CN110257435B

CN110257435B - Construction method and application of PROM1-KO mouse model

Info

Publication number: CN110257435B
Application number: CN201910593045.7A
Authority: CN
Inventors: 梁坚
Original assignee: Shanghai Langsheng Biotechnology Co ltd
Current assignee: Shanghai Langsheng Biotechnology Co.,Ltd.
Priority date: 2019-07-03
Filing date: 2019-07-03
Publication date: 2021-10-08
Anticipated expiration: 2039-07-03
Also published as: CN110257435A

Abstract

The invention relates to a construction method and application of a PROM1-KO mouse model, wherein the mouse model is a PROM1 gene knock-out mouse. The invention relates to a PROM1 gene knockout mouse model constructed based on CRISPR/Cas9gene knockout technology, which comprises the following steps: designing sgRNA and Cas9mRNA and transcribing the sgRNA and Cas9mRNA into mRNA in vitro, and microinjecting the active sgRNA and Cas9mRNA into mouse fertilized eggs to obtain PROM1-KO mice; and step two, identifying the PROM1-KO mouse animal model. The invention firstly constructs a PROM1-KO mouse animal model based on CRISPR/Cas9gene knockout technology, and provides a convenient, reliable and economic animal model for researching the relation between PROM1 and hereditary retinopathy, tumor and other diseases.

Description

Construction method and application of PROM1-KO mouse model

Technical Field

The invention relates to the technical field of molecular biology and biomedicine, in particular to a construction method and application of a PROM1-KO mouse model.

Background

The CRISPR/Cas system is developed from an adaptive immune system of bacteria and archaea for resisting foreign viruses or plasmids and comprises three different types, wherein the DNA endonuclease Cas9 of the type TypeII CRISPR/Cas system only has one subunit, has the simplest structure and is most widely applied. In addition to the Cas9 protein, the system also includes two short CRISPR RNAs (crRNAs) and trans-activating crRNAs (tracrRNA). The mature crRNA-tracrRNA complex can guide the Cas9 protein to a target sequence through base complementary pairing, and specifically cut a DNA double strand near PAM (proto-acredjacent motif) to form DSB (double strand break). DSB can be repaired in two ways, one is Non-Homologous recombination End joining (Non-Homologous joining NHEJ) DNA repair mode, and the other is Homologous recombination repair (Homology Directed repair HDR) mode. The NHEJ repair may result in base insertions or deletions resulting in frame shift mutations or may also result in stop codons, all of which alter the open reading frame of the gene of interest; the HDR approach requires a template fragment homologous to the cleaved fragment to repair the DSB by copying the sequence of the homologous fragment used as a template into the target gene, so that the specific gene fragment can be introduced into the target gene by using the repair method.

Stargardt-like macular dystrophy 4(STGD4) is a rare macular dystrophy characterized by central atrophy of the macula and underlying retinal pigment epithelium. Patients with STGD4 often have central vision loss characteristics that often lead to severe vision loss. The PROM1 gene encodes protamine-1, a 5-transmembrane glycoprotein also known as CD133, and is primarily associated with morphogenesis in the photoreceptor optic disc. PROM1 mutations have been identified as a major genetic factor responsible for STGD4 and other retinal degenerations such as retinitis pigmentosa.

There are two PROM1 genetically engineered mice reported in the prior art in related literature:

the first method is to construct the human WT PROM1cDNA and 1117C > T (R373C) mutant cDNA into pcDNA3.1 (-) vector, to inject the vector into mouse embryo by micro-injection, and finally to implant the embryo into the uterus of pregnant mother mouse. The method has the following defects: the PROM1 of human origin is introduced, and the human PROM1 gene and the mouse PROM1 gene have certain difference, so that the pathological expression cannot be completely expressed.

The second method comprises the following steps: the pPNT targeting vector is constructed, a 7.7kb fragment of the murine PROM1 gene is contained, a second exon is replaced by neo and is introduced into an embryo, and the embryo is finally implanted into the uterus of a pregnant female mouse. Unfortunately, this approach integrates non-mouse endogenous genomic neo fragments into the mouse genome, potentially having unpredictable effects on the endogenous genes.

Aiming at the defects, the inventor designs a construction method and application of a PROM1-KO mouse model, the PROM1 gene knockout mouse model is constructed based on CRISPR/Cas9gene knockout technology, and about 150bp fragments of an endogenous PROM1 gene are cut at a second exon shared by all transcripts of the PROM1 gene, so that the gene knockout effect is achieved. Has the advantages of high knockout rate and no introduction of exogenous gene segments. There are no reports on the present invention.

Disclosure of Invention

The first purpose of the invention is to provide a method for establishing a PROM1 gene knockout mouse model aiming at the defects in the prior art.

The second purpose of the invention is to provide an application of the method for establishing the PROM1 gene knockout mouse model.

The third purpose of the invention is to provide a cell for knocking out PROM1 gene.

The fourth purpose of the invention is to provide sgRNA for constructing a PROM1 gene knockout mouse model based on CRISPR/Cas9gene knockout technology.

A fifth object of the present invention is to provide use of the sgRNA described above.

In order to achieve the first purpose, the invention adopts the technical scheme that:

a construction method of a PROM1-KO mouse model is used for establishing the PROM1-KO mouse model based on CRISPR/Cas9gene knockout technology, and comprises the following steps:

step one, determining specific target sites sgRNA1 and sgRNA2 of a PROM1 mouse gene to be knocked out, and transcribing the specific target sites sgRNA1 and sgRNA2 and a Cas9 nuclease into mRNA in vitro;

injecting the active sgRNA and Cas9mRNA into a mouse fertilized egg to obtain a PROM1 knockout mouse; the sgRNA1 is shown in SEQ ID NO. 1, and the sgRNA2 is shown in SEQ ID NO. 2.

As a preferred embodiment of the present invention, the second step in the method is the following steps:

(1) ovulation promotion and in-vitro fertilization of a mouse, and fertilized egg cultivation;

(2) microinjecting the active sgRNA and Cas9mRNA into mouse zygotes;

(3) fertilized egg in vitro culture, receptor implantation and targeted gene modified animal culture.

As a preferred embodiment of the present invention, the method comprises the steps of:

(1) determining a target point to be knocked out of PROM1 gene, and transcribing sgRNA and Cas9 nuclease mRNA in vitro;

(2) ovulation promotion, in-vitro fertilization and fertilized egg microinjection of a mouse;

(3) transplanting the fertilized eggs survived after injection into a pseudopregnant female mouse to produce a mouse, namely an F0 generation mouse;

(4) extracting tail DNA of the F0 mouse, amplifying by PCR and sequencing the product;

(5) mating the positive mouse with a wild type heteromouse to obtain an F1 generation heterozygote mouse;

(6) and hybridizing the heterozygote mice of the F1 generation to obtain homozygote mice of the F2 generation, namely the mouse animal model.

As a preferred embodiment of the present invention, the method further comprises a third step, wherein the third step is: PROM1 knock-out mouse animal models were identified.

As a preferred embodiment of the present invention, the step three specifically is:

(1) transplanting the fertilized eggs survived after injection into a pseudopregnant female mouse to produce a mouse, namely an F0 generation mouse;

(2) extracting tail DNA of the F0 mouse, amplifying by PCR and sequencing the product;

(3) mating the positive mouse with a wild type heteromouse to obtain an F1 generation heterozygote mouse;

(4) and hybridizing the heterozygote mice of the F1 generation to obtain homozygote mice of the F2 generation, namely the mouse animal model.

In order to achieve the second object, the invention adopts the technical scheme that:

the method is applied to the research of retinal diseases and tumors.

In order to achieve the third object, the invention adopts the technical scheme that:

cells of the mouse animal model obtained as described above in which the PROM1 gene was knocked out.

In order to achieve the fourth object, the invention adopts the technical scheme that:

an sgRNA for constructing a PROM1 gene knockout mouse model based on CRISPR/Cas9gene knockout technology comprises sgRNA1-2, wherein sgRNA1 is shown as SEQ ID NO. 1, and sgRNA2 is shown as SEQ ID NO. 2.

In order to achieve the fifth object, the invention adopts the technical scheme that:

the sgRNA as described above is used to establish a gene-deficient mouse.

According to the invention, a fragment of about 150bp of an endogenous PROM1 gene is cut from a second exon shared by all transcripts of the PROM1 gene, so that the gene knockout effect is achieved.

The invention has the advantages that:

1. the invention analyzes and detects the inhibition effect of sgRNA on tumor cell proliferation and the apoptosis effect of cancer cells in a tumor cell model, and screens effective target sites.

2. The mouse animal model with the PROM1 gene knockout is established for the first time by adopting CRISPR/Cas9gene knockout technology. The experimental results show that: the sgRNA1-2 and the method designed by the invention have the advantages of high knockout efficiency and no exogenous gene interference.

3. The invention provides a convenient, reliable and economic animal model for researching the relation between PROM1 and hereditary retinopathy, tumor and other diseases, and provides a reliable theoretical basis for tumor research.

Drawings

FIG. 1 is a CRISPR/Cas9genetargeting schematic.

FIG. 2 is a schematic representation of a systemic knockout/knockout mouse breeding scheme.

FIG. 3 shows the target band obtained by PCR amplification followed by electrophoresis.

FIG. 4 is PROM1-KO mouse PROM1 protein assay, A: PROM1 protein is mainly expressed in outer segment layer of WT mouse retina photoreceptor cell, and PROM1-KO mouse retina has no PROM1 protein expression. B: WT and PROM1-KO mouse retina protein are extracted to carry out WesternBlot detection, and the result shows that PROM1-KO mouse retina does not express PROM1 protein.

Fig. 5 is a graph showing the results of the knockdown rate of sgRNA designed according to the present invention against a target gene.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

The invention relates to a method for establishing a PROM1 gene knockout mouse model based on CRISPR/Cas9gene knockout technology, wherein a fragment of about 150bp of an endogenous PROM1 gene is cut at a second exon shared by all transcripts of a PROM1 gene, so that the gene knockout effect is achieved, and a technical scheme is shown in figure 1.

Example 1 construction and identification of PROM1-KO mouse model

Materials (I) and (II)

The mice used in this example were: c57BL/6J, the surrogate mother mouse is C57BL/6J, purchased from Shanghai Slek laboratory animals Co., Ltd, and the mice were randomly divided into control groups and experimental groups.

Second, method

Experimental groups experiments were performed as follows:

1. searching for suitable target sequence in target gene intron

Through an online design tool (http:// crispr. mit. edu /) and a design principle of the gRNA, a gRNA is designed by evaluating a target site with higher score on a mouse PROM1 gene sequence, and the target site sequence is SEQ ID NO:1-SEQ ID NO: 2.

The gene sequence and sgRNA information are as follows:

sgRNA1：CTTCAGTGCCCTGCTGTTACTGG(SEQ ID NO:1)；

sgRNA2：CATTCGGCTGGACCACGTTGAGG(SEQ ID NO:2)；

the gene sequence is shown in SEQ ID NO: 3:

---ctattccgtggctgggggcctctttggcaccagccggagcttgggaggacaagccggagcatcag agccctgctgtgctggtcagatggactctaagggaaaagactgagttgaattctggccagactgtactaaaatccc ttctgcacatgttccgtcatctataaggtgggattatgccttaagagcatgtggaatgtacctggaattcagtgag tgatgaacatctgtttccaagaataaacagcttcctgaaccgtgcctggtgtttcttgatcctggaaaggactgtc ctctgaatcatcctatcttggaggaaggagcctcttcctagtccctacctgtagctccggagcaggctgttagcttgggttccacctcgttaggcctaagtgtttaattctaagccatgtcacttcctctggggtgggctcctggggaactg gttgtctctgagtaatcagtgttctttctctccctcccagGGATGGTACTTTGAGTGAATGACCACCTTGGAGACCGTTCTTCTGTTTCCCTTGTTACCAGCCAGGAGGCAGAAGAGTCCACCGGTCCAGGAAAGACCCATTTCCCTTGAGTTTCCAGAAAGTACCTCATGCTTGAGAGATCAGGCCAACAACTATGGCTCTCGTCTTCAGTGCCCTGCTGTTACTGG GGCTGTGTGGAAAGATCTCTTCAGAAGGTCAGCCTGCATTCCATAACACTCCTGGGGCTATGAATTATGAATTGCC TACCACCAAATATGAGACCCAAGATACCTTCAATGCTGGGATTGTTGGCCCTCTCTACAAAATGGTGCACATCTTC CTCAACGTGGTCCAGCCGAATGACTTCCCTCTAGgtgagtgttgaccatggggagcattgatttgccactcttggt tgcttcatgaccacgctatccagacagaaggcaatcagcactttacaaggttcagaagcactgattatacacgggc aaaaaaatatttatatagaatttagaaagatgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtctgtttaag ccaagtgagaaacaaaattgaatctttctgcctggttgcttttccattttatcttgctttgttttgcttagagcaa cacattgcttcaaattgcgtgtgcgtgtatacacacaaagtgaaaattgggataaaaaaaatgcaagttccttgtc atgaaatgagaggttgatgatcaccaggcaactgctggttatatgtcacttaagaatttaactcttctctactcac catgaaaacagctttgataccctccttagggcaagggcagacatgtctagagttacttccacaattaactgtgcca agagattcgtgtggggaagggttggtcattctcaggtagagggcattttgtggtcagggttcttgttctctgtaca caatactgctgcttctttgacgctgtctgtaattctttaacctggaagccctctggtgtgtgtgatcacgaagaca gtcctgggaagaggacagtggtccctgtgacgaatctccagttggcctgcatttctgatgaacttcagttggacct ctcctaagcctgtctttaaactacagagatttgaatactggtattggttattcagttggtggcagtccaaaagatg aatgtggaagacactgaaataagggtcgcttagatggt---(SEQ ID NO:3)

the upper underlined part of the above gene sequence represents the coding region; the upper case non-underlined section represents the non-coding region and the lower case underlined section represents the intron region.

2. Cas9mRNA preparation

Linearized and purified DNA was transcribed in vitro with Cas9 nuclease to mRNA: the sgrnas were purified to the purity of appropriate transgene injections.

3. Fertilized egg injected microscopically

And uniformly mixing the sgRNA and the Cas9mRNA according to the proportion of ((25ng/uL and 50ng/uL) by using a microinjection instrument, injecting the mixture into a cytoplasmic part of a fertilized egg of an in vitro fertilized mouse to construct and form a specific mouse embryo cell (fertilized egg), transplanting the surviving fertilized egg into an oviduct of a pseudopregnant female mouse after in vitro culture for 1-2 hours, and obtaining the mouse with the embryo transplantation as the F0 generation mouse.

4. Mouse genotype identification

(1) Extracting the genomic DNA of the offspring mice obtained in the step for genotype identification;

(2) and (3) hybridizing the mice with the knocked-out target genes with wild mice respectively.

The specific scheme is shown in figure 2.

5. Genotyping

5.1, extracting genome DNA:

A. digestion: within about one week of the birth of the mouse, 0.5cm of the toe of the mouse is cut, the toe is put into a 1.5ml EP tube, after slight centrifugation, 500ul of lysate (formulation: 100mM Tris pH8.0, 5mM EDTA ApH8.0, 0.5% SDS, NaCl1.17g/100ml) and 0.5ul of proteinase K (concentration: 20mg/ml, dissolved in pH7.4,20mM Tris and 1mM CaCl2, 50% glycerol buffer solution is stored at-20 ℃), mixed and digested overnight in a water bath at 55 ℃;

B. phenol chloroform extraction:

1) taking out the EP tube, reversing and mixing evenly, and centrifuging at 1,2000rpm for 10 min;

2) sucking 400ul of supernatant into a new EP tube, adding equal volume of phenol/chloroform, turning upside down, mixing for 3min, slightly standing, and centrifuging at 1,2000rpm for 5 min;

3) gently pipette the supernatant into a new 1.5ml EP tube (not pipette the lower phenol or precipitate), add an equal volume of chloroform to the supernatant, mix the mixture by inversion for 3min, centrifuge at 1,2000rpm for 3min after a little standing;

4) sucking the supernatant into another new 1.5ml EP tube, adding 1/10 volumes of sodium acetate solution and 2.5 times volume of absolute ethyl alcohol, shaking, and standing at-20 deg.C for about 30 min;

5) centrifuging at 1,2000rpm for 10min at 4 deg.C, removing supernatant, and placing EP tube on absorbent paper upside down to suck dry ethanol;

6) after the DNA had dried, 30ul of sterile ddH2O was added and dissolved, and the solution was concentrated and stored at-20 ℃.

The step of designing sgRNA is not carried out in a control group, a target gene is directly knocked out, and the rest steps are the same as those in an experimental group.

5.2, PCR identification:

forward and reverse PCR primers are respectively designed aiming at the upstream and downstream regions of about 200-300 bp.

5.2.1 primer information results are shown in Table 1.

TABLE 1

5.2.2 PCR reaction system see Table 2.

TABLE 2

Note: if the sequence is complex, PCR may be performed by replacing other enzymes, and 5% DMSO or the like may be added.

5.3 sequencing analysis

A. Direct sequencing of PCR products (50ul system with primers S): if a large fragment is deleted or inserted, the sequence of the target band can be directly seen through an electrophoretogram;

B. or the PCR product is reclaimed by tapping, is connected with a PMD18T vector, is coated after DH5 alpha is transformed and is directly sent to a plate for sequencing (Taq enzyme is recommended to be used in PCR);

C. the sequencing result is seen through a chromas color chart; if the single peak is present, directly comparing with the WT sequence (DNAMAN software), and analyzing to obtain a WTOR homozygote; color plots are later bimodal, and the sequence of mutant was analyzed by comparing the screenshots with the WT sequence.

Three, result in

The key of gene knockout lies in target selection, and when acting on a correct target, the function of the gene can be deleted through sequence mutation. By analyzing the structure of mouse PROM1 gene, sgRNA is designed in the second exon sequence shared by all transcripts of PROM1 gene, and sgRNA1-2 with highest knockout efficiency is selected.

3.1 Founder information

Information of 3.1.1, F0 generation

2018.1.30 injection, 2.19 raw, 2.24 cut 1-34#

17#:

ATGGCTCTCGTCTTCAGTGCCCTGCTG--------------(184)---------------------TCCCTCTAG(SEQ ID NO:6)-184bp

23#:

CTTGGAGACCGTTCTTCT-------------(306)-----------------TCCAGCCGAATGACTTCCCTCTAG(SEQ ID NO:7)-306bp(-ATG)

ATGGCTCTCGTCTTCAGTGCCCTGCT--------(166)----------GTGGTCCAGCCGAATGACTTCCCTCTAG(SEQ ID NO:8)-166bp

26#:

ATGGCTCTCGTCTTCAGTGCCCTGCT-----------(169)------GTCCAGCCGAATGACTTCCCTCTAG(SEQ ID NO:9)-169bp

The results are shown in FIG. 3.

3.2, PROM1-KO mouse PROM1 protein detection is shown in FIG. 4.

The PROM1 protein was found to be absent by retinal staining. A: PROM1 protein is mainly expressed in outer segment layer of WT mouse retina photoreceptor cell, and PROM1-KO mouse retina has no PROM1 protein expression. B: WT and PROM1-KO mouse retina protein are extracted to carry out WesternBlot detection, and the result shows that PROM1-KO mouse retina does not express PROM1 protein.

3.3 knock-out efficiency is shown in FIG. 5.

In fig. 5 is shown: the sgRNA knockout rate designed by the invention is 82%, and the knockout rate of a control group is 0.

Most genetically modified mice are constructed to study the effects of gene expression deletions, additions or target gene sequence changes on body functions; biological processes (reporter strains) can also be monitored by designing genetically modified mice to label specific cell populations (reporter staining), and previous methods for making genetically modified animal models include: the method comprises the following steps: by constructing human WTPROM1cDNA and 1117C > T (R373C) mutant cDNA into pcdna3.1 (-) vector, by microinjection into mouse embryos, and finally implanting the embryos into the uterus of pregnant mother mice, and: the second method comprises the following steps: the pPNT targeting vector is constructed, a 7.7kb fragment of the murine PROM1 gene is contained, a second exon is replaced by neo and is introduced into an embryo, and the embryo is finally implanted into the uterus of a pregnant female mouse. The two methods have defects: defect one: the PROM1 of human origin is introduced, and the human PROM1 gene and the mouse PROM1 gene have certain difference, so that the pathological expression cannot be completely expressed. And: and defect two: integration of a non-mouse endogenous genomic neo fragment into the mouse genome can have unpredictable effects on the endogenous gene.

And CRISPR/Cas9 is a new generation of gene editing technology that has recently been developed. When the PROM1 gene knockout mouse model is constructed, various factors are considered, and finally, a simpler and more efficient CRISPR/Cas9 technology is selected. The CRISPR/Cas9 technology has high targeting efficiency, convenient design and simple steps, and has profound influence on the technical development in the field of biology.

The mouse animal model with the PROM1 gene knockout is established for the first time by adopting CRISPR/Cas9gene knockout technology. The optimal sgRNA1-2 is selected, and the sgRNA1-2 and the method designed by the invention have the advantages of high knockout efficiency and no exogenous gene interference.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> first-person hospital in Shanghai City

<120> construction method of PROM1-KO mouse model and application thereof

<130> /

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 23

<212> DNA

<213> Artificial sequence

<400> 1

cttcagtgcc ctgctgttac tgg 23

<210> 2

<211> 23

<212> DNA

<213> Artificial sequence

<400> 2

cattcggctg gaccacgttg agg 23

<210> 3

<211> 1699

<212> DNA

<213> Artificial sequence

<400> 3

ctattccgtg gctgggggcc tctttggcac cagccggagc ttgggaggac aagccggagc 60

atcagagccc tgctgtgctg gtcagatgga ctctaaggga aaagactgag ttgaattctg 120

gccagactgt actaaaatcc cttctgcaca tgttccgtca tctataaggt gggattatgc 180

cttaagagca tgtggaatgt acctggaatt cagtgagtga tgaacatctg tttccaagaa 240

taaacagctt cctgaaccgt gcctggtgtt tcttgatcct ggaaaggact gtcctctgaa 300

tcatcctatc ttggaggaag gagcctcttc ctagtcccta cctgtagctc cggagcaggc 360

tgttagcttg ggttccacct cgttaggcct aagtgtttaa ttctaagcca tgtcacttcc 420

tctggggtgg gctcctgggg aactggttgt ctctgagtaa tcagtgttct ttctctccct 480

cccagggatg gtactttgag tgaatgacca ccttggagac cgttcttctg tttcccttgt 540

taccagccag gaggcagaag agtccaccgg tccaggaaag acccatttcc cttgagtttc 600

cagaaagtac ctcatgcttg agagatcagg ccaacaacta tggctctcgt cttcagtgcc 660

ctgctgttac tggggctgtg tggaaagatc tcttcagaag gtcagcctgc attccataac 720

actcctgggg ctatgaatta tgaattgcct accaccaaat atgagaccca agataccttc 780

aatgctggga ttgttggccc tctctacaaa atggtgcaca tcttcctcaa cgtggtccag 840

ccgaatgact tccctctagg tgagtgttga ccatggggag cattgatttg ccactcttgg 900

ttgcttcatg accacgctat ccagacagaa ggcaatcagc actttacaag gttcagaagc 960

actgattata cacgggcaaa aaaatattta tatagaattt agaaagatgt gtgtgtgtgt 1020

gtgtgtgtgt gtgtgtgtgt gtgtctgttt aagccaagtg agaaacaaaa ttgaatcttt 1080

ctgcctggtt gcttttccat tttatcttgc tttgttttgc ttagagcaac acattgcttc 1140

aaattgcgtg tgcgtgtata cacacaaagt gaaaattggg ataaaaaaaa tgcaagttcc 1200

ttgtcatgaa atgagaggtt gatgatcacc aggcaactgc tggttatatg tcacttaaga 1260

atttaactct tctctactca ccatgaaaac agctttgata ccctccttag ggcaagggca 1320

gacatgtcta gagttacttc cacaattaac tgtgccaaga gattcgtgtg gggaagggtt 1380

ggtcattctc aggtagaggg cattttgtgg tcagggttct tgttctctgt acacaatact 1440

gctgcttctt tgacgctgtc tgtaattctt taacctggaa gccctctggt gtgtgtgatc 1500

acgaagacag tcctgggaag aggacagtgg tccctgtgac gaatctccag ttggcctgca 1560

tttctgatga acttcagttg gacctctcct aagcctgtct ttaaactaca gagatttgaa 1620

tactggtatt ggttattcag ttggtggcag tccaaaagat gaatgtggaa gacactgaaa 1680

taagggtcgc ttagatggt 1699

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

gcttgggttc cacctcgtta 20

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence

<400> 5

tgccctaagg agggtatcaa a 21

<210> 6

<211> 36

<212> DNA

<213> Artificial sequence

<400> 6

atggctctcg tcttcagtgc cctgctgtcc ctctag 36

<210> 7

<211> 42

<212> DNA

<213> Artificial sequence

<400> 7

cttggagacc gttcttcttc cagccgaatg acttccctct ag 42

<210> 8

<211> 54

<212> DNA

<213> Artificial sequence

<400> 8

atggctctcg tcttcagtgc cctgctgtgg tccagccgaa tgacttccct ctag 54

<210> 9

<211> 51

<212> DNA

<213> Artificial sequence

<400> 9

atggctctcg tcttcagtgc cctgctgtcc agccgaatga cttccctcta g 51

Claims

1. A construction method of a PROM1-KO mouse model is used for establishing a PROM1-KO mouse model based on CRISPR/Cas9gene knockout technology, and is characterized by comprising the following steps:

step one, determining specific target sites sgRNA1 and sgRNA2 corresponding to a PROM1 mouse gene to be knocked out, and transcribing the target sites sgRNA1 and sgRNA2 with a Cas9 nuclease in vitro to form mRNA, wherein the sgRNA1 and the sgRNA2 are used for site-directed knockout of a second exon of a PROM1 gene;

injecting the active sgRNA and Cas9mRNA into a mouse fertilized egg to obtain a PROM1 knockout mouse; and the second step is the following steps:

(2) microinjecting the active sgRNA and Cas9mRNA into mouse zygotes;

(3) fertilized egg in-vitro culture, receptor implantation and targeted gene modification animal culture are carried out, so as to obtain PROM1-KO mouse model with the second exon of PROM1 gene knocked out at fixed point

And the sgRNA1 is shown in SEQ ID NO. 1, and the sgRNA2 is shown in SEQ ID NO. 2:

sgRNA1：CTTCAGTGCCCTGCTGTTACTGG(SEQ ID NO:1)；

sgRNA2：CATTCGGCTGGACCACGTTGAGG(SEQ ID NO:2)。

2. the method of constructing a PROM1-KO mouse model according to claim 1, comprising the steps of:

3. The method for constructing the PROM1-KO mouse model according to claim 1, wherein the method further comprises a third step, the third step is: PROM1 knock-out mouse animal models were identified.

4. The method according to claim 3, wherein the third step is specifically:

5. The method for constructing the PROM1-KO mouse model according to claim 2 or 4, wherein the primers used in the PCR amplification are SEQ ID Nos. 4 and 5:

SEQ ID No:4：GCTTGGGTTCCACCTCGTTA 20

SEQ ID No:5：TGCCCTAAGGAGGGTATCAAA 21。

6. an sgRNA of a PROM1 knockout mouse model is constructed based on CRISPR/Cas9gene knockout technology, and is characterized by comprising sgRNA1 and sgRNA2, wherein the sgRNA1 is shown in SEQ ID NO:1, the sgRNA2 is shown in SEQ ID NO:2, and the sgRNA1 and the sgRNA2 are used for site-directed knockout of a second exon of PROM1 gene.

7. Use of the sgRNA of claim 6 in the preparation of a reagent for establishing a gene-deficient mouse.