CN113801893A - Construction method and application of Psme3 conditional gene knockout mouse model - Google Patents

Abstract

The invention discloses a construction method and application of a Psme3 conditional gene knockout mouse model. The construction method of the Psme3 conditional gene knockout mouse model is characterized in that the 3 rd to 5 th exons of the Psme3 gene are knocked out by using a Cas9/RNA system gene targeting technology, so that the inactivation of the whole gene is realized, and the Psme3 gene conditional knockout mouse model is obtained. The Psme3 acts as a proteasome activator, regulates the hydrolytic activity of the 20S proteasome, and regulates the degradation of various tumor-associated factors. The Psme3 gene conditional knockout mouse model established by the invention lays a foundation for further researching the biological function of the Psme3 gene, and can provide an ideal model for an action mechanism in the occurrence and development of Psme3 high-expression solid tumors.

Description

Construction method and application of Psme3 conditional gene knockout mouse model

Technical Field

The invention belongs to the field of medicines, and particularly relates to a construction method and application of a Psme3 conditional gene knockout mouse model.

Background

REG γ, also known as 11S γ, PA28 γ, PSME3 or Ki (GeneID: 10197), is a member of the 11S proteasome activator family, originally found in the serum of systemic lupus erythematosus patients and designated as Ki antigen, but its relationship to systemic lupus erythematosus patients has not been fully established to date. Later studies found that the Ki antigen, like REG α and REG β, belongs to a member of the 11S proteasome activator family, and is named REG γ because it is found after REG α and REG β.

REG γ acts as a proteasome activator, and by binding to the α subunit of the 20S core particle, opening the 20S gate, the substrate protein is transported to the β subunit protease catalytic site of 20S, degrading the substrate protein. The REG gamma-20S protein degradation system does not need the participation of ubiquitination and ATP in the process of degrading substrate protein, and is a ubiquitin-ATP independent protein degradation system. The REG gamma-20S protein degradation system regulates a series of physiological and biochemical processes by degrading some proteins having important biological functions. For example, the generation and development of tumors are regulated and controlled by degrading tumor inhibiting factors and the like; promoting coxsackie virus replication by promoting degradation of p53 to enhance infection efficiency, and regulating myocarditis and dilated cardiomyopathy; inhibiting CK1 delta-MDM 2-p53 pathway by degrading casein kinase 1 delta (CK1 delta), thereby promoting aging; inhibiting autophagy and regulating lipid metabolism of the liver by degrading sirtuin 1(SriT 1); the activity of FoxO1 is regulated by degrading protein kinase a (pka), promoting VCAM 1-induced angiogenesis. Recent research shows that REG gamma can also regulate the activity of a nuclear factor kappa B (NF kappa B) signal channel by degrading a nuclear factor kappa B inhibitor epsilon (I kappa B epsilon), thereby promoting the occurrence of inflammatory-related colon cancer, experimental enteritis and the like. In addition to the function of degrading proteins by proteasome activation, REG γ can also regulate life activities independent of proteasome activation. For example, REG γ can act on promyelocytic leukemia Protein (PML) to cause it to accumulate in large amounts in the nucleosomes, thereby regulating the number of PML nucleosomes; REG gamma has different intracellular distribution in different mitotic stages, and has certain effect on maintaining the stability of centromere and chromosome of cells; for the damage response of DNA, REG γ, which is a targeting protein of ATM protein, can aggregate at the damage site of DNA, mediate accumulation of proteasome, thereby degrading damage-associated proteins.

In recent years, scientific research work on the relationship between REG γ and cancer is being vigorously developed. However, for a long time in the past, the scientific community generally accepted that REG γ can only degrade short peptide chains and cannot degrade intracellular intact proteins, until 2006, Xiatotoi et al found that REG γ degrades the first intracellular intact protein, steroid receptor co-activator-3 (SRC-3), in a ubiquitin and ATP independent manner. SRC-3 is one of the SRC family members of the transcriptional coactivator, is a known carcinogen, and has high-level expression in various cancers, such as ovarian cancer, prostate cancer, breast cancer, gastric cancer, pancreatic cancer, and the like. Therefore, the discovery of the degradation of SRC-3 by REG γ also suggests that REG γ is closely related to the development of tumors. Subsequently, it was found that REG γ can regulate cell cycle and apoptosis by degrading cyclin-dependent kinase inhibitors such as p21, p16, p19 and p14, and the absence of these cyclin-dependent kinase inhibitors can induce the transformation of normal cells into cancer cells, thereby causing the development of cancer. Meanwhile, REG gamma can also promote ubiquitination degradation of p53 through binding with p53 and MDM2, and p53 is a tumor suppressor which is relatively deeply studied at present and has biological functions of accelerating apoptosis and inhibiting tumorigenesis. These evidences further confirm that REG γ has an important regulatory role in the development of cancer.

Since then, the research on the expression level of REG γ in tumor tissues of human and mouse found that REG γ has higher level expression in lung cancer, colon cancer, thyroid cancer and liver cancer, and these findings confirm the important relationship between REG γ and cancer, making REG γ a potential cancer label and also indicate the direction for the deep research on REG γ regulated cancer. Then, some target proteins related to the development of cancer, such as glycogen synthase kinase 3(GSK3 β), which is a serine/threonine kinase, have the functions of regulating a plurality of signal proteins and transcription factors in addition to glycogen synthesis, regulating cell differentiation, proliferation, survival and apoptosis, and have certain inhibitory effects on the development of cancer, are sequentially discovered. Recent research shows that REG gamma can promote the expression of target genes CyclinD1 and c-Myc downstream of a Wnt/beta-catenin signal channel by degrading GSK3 beta and up-regulating the expression level of beta-catenin, thereby promoting the occurrence of skin cancer. These research results all indicate that REG gamma plays a very important regulatory role in cell activities and human diseases, and is a potential target for diagnosis and treatment of diseases such as cancer, therefore, determination of REG gamma protein and its expression level will provide a certain scientific basis for diagnosis and treatment of diseases such as cancer.

However, the mechanism of involvement of Psme3 in tumor development and development is yet to be further elucidated. Therefore, the construction of a conditional knockout mouse model is crucial to the study of the role of Pmse3 in tumor formation.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a method for constructing a Pmse3 conditional gene knockout mouse model. The construction method of the invention is characterized in that after mating a flox mouse with a Pmse3 gene with a mouse expressing Cre recombinase in a tissue specificity mode, the flox region of a flox homozygous mouse and a Cre positive mouse of the offspring is knocked out, so that the Pmse3 gene is functionally deleted in a specific tissue and a cell type, and an ideal model is provided for purposefully researching the action mechanism of the Pmse3 in the occurrence and development of a specific tumor.

The invention provides a construction method of a Psme3 conditional gene knockout mouse model, which comprises the following steps:

(1) constructing a gRNA aiming at a mouse Psme3 gene by using a Cas9/RNA system gene targeting technology;

the invention designs 1 pair of single-stranded guide RNA (sg RNA) of Psme3 gene according to the sequence of Psme3 gene; respectively as follows:

sg RNA-5S:5’-AGTGGTGATGGTTAATAGCT-3’(SEQ ID No.1)；

sg RNA-3S:5’-GGTATCCAACCAGACCCTCC-3’(SEQ ID No.2)；

using CRISPR Design tool (http:// CRISPR. mit. edu /) of Massachusetts institute of technology, a pair of oligonucleotide chain sequences with a length of 20bp for target DNA was designed according to the height of Score for preparing sgRNA.

(2) Constructing a recombinant targeting vector: designing a corresponding Donor according to the sgRNA, constructing a Donor plasmid, manufacturing a Donor vector carrying a loxp fragment, and purifying the Donor vector for injection;

preparing a recombinant plasmid (Donor vector) carrying a target site homologous region and a loxP site fragment, transforming the recombinant plasmid into a DH5a competent cell, screening and identifying positive clone plasmids through ampicillin resistance and sequencing of an insert fragment, selecting correct colony clone, extracting plasmids after amplification culture and purifying, and obtaining a Donor fragment product (recombinant targeting vector) for injection.

(3) Prokaryotic injection and transplantation: supervola of mice; injecting fertilized eggs and transplanting; obtaining F0 mouse;

the Donor vector with loxp fragment was co-injected with Cas9 system into fertilized eggs, and after the Cas9 system cut the DNA strand of interest, the loxp fragment was recombined at the target site by homologous recombination.

The method comprises the following specific steps: the transcribed sgRNA, Cas9 and purified donor fragment products are mixed and adjusted in concentration, the mixture is injected into fertilized eggs of a C57BL/6 mouse microscopically by a microinjection instrument, after a Cas9 system cuts a target DNA chain, loxp fragments are recombined to a target site through homologous recombination, the fertilized eggs are transplanted into the uterus of a C57BL/6 pseudopregnant mother mouse, the mouse is marked by a toe shearing method 5-7 days after the birth of the F0 mouse, sheared rat tail tissues are extracted by a phenol chloroform method to obtain DNA, primers designed in a target region are identified, and a PCR positive sample is selected for sequencing.

(4) Identifying and breeding F0 mouse: positive mice were verified by PCR and sequencing; then backcrossing the positive F0 mouse and the C57BL/6J background mouse to obtain an F1 mouse;

the F0 generation mice with correct PCR and sequencing are mated with wild C57BL/6 mice to generate F1 generation mice, and the F1 generation mice are identified according to the identification method of the F0 generation mice to obtain positive F1 generation heterozygote mice.

(5) Identification of F1 mouse generation: positive mice were verified by PCR and sequencing; then, carrying out brother and sister matching and propagation on the heterozygote mice of the positive F1 generation to obtain mice of the F2 generation, then carrying out PCR screening, firstly identifying by using a primer pair No.1, screening the mice of the positive F2 generation, and then identifying by using primer pairs No. 5, 7 and 8 to distinguish heterozygote mice;

wherein, the primer No.1 is a primary screening primer, and can judge a heterozygosis mouse. The No. 7 primer amplifies the end 5, the No.8 primer amplifies the end 3, and the No. 5 primer judges whether two loxps are in the same allele.

Wherein, when PCR identification is carried out, the primer sequences are respectively as follows:

primer pair No. 1:

Psme3-loxPtF1：5’-AATTTCAAGGTGAGGGCGAGACAG-3’(SEQ ID No.3)，

Psme3-loxPtR1：5’-AATAAACGTGGGACAGTCCCTCACT-3’(SEQ ID No.4)，

the target fragment is Fl: 307bp Wt: 216 bp;

the touch down PCR reaction conditions are as follows: 95 ℃ for 5min, 98 ℃ for 30s, 65 ℃ for 30s (-0.5 ℃), 72 ℃ for 45s (98 ℃ -72 ℃, 20 cycles), 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s (95 ℃ -72 ℃, 20 cycles), 72 ℃ for 5min, 10 ℃ hold.

Wherein, when PCR identification is carried out, the 5 primer pair sequences of the 3' allele are respectively as follows:

PNF：5’-ATCCGGGGGTACCGCGTCGAG-3’(SEQ ID No.5)，

Psme3-D-3in-tR1：5’-ACCACCTCAGATCACAA-3’(SEQ ID No.6)，

the fragment of interest was Flox: 3582bp Wt: none;

the touch down PCR reaction conditions are as follows: 95 ℃ for 5min, 98 ℃ for 30s, 65 ℃ for 30s (-0.5 ℃), 72 ℃ for 45s (98 ℃ -72 ℃, 20 cycles), 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s (95 ℃ -72 ℃, 20 cycles), 72 ℃ for 5min, 10 ℃ hold.

Wherein, when PCR identification is carried out, the sequence of the primer pair No. 7 of the 5' arm is respectively as follows:

Psme3-D-5in-tF2：5’-ACTGAGTATCAGAGCGGCTGCGC-3’(SEQ ID No.7)，

Common En2-R：5’-CCAACTGACCTTGGGCAAGAACAT-3’(SEQ ID No.8)，

the fragment of interest was Flox: 1204bp Wt: none;

the touch down PCR reaction conditions are as follows: 95 ℃ for 5min, 98 ℃ for 30s, 65 ℃ for 30s (-0.5 ℃), 72 ℃ for 45s (98 ℃ -72 ℃, 20 cycles), 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s (95 ℃ -72 ℃, 20 cycles), 72 ℃ for 5min, 10 ℃ hold.

Wherein, when PCR identification is carried out, the primer sequences No.8 of the 3' arm are respectively as follows:

ZMK2F4：5’-GCATCGCATTGTCTGAGTAGGTG-3’(SEQ ID No.9)，

Psme3-D-3in-tR2：5’-GTAGGGAGGCTAGTCT-3’(SEQ ID No.10)，

the fragment of interest was Flox: 1138 bpWt: none;

the touch down PCR reaction conditions are as follows: 95 ℃ for 5min, 98 ℃ for 30s, 65 ℃ for 30s (-0.5 ℃), 72 ℃ for 45s (98 ℃ -72 ℃, 20 cycles), 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s (95 ℃ -72 ℃, 20 cycles), 72 ℃ for 5min, 10 ℃ hold.

(6) And (3) carrying out brother and sister matching after the heterozygote of the F2 generation, and identifying by using the primer pair No.1 in the step (5) to obtain an F3 generation homozygote mouse, namely a mouse model with Psme3 Cas9-CKO conditional gene knockout.

The method also comprises a step (7) after the step (6): female and male homozygous mice of generation F3 were crossed, and homozygous mouse models with Psme3 Cas9-CKO conditional gene knockout were obtained from the offspring.

The invention also provides a Psme3 conditional gene knockout mouse model obtained by the construction method.

The invention also provides application of the Psme3 conditional gene knockout mouse model in mechanism of action research, functional mechanism and regulation and control mode in specific tumor occurrence and development.

The invention also provides application of the Psme3 conditional gene knockout mouse model in preparation or screening of antitumor drugs.

Wherein the tumor comprises lung cancer, intestinal cancer, skin cancer, and osteosarcoma.

The invention also provides sgRNA shown in SEQ ID No. 1-2.

sg RNA-5S:5’-AGTGGTGATGGTTAATAGCT-3’(SEQ ID No.1)；

sg RNA-3S:5’-GGTATCCAACCAGACCCTCC-3’(SEQ ID No.2)。

The invention also provides application of the sgRNA in construction of a Psme3 conditional gene knockout mouse model.

The invention also provides a primer pair as described above.

Primer pair No. 1:

Psme3-loxPtF1：5’-AATTTCAAGGTGAGGGCGAGACAG-3’(SEQ ID No.3)，

Psme3-loxPtR1：5’-AATAAACGTGGGACAGTCCCTCACT-3’(SEQ ID No.4)；

primer set No. 5:

PNF：5’-ATCCGGGGGTACCGCGTCGAG-3’(SEQ ID No.5)，

Psme3-D-3in-tR1：5’-ACCACCTCAGATCACAA-3’(SEQ ID No.6)；

primer set No. 7:

Psme3-D-5in-tF2：5’-ACTGAGTATCAGAGCGGCTGCGC-3’(SEQ ID No.7)，

Common En2-R：5’-CCAACTGACCTTGGGCAAGAACAT-3’(SEQ ID No.8)；

primer set No. 8:

ZMK2F4：5’-GCATCGCATTGTCTGAGTAGGTG-3’(SEQ ID No.9)，

Psme3-D-3in-tR2：5’-GTAGGGAGGCTAGTCT-3’(SEQ ID No.10)。

the invention also provides application of the primer pair in construction of a Psme3 conditional gene knockout mouse model.

The invention also provides a mouse Psme3 conditional gene knockout donor plasmid obtained by the method.

The invention also provides application of the donor plasmid for conditional gene knockout of the mouse Psme3 in construction of a Psme3 conditional gene knockout mouse model.

The Psme3 conditional gene knockout mouse model established by the invention lays a foundation for further researching the biological function of the Psme3 gene, and can provide an ideal model for the action mechanism in the occurrence and development of Psme3 high-expression solid tumors. Changes in other mechanisms due to systemic knockout are reduced, and an ideal model is provided for the research of specificity aiming at the psme 3.

Drawings

Fig. 1 is a sequence diagram of sgRNA of the present invention.

FIG. 2 is a map of the targeting vector of the present invention.

FIG. 3 is a sequence diagram of the targeting vector of the present invention, wherein exons 3-5 are marked by solid line boxes, and loxp sites are marked by dashed line boxes.

FIG. 4 is a schematic diagram of the PCR strategy for screening mice heterozygous for the positive F1 generation according to the present invention.

FIG. 5 is a schematic diagram of the position of the primer of the present invention.

FIG. 6 is a PCR identification electrophoresis diagram of a positive F1 generation heterozygote mouse, wherein a is a primer identification result No. 1h, B is a primer identification result No.1, c is a primer identification result No. 5, d is a primer identification result No. 7, and e is a primer identification result No.8 (note: B6 is a negative control, which is a negative background rat tail genomic DNA, N is a blank control, no template control; P is a positive rat tail control; and a Trans2k plus band is 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100bp, M band is 2000bp \1000bp \750bp \500bp \250bp \100bp, and Fl/Wt is 56#58#60#62#145 #).

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1

1. sgRNA was constructed. Constructing a gRNA aiming at a mouse Psme3 gene by using a Cas9/RNA system gene targeting technology, and guiding a Cas9 protein to cut a DNA double strand at a specific site of a crRNA guide sequence target; and testing sgRNA cleavage efficiency in vitro.

2. Designing corresponding Donor according to the sgRNA, constructing Donor plasmid, and making the Donor vector carrying loxp fragment.

3. Prokaryotic injection and transplantation: supervola of mice; injecting fertilized eggs and transplanting; f0 mouse generations were obtained.

Identification and breeding of F0 mouse generation: positive mice were verified by PCR and sequencing; positive F0 mice and C57BL/6J mice were backcrossed to give F1 mice.

Identification of F1 mouse generation: positive mice were verified by PCR and sequencing.

Genotyping of F2 mouse:

(1) obtaining a mouse genome: adding 80 μ L of extract A (2M NaOH,0.5M EDTA) into a centrifuge tube containing mouse tail, and heating the centrifuge tube in a metal bath at 100 deg.C for 30-40 min. Add 80. mu.L of extract B (1M Tris pH8.0) to the heated tube, mix by inversion, and centrifuge at 12000rpm for 1 min.

(2) PCR amplification of the target gene: the target gene primers are synthesized by Shanghai platane biotechnology company, the concentration of the target gene primers is 10 mu M, and the sequences are as follows:

primer pair No.1 of preliminary screening:

Psme3-loxPtF1：5’-AATTTCAAGGTGAGGGCGAGACAG-3’(SEQ ID No.3)

Psme3-loxPtR1：5’-AATAAACGTGGGACAGTCCCTCACT-3’(SEQ ID No.4)；

primer pair No. 5 for 3' allele:

PNF：5’-ATCCGGGGGTACCGCGTCGAG-3’(SEQ ID No.5)

Psme3-D-3in-tR1：5’-ACCACCTCAGATCACAA-3’(SEQ ID No.6)；

primer pair No. 7 of 5' arm:

Psme3-D-5in-tF2：5’-ACTGAGTATCAGAGCGGCTGCGC-3’(SEQ ID No.7)

Common En2-R：5’-CCAACTGACCTTGGGCAAGAACAT-3’(SEQ ID No.8)；

primer pair No.8 of 3' arm:

ZMK2F4：5’-GCATCGCATTGTCTGAGTAGGTG-3’(SEQ ID No.9)

Psme3-D-3in-tR2：5’-GTAGGGAGGCTAGTCT-3’(SEQ ID No.10)；

the PCR reaction system for genotype identification is shown in Table 1:

TABLE 1

The touch down PCR program for genotype identification is shown in Table 2:

TABLE 2

Agarose gel (2%) electrophoresis analysis of genotype: the agarose was poured into 0.5 XTBE buffer and boiled with microwave heating. And after the agarose is completely dissolved, taking out the agarose, cooling the agarose to 50-60 ℃ at room temperature, adding a proper amount of ethidium bromide solution, uniformly mixing, fixing comb teeth in a clamping groove of a gel making mold, and pouring gel solution into the gel making mold to solidify the gel at room temperature. The prepared agarose gel is put into an electrophoresis tank containing 0.5 XTBE buffer solution and is electrophoresed for 30min at a constant voltage of 130V. After electrophoresis, the gel is placed into an imager for photographing and analysis. The target fragment of the F1 generation positive mouse identified by the primary screening primer No.1 is Fl: 307bp Wt: 216 bp; the fragment of interest of F1 generation positive mice identified with 3' allele primer No. 5 was Flox: 3582bp Wt: none; the fragment of interest of the F1 generation positive mouse identified with the 5' arm primer No. 7 was Flox: 1204bp Wt: none; the target fragment of the F1 generation positive mouse identified by the 3' arm primer No.8 is Flox: 1138bp Wt: none. Taking F1 generation positive mice to self-breed to generate a progeny F2 generation, then carrying out brother and sister matching, carrying out rechecking by using No.1, 5, 7 and 8 primer pairs and identifying pure heterozygosity.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

SEQUENCE LISTING

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<170> PatentIn version 3.3

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Claims (15)

1. A method for constructing a Psme3 conditional gene knockout mouse model is characterized by comprising the following steps:

(1) constructing a gRNA aiming at a mouse Psme3 gene by using a Cas9/RNA system gene targeting technology;

(2) constructing a recombinant targeting vector: designing a corresponding Donor according to the sgRNA, constructing a Donor plasmid, making a Donor plasmid carrying a loxp fragment, and purifying the Donor plasmid for injection;

(3) prokaryotic injection and transplantation: supervola of mice; injecting fertilized eggs and transplanting; obtaining F0 mouse;

(4) identifying and breeding F0 mouse: positive F0 mice were screened by PCR and sequencing validation; backcrossing positive F0 mice with C57 BL/6J;

(5) identification of F1 mouse generation: positive mice are verified and screened by PCR and sequencing; then carrying out intercross propagation on the F1 generation mouse and a C57BL/6J background mouse to obtain an F2 generation, firstly identifying by using a primer pair No.1, screening a positive F2 generation mouse, and then carrying out PCR identification by using primer pairs No. 5, 7 and 8;

(6) and (3) carrying out brother and sister matching after heterozygotes of the F2 generation, and carrying out PCR identification by using the primer pair No.1 in the step (5) to obtain homozygote mice of the F3 generation, namely the mice model with Psme3 Cas9-CKO conditional gene knockout.

2. The method for constructing a Psme3 conditional gene knockout mouse model according to claim 1, wherein in step (1), a single-stranded guide RNA of 1 to Psme3 gene is designed according to the sequence of Psme3 gene;

sg RNA-5S:5’-AGTGGTGATGGTTAATAGCT-3’(SEQ ID No.1)；

sg RNA-3S:5’-GGTATCCAACCAGACCCTCC-3’(SEQ ID No.2)。

3. the method for constructing the Psme3 conditional gene knockout mouse model according to claim 1, wherein in the step (2), the recombinant targeting vector is constructed by the following steps:

preparing a recombinant plasmid Donor vector carrying a target site homologous region and a loxP site fragment, transforming the recombinant plasmid into a DH5a competent cell, screening and identifying positive clone plasmids through ampicillin resistance and sequencing of an insert fragment, selecting correct colony clone, extracting plasmids after amplification culture and purifying, and using the obtained Donor fragment product for injection.

4. The method for constructing a Pmse3 conditional gene knockout mouse model according to claim 1, wherein in step (3), the Donor vector with the loxp fragment is co-injected with the Cas9 system into a fertilized egg, and after the Cas9 system cuts the DNA strand of interest, the loxp fragment is recombined onto the target site by homologous recombination.

5. The method for constructing the Psme3 conditional gene knockout mouse model according to claim 1, wherein in the step (5), the sequences of the primer pair No.1 are respectively:

Psme3-loxPtF1：5’-AATTTCAAGGTGAGGGCGAGACAG-3’(SEQ ID No.3)，

Psme3-loxPtR1：5’-AATAAACGTGGGACAGTCCCTCACT-3’(SEQ ID No.4)，

the target fragment is Fl: 307bp Wt: 216 bp;

the touch down PCR reaction conditions are as follows: 95 ℃ for 5min, 98 ℃ for 30s, 65 ℃ for 30s (-0.5 ℃), 72 ℃ for 45s (98 ℃ -72 ℃, 20 cycles), 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s (95 ℃ -72 ℃, 20 cycles), 72 ℃ for 5min, 10 ℃ hold.

6. The method of constructing a Psme3 conditional gene knockout mouse model according to claim 1,

in the step (5), when PCR identification is carried out, the sequences of primer pair No. 5 of 3' allele are respectively as follows:

PNF：5’-ATCCGGGGGTACCGCGTCGAG-3’(SEQ ID No.5)，

Psme3-D-3in-tR1：5’-ACCACCTCAGATCACAA-3’(SEQ ID No.6)，

the fragment of interest was Flox: 3582bp Wt: the non(s) are (are),

the touch down PCR reaction conditions are as follows: 95 ℃ for 5min, 98 ℃ for 30s, 65 ℃ for 30s (-0.5 ℃), 72 ℃ for 45s (98 ℃ -72 ℃, 20 cycles), 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s (95 ℃ -72 ℃, 20 cycles), 72 ℃ for 5min, 10 ℃ hold.

7. The method of constructing a Psme3 conditional gene knockout mouse model according to claim 1,

in the step (5), during PCR identification, the primer pair No. 7 sequences of the 5' arm are respectively as follows:

Psme3-D-5in-tF2：5’-ACTGAGTATCAGAGCGGCTGCGC-3’(SEQ ID No.7)，

Common En2-R：5’-CCAACTGACCTTGGGCAAGAACAT-3’(SEQ ID No.8)，

the fragment of interest was Flox: 1204bp Wt: the non(s) are (are),

the touch down PCR reaction conditions are as follows: 95 ℃ for 5min, 98 ℃ for 30s, 65 ℃ for 30s (-0.5 ℃), 72 ℃ for 45s (98 ℃ -72 ℃, 20 cycles), 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s (95 ℃ -72 ℃, 20 cycles), 72 ℃ for 5min, 10 ℃ hold.

8. The method of constructing a Psme3 conditional gene knockout mouse model according to claim 6,

in the step (5), when PCR identification is carried out, the sequences of the primer pair No.8 of the 3' arm are respectively as follows:

ZMK2F4：5’-GCATCGCATTGTCTGAGTAGGTG-3’(SEQ ID No.9)，

Psme3-D-3in-tR2：5’-GTAGGGAGGCTAGTCT-3’(SEQ ID No.10)，

the fragment of interest was Flox: 1138bp Wt: the non(s) are (are),

the touch down PCR reaction conditions are as follows: 95 ℃ for 5min, 98 ℃ for 30s, 65 ℃ for 30s (-0.5 ℃), 72 ℃ for 45s (98 ℃ -72 ℃, 20 cycles), 95 ℃ for 30s, 55 ℃ for 30s, 72 ℃ for 45s (95 ℃ -72 ℃, 20 cycles), 72 ℃ for 5min, 10 ℃ hold.

9. The application of the Psme3 conditional gene knockout mouse model constructed according to the method of claim 1 in mechanism of action research, functional mechanism and regulation and control mode in specific tumorigenesis and development.

The sgRNA is characterized in that the sgRNA has a sequence shown in SEQ ID No.1 or SEQ ID No. 2:

sg RNA-5S:5’-AGTGGTGATGGTTAATAGCT-3’(SEQ ID No.1)；

sg RNA-3S:5’-GGTATCCAACCAGACCCTCC-3’(SEQ ID No.2)。

11. the use of the sgRNA of claim 10 to construct a Psme3 conditional gene knockout mouse model.

12. A primer pair, characterized by comprising the following primer pairs:

primer pair No. 1:

Psme3-loxPtF1：5’-AATTTCAAGGTGAGGGCGAGACAG-3’(SEQ ID No.3)，

Psme3-loxPtR1：5’-AATAAACGTGGGACAGTCCCTCACT-3’(SEQ ID No.4)，

primer set No. 5:

PNF：5’-ATCCGGGGGTACCGCGTCGAG-3’(SEQ ID No.5)；

Psme3-D-3in-tR1：5’-ACCACCTCAGATCACAA-3’(SEQ ID No.6)；

primer set No. 7:

Psme3-D-5in-tF2：5’-ACTGAGTATCAGAGCGGCTGCGC-3’(SEQ ID No.7)；

common En 2-R: 5'-CCAACTGACCTTGGGCAAGAACAT-3' (SEQ ID No. 8); primer set No. 8:

ZMK2F4：5’-GCATCGCATTGTCTGAGTAGGTG-3’(SEQ ID No.9)；

Psme3-D-3in-tR2：5’-GTAGGGAGGCTAGTCT-3’(SEQ ID No.10)。

13. use of the primer pair of claim 12 in the construction of a Psme3 conditional gene knockout mouse model.

14. The recombinant targeting vector is characterized in that the construction step of the recombinant targeting vector comprises the following steps:

preparing a recombinant plasmid Donor vector carrying a target site homologous region and a loxP site fragment, transforming the recombinant plasmid into a DH5a competent cell, screening and identifying positive clone plasmids through ampicillin resistance and sequencing of an insert fragment, selecting correct colony clone, extracting plasmids after amplification culture and purifying, and using the obtained Donor fragment product for injection.

15. Use of the recombinant targeting vector of claim 14 for constructing a Psme3 conditional gene knockout mouse model.

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