[0056] Publication:CCAGTGCAGCAGGATGGTTAA(SEQ ID NO.12)

[0057] Hmox1-L5:TCAGTCATTACTAT GGG (SEQ ID NO.13) ；

[0058] Publication: CCCATAGTACTTAATGACTGA(SEQ ID NO.14)

[0059] Hmox1-L6:TATTGTCCCCAAAACCAC AGG (SEQ ID NO.15) ；

[0060] Publication: CCTGTGGTTTTGGGGACAATA(SEQ ID NO.16)

[0061] Hmox1-L7:TAAAGGGGAGGCAAGGTA TGG (SEQ ID NO.17) ；

[0062] Publication: CCATACCTTGCCTCCCCTTTA(SEQ ID NO.18)

[0063] Hmox1-L8:CCAACCACTATGCTCAAA GGG (SEQ ID NO.19) ；

[0064] Reverse complement: CCCTTTGAGCATAGTGGTTGG (SEQ ID NO. 20);

[0065] Hmox1-R5:CTTATTACATTGATACTG TGG (SEQ ID NO.21) ；

[0066] Hmox1-R6:GAGAAGATGTGATGCGGG TGG (SEQ ID NO.22) ；

[0067] Hmox1-R7:AGTGCAGCAGGATGGTTA AGG (SEQ ID NO.23) ；

[0068] Hmox1-R8:GGAACATGTATTCTGAGT TGG (SEQ ID NO.24) ；

[0069] The designed Cas9 / gRNA was used to measure the target activity using Beijing Weishanglide Cas9 / gRNA Target Efficiency Detection Kit (Cat. No.: VK007). The results are as follows Figure 2 As shown: The cutting efficiency of Hmox1-L5 and Hmox1-R8 can reach more than 98%, which can be used for the subsequent construction of Hmox1 gene knockout mouse model.

[0070] Example 2 Construction of Hmox1 gene knockout mouse model

[0071] 1. Model construction: Based on the activity results of Cas9 / gRNA, targets with higher activity (Hmox1-L5 and Hmox1-R8) were selected for subsequent experiments.

[0072] Hmox1-L5:TCAGTCATTAAGTACTATGGG

[0073] Reverse complement: CCCATAGTACTTAATGACTGA

[0074] Hmox1-R8:GGAACATGTATTCTGAGTTGG

[0075] Cas9, Hmox1-L5 and Hmox1-R8 were transcribed into mRNA and RNA in vitro as follows:

[0076] After Cas9, Hmox1-L5 and Hmox1-R8 were transcribed into mRNA and RNA in vitro, they were added with the DonorDNA plasmid constructed in Example 1 and microinjected into mouse fertilized eggs to efficiently obtain gene knock-in Hmox1-FloxP mice.

[0077] 2. Identification method: After Cas9, Hmox1-L5 and Hmox1-R8 were transcribed into mRNA and RNA in vitro, the Donor DNA plasmid constructed in Example 1 was added and microinjected into the fertilized eggs of mice, and the fertilized eggs were transplanted into mice. The mice gave birth to offspring, namely F0 generation mice, and the mice were genotyped by PCR and sequencing. The Hmox1-flox / + mice obtained by F0 generation identification were used as founders, mated with wild-type C57BL / 6 mice to obtain F1 generation mice, and the mice were genotyped by PCR and sequencing.

[0078] Its identification strategy is as follows Figure 3 As shown, two pairs of PCR primers were designed based on the homologous recombination positive genome and negative genome (wild type) to identify F0 generation mice.

[0079] (1) PCR agarose gel was used to detect the floxp insertion at the 5' end using loxp1-F and loxp1-R primers, and PCR agarose gel was used to detect the floxp insertion at the 3' end using loxp2-F and loxp2-R primers. PCR sequencing was used to confirm the floxp insertion at the 5' and 3' ends using Hmox1-F and Hmox1-R primers.

[0080] 1) PCR primer information is as follows:

[0081] Table 1 Primer sequences

[0082]

[0083] 2) The reaction system is as follows:

[0084] Table 2 PCR reaction system

[0085]

[0086]

[0087] 3) The reaction conditions are as follows:

[0088] KODFXNEO (TOYOBO, KFX-201)

[0089] 94℃for 2min; 35×(98℃for 10s, 60℃for 30s, 68℃for 20s); 68℃for 10min; hold at16℃.

[0090] (2) PCR identification results of homologous recombination positive F0 generation mice:

[0091] The results of long fragment PCR electrophoresis of F0 generation mice with positive double-arm homologous recombination are as follows Figure 4 The sequencing results are shown in Figure 5 As shown, it can be seen that 1#, 4#, and 9# are mice with floxp inserted at both the 5' and 3' ends, and are Hmox1-flox / + mice.

[0092] (3) PCR identification of 5' and 3' homology arms of F1 mice

[0093] Then, 9# mice were selected as founders (Homx1-flox / + mice) and mated with wild-type C57BL / 6 mice to obtain F1 mice. The PCR identification strategy and method were the same as those of the F0 mice. The electrophoresis results of the PCR identification of the 5' and 3' homology arms of the F1 mice were shown in Figure 2. Figure 6 As shown, it can be seen that 9-4# (indicating F1 generation 4# mouse obtained by mating F0 generation 9# mouse with wild mice), 9-5#, 9-8# and 9-9# are mice with floxp inserted at both the 5' and 3' ends, and are F1 generation heterozygous (Hmox1-flox / +) mice, among which 9-4# and 9-5# are female mice, and 9-8# and 9-9# are male mice.

[0094] (4) Obtaining F2 homozygous mice

[0095] The F1 generation mice 9-4# and 9-5# obtained above were mated with 9-8# and 9-9# mice respectively, and the F2 generation mice obtained were identified by PCR and sequencing to obtain F2 generation homozygous (Homx1-flox / flox) mice.

[0096] (5) Obtaining mice with inducible conditional knockout of Hmox1 in type II alveolar epithelial cells and identifying their genotype

[0097] F2 homozygous (Homx1-flox / flox) mice were mated with sftpc-cre (the 3'-UTR region of the sftpc gene carries an IRES-cre sequence, which is specifically expressed in type II alveolar epithelial cells) tool mice to obtain F3 mice. The F3 mice were interbred to obtain F4 mice. Homx1-flox / flox was identified by the PCR method and sequencing of step (1), and the insertion of sftpc-cre was also identified. The identification results are as follows: Figure 7As shown, the upper row detects the insertion of Homx1 floxp, and the lower row detects the insertion of sftpc-cre. The results show that 1#, 2#, and 3# are sftpc-cre positive and Homx1-flox / flox positive mice, namely type II alveolar epithelial cell Hmox1 inducible conditional gene knockout mice (hereinafter referred to as HO-1cKO mice). 4#, 5#, and 6# are sftpc-cre negative mice, namely Homx1-flox / flox mice. In the homozygous mice obtained in the F4 generation, the Hmox1 gene was knocked out in the genome of type II alveolar epithelial cells after tamoxifen induction, namely HO-1cKO mice.

[0098] The method for identifying the insertion of SFTPC-Cre is as follows:

[0099] 1) PCR primer information:

[0100] Table 3 Primer sequences

[0101]

[0102] PCR product size: sftpc-cre is a band of 334 bp, while the wild type has no band.

[0103] 2) The reaction system is as follows:

[0104] Table 4 PCR reaction system

[0105]

[0106] 3) The reaction conditions are as follows:

[0107] KODFXNEO (TOYOBO, KFX-201)

[0108] 94℃for 2min; 35×(98℃for 10s, 60℃for 30s, 68℃for 45s); 68℃for 10min; 16℃for 2min.

[0109] Example 3 Verification of Hmox1-induced conditional mice in type II alveolar epithelial cells

[0110] The type II alveolar epithelial cell Hmox1 inducible conditional knockout (hereinafter referred to as HO-1cKO) mice constructed in Example 2 and the control Homx1-flox / flox (hereinafter referred to as HO-1 loxp / loxp) The mice were placed in centrifuge tubes with smooth inner walls (there were holes at both ends of the centrifuge tubes, and the mice were placed in the tubes and filled with water. This step was performed in a bucket filled with water. The water surface was 10 cm away from the upper edge of the centrifuge tubes). They were allowed to swim freely and carefully observed. When they choked on water for the first time, a stopwatch was used to time the mice until they drowned. After drowning, the mice were completely taken out of the water to establish a mouse drowning model. Signs of mouse drowning: obvious paleness of the oral and lip mucosa, rigidity of the trunk, no swimming movements of the limbs, the head could not float above the water, and then the body gradually sank to the bottom of the container. There was no resistance when being grabbed.

[0111] The results are as follows Figure 8 As shown, HO-1cKO mice and HO-1 loxp / loxp After mice were drowned in seawater, the cell count in the bronchoalveolar lavage fluid of mice increased, the protein concentration increased, the lung pathological damage was significantly aggravated, and the oxidative stress damage was aggravated. loxp / loxp Compared with HO-1cKO mice, HO-1cKO mice had aggravated lung injury, with the number of cells in bronchoalveolar lavage fluid increasing by 1.32 times, LDH activity increasing by 0.14 times, and MPO activity increasing by 0.75 times.

[0112] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for constructing an Hmox1 inducible gene knockout mouse animal model, characterized in that: The method is: (1) Designing Cas9 / gRNA and Donor DNA plasmids for the FloxP site, inserting the FloxP site at both sides of the second exon, the amino acid sequence of the second exon is shown in SEQ ID NO.28, and the sequence of the gRNA is shown in SEQ ID NO.25 or SEQ ID NO.26; (2) Implanting the Donor DNA plasmid and Cas9 / gRNA into the fertilized eggs of wild-type mice, and then transplanting the fertilized eggs into wild-type mice, which give birth to offspring, namely F0 generation mice; (3) Crossbreeding F0 mice with wild-type mice to obtain F1 heterozygous mice; (4) The F1 generation heterozygotes are mated to obtain F2 generation mice, which are Hmox1-FloxP mice; (5) Hmox1-FloxP mice were crossed with mice expressing Cre in a tissue-specific manner to obtain stable genetic offspring.

2. The method according to claim 1, characterized in that In step (2), the DonorDNA plasmid and Cas9 / gRNA are implanted into the fertilized eggs of wild-type mice using microinjection technology.

3. The method according to claim 1, characterized in that In steps (2) and (3), the wild-type mouse strain is C57BL / 6.

4. Use of the mouse constructed according to the method of any one of claims 1 to 3 in studying diseases associated with Hmox1 gene deficiency, wherein the purpose of the use is not to diagnose or treat the disease.

5. Use of the mouse constructed according to the method of any one of claims 1 to 3 in screening drugs for treating or preventing diseases related to the Hmox1 gene, wherein the purpose of the use is not to diagnose or treat the disease.

6. Use of the mouse constructed according to the method of any one of claims 1 to 3 in studying the functions and mechanisms of action of the Hmox1 gene.

7. The use according to claim 6, characterized in that: The application is to study the correlation mechanism between Hmox1 gene and seawater drowning lung injury.

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