CN112011538B - Method for constructing Mutyh gene conditional knockout mouse model - Google Patents
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Abstract
The invention discloses a method for constructing a Mutyh gene conditional knockout mouse model, which is characterized in that a CRISPR/Cas9 technology is utilized, a Mutyh gene is subjected to flox modification by the principle of homologous recombination, exons 3 to 15 of the Mutyh gene can be knocked out after the flox mouse is mated with a specific Cre tool mouse, a gene tissue or cell specific knockout model mouse is obtained, MUTYH gene variation can become a risk factor for diseases related to energy metabolism disorder by inducing mitochondrial dysfunction, and myocardial energy metabolism disorder is an important factor for causing and promoting the generation and development of heart failure. The Mutyh gene conditional knockout mouse model has good stability, and provides an economic, simple and reliable animal model for further researching the pathogenesis and gene therapy of heart failure.
Description
Technical Field
The invention relates to a method for constructing a mouse model, in particular to a method for constructing a Mutyh gene conditional knockout mouse model.
Background
Chronic heart failure is the final outcome of the development of various cardiovascular diseases, and is clinically characterized by cardiac hypofunction and various arrhythmia. The psychological remodeling of heart disease is the most important pathophysiological basis for the progress of various cardiovascular diseases into chronic heart failure, and how to prevent the psychological remodeling of heart disease is the key and difficult point for the treatment of various cardiovascular diseases.
Mitochondria are the primary site of energy production by cardiomyocytes. Mitochondrial sustained stress, the production of oxygen free Radicals (ROS), causes oxidative damage to mitochondrial dna (mtdna) and mitochondrial proteins; ROS increase to enter a vicious circle, which can cause accumulation of abnormal mtDNA and damaged mitochondria in cells, possibly cause energy metabolism disorder of cardiac muscle cells, and promote rational remodeling of heart disease and development of heart failure; however, the exact molecular mechanism has not been fully elucidated, and thus an effective target for intervention is lacking.
MUTYH is located at 1p34.3-32.1, has a total length of about 11.2kb, contains 16 exons, and encodes A/G specific adenine DNA glycosylase. The major function of the MUTYH protein is to remove the G → T mutation possibly caused by oxidative damage by excising adenine mismatched with the oxidative damage base 8-hydroxydeoxyguanosine (8-OHdG) in the DNA chain, to repair the oxidative damage and maintain the stability of the genome. MUTYH produces primarily an alpha transcript with the full coding sequence of the gene, and a beta transcript, which translates to the type 1 peptide chain of the gene (MUTYH 1, CCDS41320.1), containing the mitochondrial localization sequences, primarily localized to the mitochondria; the beta transcript lacks the Exon1 sequence of MUTYH, the start code is within Exon2, and its translated peptide chain (MUTYH2, CCDS41322.1) lacks the mitochondrial localization sequence, and is localized to the nucleus. MUTYH1 and MUTYH2 are involved in oxidative damage repair of mtDNA and nuclear genomes, respectively, whose dysfunction would result in reduced genome stability. MUTYH variation is a risk factor for the occurrence of diseases associated with disorders of energy metabolism, but to date, the function of the expression product of the MUTYH gene and the molecular mechanism of heart failure caused by gene mutation have not been elucidated.
Therefore, the Mutyh gene conditional knockout animal model is used for researching the heart failure, and theoretical basis can be provided for the diagnosis and treatment of the human heart failure diseases.
Disclosure of Invention
The invention aims to provide a method for constructing a Mutyh gene conditional knockout mouse model, which is characterized in that a CRISPR/Cas9 technology is utilized, a Mutyh gene is subjected to flox modification by the principle of homologous recombination, exons 3 to 15 of the Mutyh gene can be knocked out after the flox mouse is mated with a specific Cre tool mouse, a gene tissue and cell specific knockout model mouse is obtained, Muthy gene variation can become a risk factor of diseases related to energy metabolism disorder by inducing mitochondrial dysfunction, myocardial energy metabolism disorder is an important factor for causing and promoting the occurrence and development of heart failure, and the Mutyh gene conditional knockout mouse model has good stability and provides an economic, simple and reliable animal model for further researching the pathogenesis and gene therapy of the heart failure.
The purpose of the invention can be realized by the following technical scheme:
a method for constructing a Mutyh gene conditional knockout mouse model is characterized in that a CRISPR/Cas9 system of a Mutyh gene designs and extracorporeally transcribes gRNA, and flox modification is carried out on the Mutyh gene to construct a homologous recombination vector (Donor vector).
Further, in the CRISPR/Cas9 system for conditional knockout of mouse Mutyh gene, Cas9 protein binds to a target site under the guidance of gRNA to cause DNA double strand break, the gRNA action site is positioned at two sides of 3-15 exon of Mutyh gene, and the DNA sequence of the gRNA action site is (SEQ NO.1)5 '-CTAAGATCAGAAATTTGGTC-3' and (SEQ NO.2)5 '-TGAGTAGCTTCCTTCAGCTT-3'.
Further, the construction of the homologous recombination vector was performed by flox modification of the Mutyh gene, and the DNA sequences of the flox modification site were (SEQ NO.3)5 '-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-3' and (SEQ NO.4)5 '-ATAACTTCGTATAATGT ATGCTATACGAAGTTAT-3'.
Furthermore, the Mutyh gene is modified by flox, and after the flox is combined with specific Cre, exons 3 to 15 of the Mutyh gene can be conditionally knocked out.
Further, the CRISPR/Cas9 system and the homologous recombination vector are applied to preparing a Mutyh gene conditional knockout cell line.
Further, the CRISPR/Cas9 system and the homologous recombination vector transfect cells, and the obtained mid-target positive cell clone is a Mutyh conditional knockout cell line.
Further, the cell line is applied in the preparation of a Mutyh conditional gene knockout mouse model.
Further, the operating step includes:
1) designing a gRNA targeting a Mutyh gene based on a CRISPR/Cas9 system; simultaneously, flox modification is carried out on the target gene to construct a homologous recombinant vector;
2) cas9, gRNA and a homologous recombination vector are simultaneously injected into a fertilized egg of a mouse;
3) transplanting the fertilized eggs into a pseudopregnant female mouse body to generate FO generation, and carrying out PCR identification on the FO generation;
4) mating the positive FO generation with wild type mice to obtain F1 generation heterozygotes;
5) breeding heterozygotes of the F1 generation with a specific Cre tool mouse to obtain mice with flox deleted regions and Cre positive, mating with wild background mice to obtain mice with flox deleted regions and Cre negative, breeding brothers and sisters after the mice are sexually mature, obtaining homozygotes with flox deleted regions and Cre negative after offspring generation, and obtaining a model mouse with gene tissues and cell specificity knocked out.
Further, the specific primers for PCR identification:
(SEQ NO.5)Mutyh—F1：5'—TAGGGATGCCCCTTGCATAGTA—3'
(SEQ NO.6) Mutyh-R1: 5 '-ACAATCTGTTCCCACTCAGGGC-3' and
(SEQ NO.7)Mutyh—F2：5'—AGGCTTGCTGTGAACCAAGGA—3'
(SEQ NO.8) Mutyh-R2: 5 '-CCAACTGACCTTGGGCAAGAACAT-3' and
(SEQ NO.9)Mutyh—F3：5'—TCTGAGGCGGAAAGAACCAG—3'
(SEQ NO.10)Mutyh—R3：5'—TCTTCCAGCCTCAATGGGCAC—3'
the size of an amplification product corresponding to the Mutyh-F1 is 204 bp; the size of an amplification product corresponding to Mutyh-R1 is 302 bp; the sizes of the amplification products corresponding to Mutyh-F2 and Mutyh-R2 are both 368 bp; the sizes of the amplification products corresponding to Mutyh-F3 and Mutyh-R3 are 338 bp.
The invention has the beneficial effects that:
the building method of the mouse model utilizes CRISPR/Cas9 technology and homologous recombination principle to perform flox modification on the Mutyh gene, the flox mouse can knock out 3 rd to 15 th exons of the Mutyh gene after mating with a specific Cre tool mouse, and becomes a risk factor for the occurrence of diseases related to energy metabolism disorder by inducing mitochondrial dysfunction after the Mutyh gene is knocked out.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 is a schematic diagram of a Mutyh gene selective knockout mouse model of the method for constructing a mouse model of the present invention;
FIG. 2 is a schematic diagram of a Mutyh knockout and control mouse genotype identification strategy in the method of constructing a mouse model of the present invention;
FIG. 3 is an electrophoretogram for identifying Mutyh knockout and control mouse genotypes by the method for constructing the mouse model of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
firstly, constructing a Mutyh gene selective knockout mouse model:
1. gRNAs were designed based on the CRISPR/Cas9 system based on the 3 rd to 15 th exon flanking sequence of the mouse Mutyh gene (knockout gene name (NCBI number): 70603) and tested for gRNA in vitro activity, as shown in FIG. 1, the gRNA sequences of the Mutyh gene are as follows:
recognition site for exon 3-sided sequence (SEQ NO. 1): 5 '-CTAAGATCAGAAATTTGGTC-3'
Recognition site for exon 15-flanking sequence (SEQ NO. 2): 5 '-TGAGTAGCTTCCTTCAGCTT-3'
2. According to the principle of homologous recombination, the Mutyh gene is subjected to flox modification in vitro, and a homologous recombination vector (Donor vector) has the following DNA sequence of a flox modification site of the Mutyh gene:
recognition site for exon 3-sided sequence (SEQ NO. 3): 5 '-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-3'
Recognition site for exon 15-flanking sequence (SEQ NO. 4): 5 '-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-3'
3. PMSG treatment C57/BL6 female mice (3 weeks old, average weight 15g), hCG injection after 46 hours, mating with male mice coop, taking fertilized eggs for microinjection the next day, after in vitro transcription of gRNA (100ng/ml) of step 1 and mRNA of Cas9 nuclease (50ng/ml), and injecting homologous recombination vector (Donor vector) into the fertilized eggs, transplanting the fertilized eggs surviving the injection into pseudopregnant female mice, and producing mice, namely F0 generation mice.
4. Extracting tail DNA of the F0 mouse, sequencing the PCR amplification product, and identifying the genotype.
5. And (3) mating the male mice 7 weeks old and the female mice 4 weeks old to the positive F0 generation with wild-type heterozygote mice to obtain F1 generation mice, carrying out PCR identification 20 days after the mice come out, and if positive mice come out, indicating that the transgenes are integrated into germ cells.
6. If homozygote with the flox region removed needs to be obtained, firstly, breeding heterozygote of the F1 generation with a specific Cre tool mouse to obtain mice with the flox region deleted and Cre positive, then, mating with wild background mice to obtain mice with the flox region deleted and Cre negative, breeding brothers and sisters after the mice are sexually mature, and obtaining homozygote with the flox region removed and Cre negative by progeny with a probability of 25%. As a result, the Mutyh knockout efficiency of cre-specific organs or tissues was verified by PCR.
7. A mouse model with stable genetic characters obtained from F3 generations and later is selected for experiments.
8. And (5) mouse genotype identification.
Secondly, the mouse genotype PCR identification result is shown in FIG. 2 and FIG. 3:
wild type: firstly, PCR reaction can obtain a single 204bp band;
heterozygote, wherein a PCR reaction can obtain bands of 204bp and 302 bp;
a homozygote, a single 302bp band can be obtained by PCR reaction.
The PCR identification result shows that 204bp and 302bp band types appear, wherein 302bp is a mutant allele band, 204bp is a wild type allele band, and the two bands appear simultaneously, which indicates that the mouse carries wild type and mutant genes simultaneously, i.e. the mouse is a heterozygote Mutyh +/-mouse.
Wherein, the specific primers for PCR identification comprise:
(SEQ NO.5)Mutyh—F1：5'—TAGGGATGCCCCTTGCATAGTA—3'
(SEQ NO.6) Mutyh-R1: 5 '-ACAATCTGTTCCCACTCAGGGC-3' and
(SEQ NO.7)Mutyh—F2：5'—AGGCTTGCTGTGAACCAAGGA—3'
(SEQ NO.8) Mutyh-R2: 5 '-CCAACTGACCTTGGGCAAGAACAT-3' and
(SEQ NO.9)Mutyh—F3：5'—TCTGAGGCGGAAAGAACCAG—3'
(SEQ NO.10)Mutyh—R3：5'—TCTTCCAGCCTCAATGGGCAC—3'
the size of an amplification product corresponding to Mutyh-F1 is 204 bp; the size of an amplification product corresponding to Mutyh-R1 is 302 bp; the sizes of the amplification products corresponding to Mutyh-F2 and Mutyh-R2 are both 368 bp; the sizes of the amplification products corresponding to Mutyh-F3 and Mutyh-R3 are 338 bp.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed.
Sequence listing
<110> affiliated Yifu hospital of Nanjing medical university
<120> construction method of Mutyh gene conditional knockout mouse model
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<212> DNA
<213> Artificial sequence (C57/B6 mouse)
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tgagtagctt ccttcagctt 20
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<213> Artificial sequence (C57/B6 mouse)
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<213> Artificial sequence (C57/B6 mouse)
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ataacttcgt ataatgtatg ctatacgaag ttat 34
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<213> Artificial sequence (C57/B6 mouse)
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tagggatgcc ccttgcatag ta 22
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<213> Artificial sequence (C57/B6 mouse)
<400> 6
acaatctgtt cccactcagg gc 22
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<213> Artificial sequence (C57/B6 mouse)
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aggcttgctg tgaaccaagg a 21
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<213> Artificial sequence (C57/B6 mouse)
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ccaactgacc ttgggcaaga acat 24
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<212> DNA
<213> Artificial sequence (C57/B6 mouse)
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tctgaggcgg aaagaaccag 20
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<212> DNA
<213> Artificial sequence (C57/B6 mouse)
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tcttccagcc tcaatgggca c 21
Claims (9)
1. A method for constructing a Mutyh gene conditional knockout mouse model is characterized in that a CRISPR/Cas9 system of the Mutyh gene is used for designing and extracorporeally transcribing gRNA, and simultaneously flox modification is carried out on the Mutyh gene to construct a homologous recombinant vector;
in a CRISPR/Cas9 system for conditional knockout of mouse Mutyh gene, Cas9 protein is combined to a target site under the guidance of gRNA to cause DNA double strand break, and the DNA sequences of the action site of the gRNA are (SEQ NO.1)5 '-CTAAGATCAGAAATTTGGTC-3' and (SEQ NO.2)5 '-TGAGTAGCTTCCTTCAGCTT-3'.
2. The method for constructing a Mutyh gene conditional knockout mouse model according to claim 1, wherein the gRNA site is flanked by exons 3 to 15 of Mutyh gene.
3. The method for constructing a Mutyh gene conditional knockout mouse model according to claim 2, wherein the homologous recombination vector is constructed by flox modification of the Mutyh gene, and the DNA sequences of the flox modification site are (SEQ NO.3)5 '-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-3' and (SEQ NO.4)5 '-ATAACTTCGTATAATGT ATGCTATACGAAGTTAT-3'.
4. The method for constructing a Mutyh gene conditional knockout mouse model according to claim 1, wherein the Mutyh gene is flox-modified, and after flox is combined with specific Cre, exons 3 to 15 of the Mutyh gene can be conditionally knocked out.
5. The method for constructing a Mutyh gene conditional knockout mouse model according to claim 2, wherein the CRISPR/Cas9 system and the homologous recombination vector are applied to preparation of a Mutyh gene conditional knockout cell line.
6. The method for constructing a Mutyh gene conditional knockout mouse model according to claim 3, wherein the CRISPR/Cas9 system and the homologous recombination vector transfect cells to obtain a target-positive cell clone, namely a Mutyh conditional knockout cell line.
7. The method for constructing a Mutyh gene conditional knockout mouse model according to claim 5, wherein the cell line is applied in preparing the Mutyh conditional knockout mouse model.
8. The method for constructing a Mutyh gene conditional knockout mouse model according to claim 2, wherein the operation steps comprise:
1) designing gRNA of a targeted Mutyh gene based on a CRISPR/Cas9 system, and simultaneously carrying out flox modification on a target gene to construct a homologous recombinant vector;
2) cas9, gRNA and a homologous recombination vector are simultaneously injected into a fertilized egg of a mouse;
3) transplanting the fertilized eggs into a pseudopregnant female mouse body to generate FO generation, and carrying out PCR identification on the FO generation;
4) mating the positive FO generation with wild type mice to obtain F1 generation heterozygotes;
5) breeding heterozygotes of the F1 generation with a specific Cre tool mouse to obtain mice with flox deleted regions and Cre positive, mating with wild background mice to obtain mice with flox deleted regions and Cre negative, breeding brothers and sisters after the mice are sexually mature, obtaining homozygotes with flox deleted regions and Cre negative after offspring generation, and obtaining a gene tissue and cell specificity knockout model mouse.
9. The method for constructing a Mutyh gene conditional knockout mouse model according to claim 8, wherein the specific primers for PCR identification comprise:
（SEQ NO.5）Mutyh—F1：5'—TAGGGATGCCCCTTGCATAGTA—3'
(SEQ NO.6) Mutyh-R1: 5 '-ACAATCTGTTCCCACTCAGGGC-3' and
（SEQ NO.7）Mutyh—F2：5'—AGGCTTGCTGTGAACCAAGGA—3'
(SEQ NO.8) Mutyh-R2: 5 '-CCAACTGACCTTGGGCAAGAACAT-3' and
（SEQ NO.9）Mutyh—F3：5'—TCTGAGGCGGAAAGAACCAG—3'
（SEQ NO.10）Mutyh—R3：5'—TCTTCCAGCCTCAATGGGCAC—3'。
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