CN111304258B - Ndufs2 gene conditional point mutation mouse model and construction method and application thereof - Google Patents
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Abstract
The invention provides a Ndufs2 gene conditional point mutation mouse model, a construction method and application thereof, wherein lysine at the 425 th site of the Ndufs2 gene of the mouse model is mutated into arginine. The mouse model constructed by the invention provides an effective experimental animal model for preparing the medicine for preventing or treating the cardiovascular and cerebrovascular diseases, has good medical clinical application prospect, and has great application value in preparing the medicine for preventing/treating the cardiovascular and cerebrovascular diseases.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a Ndufs2 gene conditional point mutation mouse model, and a construction method and application thereof.
Background
The establishment and application of the gene mutation mouse model have milestone significance on the development of life science and basic medicine, and have great promotion effect on the understanding and treatment of clinical diseases. The abnormal function of the mitochondrial respiratory chain directly or indirectly causes a plurality of serious physiological defects, and in order to overcome the medical problems, the structural information and the functional mechanism of the respiratory chain are important to be clarified by using a gene mutation mouse model. Of the four respiratory chain CI-CIV complexes, the most complex and pathogenic mutation sites of complex CI have been found to be associated with disease to 100 more mutation sites of CI to date. Non-insulin dependent diabetes mellitus, MELAS syndrome, leigh's encephalopathy (subacute necrotic encephalopathy), and the development of various cancers are all associated with mutations in CI. The complex I consists of 45 proteins and has two membrane arms, and Ndufs2 is a subunit of the complex I, is positioned at the hydrophilic membrane arm of the complex I, is close to the mitochondrial base, and belongs to a hydrophilic protein. The subunits of NADH dehydrogenase constitute the catalytic core of complex I, and Ndufs2 is one of the catalytic cores.
Scientific research on energy metabolism is an interesting and significant topic in the field of biology. Over a century of effort, human research into the mitochondrial respiratory chain has enjoyed promising success. Scientists have recognized that the complex and ordered respiratory chain organization form "subunit-respiratory complex-super complex" has made a deeper understanding of the molecular mechanisms of electron transfer and energy conversion in the respiratory body, but it is not clear how the effect of point mutation of the respiratory body subunit Ndufs2 gene on the binding and separation of super complex has specific biological significance for adapting to different cell states.
Disclosure of Invention
In view of the above, the invention aims to provide an Ndufs2 gene conditional point mutation mouse model, a construction method and an application thereof, and provides a new strategy for preparing a medicament for preventing or treating cardiovascular and cerebrovascular diseases.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a Ndufs2 gene conditional point mutation mouse model, wherein the 425 th lysine of the Ndufs2 gene is mutated into arginine.
The construction method of the Ndufs2 gene conditional point mutation mouse model comprises the following steps:
s1: transcribing the sgRNA in vitro;
s2: constructing a Cas9 targeting vector and a donor vector;
s3: microinjecting a Cas9 targeting vector, sgRNA and a donor vector into fertilized eggs of a C57BL/6J mouse for homologous recombination to obtain an F0 generation mouse;
s4: f0 generation mice which are positive through PCR and sequencing verification are mated with C57BL/6J mice to obtain a F1 generation mouse model which can be stably inherited.
Further, the step S1 includes the steps of: determining a target site of a gene to be knocked out of a mouse, and designing sgRNAs aiming at Ndufs2-T1 and Ndufs2-T2, wherein the sgRNA sequence of the Ndufs2-T1 is shown as SEQ ID No. 1; the sgRNA sequence of Ndufs2-T2 is shown in SEQ ID NO. 2.
Further, the step S2 includes the steps of:
a. artificially synthesizing target sequence oligonucleotide primers with different enzyme cutting site recognition sequences at the 5' end, directly annealing two pairs of primers through PCR to synthesize target sequence DNA short fragments carrying different viscous ends, inserting the target sequence DNA short fragments into the downstream of a promoter of a CRISPR/Cas9 expression vector pX330-U6-Chimeric _ BB-CBh-h SpCas9U6, and constructing CRISPR/Cas9 targeting vectors pX330-U6-Ndufs2T1-Chimeric _ BB-CBh-hSpCas9 and pX330-U6-Ndufs2T2-Chimeric _ BB-CBh-hSpCas9 of two target spots of a mouse Ndufs 2;
b. and (3) constructing a Donor vector, wherein the sequence of the Donor vector is shown as SEQ ID NO. 3.
The Ndufs2 gene conditional point mutation mouse model is applied to the preparation of medicines for preventing or treating cardiovascular and cerebrovascular related diseases.
Compared with the prior art, the Ndufs2 gene conditional point mutation mouse model, the construction method and the application thereof have the following advantages:
the invention provides a Ndufs2 gene conditional point mutation mouse model and a construction method and a method thereof for the first time, and no domestic and foreign literature reports exist. The mouse model constructed by the method provides an effective experimental animal model for preparing the medicament for preventing or treating the cardiovascular and cerebrovascular diseases, has good medical clinical application prospect, has great application value in preparing the medicament for preventing/treating the cardiovascular and cerebrovascular diseases, and has great social benefit.
Drawings
FIG. 1 is a schematic diagram of the principle of gene editing of Ndufs2 gene by CRISPR/Cas9 technology;
FIG. 2 is a schematic diagram of Ndufs2 targeting site;
FIG. 3 is a PCR identification electrophoretogram of homologous recombination positive F0 generation mouse;
FIG. 4 shows the result of sequencing before gene mutation in the F0 mouse;
FIG. 5 shows the result of the sequencing after the gene mutation of the F0 mouse;
FIG. 6 shows the results of sequencing of F0 mice by blast in pubMed;
FIG. 7 shows the electron microscope examination of myocardial mitochondria;
FIG. 8 shows the result of ATP detection of myocardial mitochondria;
FIG. 9 shows the results of detection of myocardial mitochondrial complex 1;
FIG. 10 shows the results of the detection of the binding between myocardial mitochondria Ndufs2 protein and SUMO1 protein.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to the following examples and accompanying drawings.
A method for constructing a mouse model with conditional point mutation of Ndufs2 gene is disclosed, the principle of which is shown in figure 1, and the method specifically comprises the following steps:
(1) Selection of Ndufs2 gene CRISPR/Cas9 targeting site
The target was selected according to the sequence of mouse Ndufs2 gene, specifically, the target was selected in exon10 and exon11, and used for knockout study of Ndufs2, and the target sequence information and the chromosomal location are shown in table 1 and fig. 2.
TABLE 1 sgRNA target sequences
Note: lower case letters representing PAM sequences
(2) Construction of CRISPR/Cas9 expression vector
The CRISPR/Cas9 expression vector construction method comprises the steps of artificially synthesizing target sequence oligonucleotide primers with different enzyme cutting site recognition sequences at the 5' end, directly annealing two pairs of primers through PCR, synthesizing target sequence DNA short fragments carrying different cohesive ends, inserting the target sequence DNA short fragments into the downstream of a promoter of a CRISPR/Cas9 expression vector pX330-U6-Chimeric _ BB-CBh-hSpCas9U6, and constructing CRISPR/Cas9 targeting vectors pX330-U6-Ndufs2T1-Chimeric _ BB-CBh-hCas Sp9 and pX330-U6-Ndufs2T2-Chimeric _ BB-CBh-hSpCas9 of two targets of mouse Ndufs 2.
The method comprises the following specific steps:
in the first step, the primers are annealed. The oligonucleotides Ndufs2T1F and Ndufs2T1R (sequences of primers are shown) were annealed directly, the two primers formed short fragments with different sticky ends, the annealing procedure was 90 ℃ 10min and 70 ℃ 10min, and the annealing reaction system was as shown in Table 2.
TABLE 2 direct primer annealing reaction System
And secondly, carrying out enzyme digestion on the vector. The CRISPR/Cas9 skeleton vector pX330-U6-Chimeric _ BB-CBh-hSpCas9 is cut by Xba I endonuclease, the procedure is that the enzyme is cut at 37 ℃ for 1h, the enzyme is inactivated at 65 ℃ for 20min, and the enzyme cutting system is shown in a table 3. The enzyme digestion product was detected with 1% agar gel and the linearized vector was recovered according to the agarose gel recovery kit instructions.
TABLE 3 digestion system
And thirdly, linking reaction. The linearized pX330-U6-Chimeric _ BB-CBh-hSpCas9 and the annealed Ndufs2T1 short fragment were ligated with T4 ligase at 16 ℃ for 12-16 h, and the ligation reaction system is shown in Table 4.
TABLE 4 ligation reaction System
And step four, converting the connecting product according to a conventional method.
And fifthly, sequencing and identifying. Randomly selecting 2-3 single colony colonies for amplification culture, extracting plasmids, sequencing and identifying by using a U6 primer, ensuring that the DNA sequence inserted into an expression vector is consistent with the designed sequence, and finally successfully obtaining the Ndufs2 targeting vector pX330-U6-Ndufs2T1-Chimeric _ BB-CBh-hSpCas9 and pX330-U6-Ndufs2T2-Chimeric _ BB-CBh-hSpCas9.
(3) Construction of Donor plasmid
Under the condition of not changing other gene base sequences of a C57 mouse, the Donor plasmid is designed by referring to the gene information and the sgRNA active target spots, and the sequence of the Donor plasmid is shown in SEQ ID NO. 3. The Do nor plasmid sequence was synthesized from the Beijing Weishangride company as a whole to a total of 1092bp.
(4) Microinjection
After a C57BL/6J male mouse and a female mouse which are 6-8 weeks old mate, fertilized eggs are obtained, a C as9 targeting vector and a donor are injected into the fertilized eggs in a microinjection mode, another 5 pseudopregnant female mice with the fallopian tubes in the same period are taken as receptors, and the injected fertilized eggs are transferred and implanted into the fallopian tubes of the pseudopregnant female mice.
(5) F0 generation mouse identification
After microinjection and embryo transfer, F0 mice were born. The correct genotypes of male mouse No.2 and male mouse No.3 were confirmed by PCR electrophoretogram (FIG. 3), sequencing identification (FIGS. 4 and 5) and alignment of sequencing results in PubMed (FIG. 6).
Ndufs2 obtained by the above construction method K425R The transgenic mouse homozygotes were each tested as follows.
Test example 1
Mixing Ndufs2 K425R Mating the gene mouse homozygote with myocardial specificity Cre mouse (Myh 6-Cre) for 2 generations to obtain Ndufs2 fl/fl Myh6 +/- Mice (Mutation) and control group Ndufs2 fl/fl Myh6 -/- Mouse (WT), 8 weeks old mice, via intraperitoneal injection tamoxifen induction (20 mg/kg) for 5 days, separation of myocardial mitochondria, electron microscope detection of mitochondria results as shown in figure 7: ndufs2 gene K425R point mutation mouse, myocardial mitochondria swelling, mitochondrial ridge fracture.
Test example 2
Mixing Ndufs2 K425R Mating the gene mouse homozygote with a cardiac muscle specific Cre mouse (Myh 6-Cre) for 2 generations to obtain Ndufs2 fl/fl Myh6 +/- The mice and the control group Ndufs2fl/fl Myh 6-/-mice, at the age of 8 weeks, were induced by intraperitoneal injection of tamoxifen (20 mg/kg) for 5 days, the myocardial mitochondria were isolated, and the ATP activity assay results are shown in FIG. 8: the ATP activity of the Ndufs2 gene K425R point mutation mouse is obviously reduced.
Test example 3
Mixing Ndufs2 K425R Mating the gene mouse homozygote with myocardial specificity Cre mouse (Myh 6-Cre) for 2 generations to obtain Ndufs2 fl/fl Myh6 +/- Mice and control group Ndufs2fl/fl Myh 6-/-mice, when 8 weeks old, were induced by intraperitoneal injection of tamoxifen (20 mg/kg) for 5 days, myocardial mitochondria were isolated, and mitochondria were complexedThe results of the activity assay for body i are shown in figure 9: the activity of the mitochondrial complex I of the Ndufs2 gene K425R point mutation mouse is obviously reduced.
Test example 4
Mixing Ndufs2 K425R Mating the gene mouse homozygote with a cardiac muscle specific Cre mouse (Myh 6-Cre) for 2 generations to obtain Ndufs2 fl/fl Myh6 +/- Mice and control group Ndufs2 fl/fl Myh6 -/- In the mice, when the mice are 8 weeks old, the mice are induced by injecting tamoxifen into the abdominal cavity (20 mg/kg) for 5 days, myocardial mitochondria are separated, and the detection result of co-immunoprecipitation is shown in fig. 10: the Ndufs2 gene K425R point mutation mouse has obviously reduced combination capacity of Ndufs2 protein and SUMO1 protein.
As can be seen from the test results of test examples 1 to 4, it was confirmed that the gene Ndufs2 plays an important role in cerebrovascular diseases in county. Therefore, the Ndufs2 gene K425R point mutation mouse model can be used as a model for researching the occurrence and development deterioration of cardiovascular and cerebrovascular related diseases, and provides convenience for the preparation of related medicaments.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (3)
- A method for constructing a mouse model with conditional point mutation of Ndufs2 gene is characterized in that: the mutation of the 425 th lysine of the mouse model Ndufs2 gene into arginine comprises the following steps:s1: transcribing the sgRNA in vitro;s2: constructing a Cas9 targeting vector and a donor vector;s3: microinjecting a Cas9 targeting vector, sgRNA and a donor vector into fertilized eggs of a C57BL/6J mouse for homologous recombination to obtain an F0 generation mouse;s4: f0 generation mice which are positive through PCR and sequencing verification are mated with C57BL/6J mice to obtain F1 generation mouse models which can be stably inherited.
- 2. The method for constructing a Ndufs2 gene conditional point mutation mouse model according to claim 1, wherein: the step S1 includes the steps of: determining a target site of a gene to be knocked out of a mouse, and designing sgRNA aiming at Ndufs2-T1 and Ndufs2-T2, wherein the sgRNA sequence of the Ndufs2-T1 is shown as SEQ ID NO. 1; the sgRNA sequence of Ndufs2-T2 is shown in SEQ ID NO. 2.
- 3. The method for constructing a Ndufs2 gene conditional point mutation mouse model according to claim 2, wherein: the step S2 includes the steps of:a. artificially synthesizing target sequence oligonucleotide primers with different enzyme cutting site recognition sequences at the 5' end, directly annealing two pairs of primers through PCR to synthesize target sequence DNA short fragments carrying different viscous ends, inserting the target sequence DNA short fragments into the downstream of a CRISPR/Cas9 expression vector pX330-U6-Chimeric _ BB-CBh-hSpCas9U6 promoter, and constructing CRISPR/Cas9 targeting vectors pX330-U6-Ndufs2T1-Chimeric _ BB-CBh-hSpCas9 and pX330-U6-Ndufs2T2-Chimeric _ BB-CBh-hSpCas9 of two target spots of a mouse Ndufs 2;b. and (3) constructing a Donor vector, wherein the sequence of the Donor vector is shown as SEQ ID NO. 3.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106929533A (en) * | 2017-03-10 | 2017-07-07 | 上海交通大学医学院附属新华医院 | The construction method of KARS point mutation mouse models and its application |
CN108977464A (en) * | 2018-08-09 | 2018-12-11 | 成都金唯科生物科技有限公司 | For improving the composition, drug and sgRNA of the blood coagulation activity of haemophilia B patient |
CN109679953A (en) * | 2018-12-28 | 2019-04-26 | 赛业(广州)生物科技有限公司 | Target sequence group, carrier and the method for point mutation animal model embryo are made using CRISPR-Cas9 system |
-
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106929533A (en) * | 2017-03-10 | 2017-07-07 | 上海交通大学医学院附属新华医院 | The construction method of KARS point mutation mouse models and its application |
CN108977464A (en) * | 2018-08-09 | 2018-12-11 | 成都金唯科生物科技有限公司 | For improving the composition, drug and sgRNA of the blood coagulation activity of haemophilia B patient |
CN109679953A (en) * | 2018-12-28 | 2019-04-26 | 赛业(广州)生物科技有限公司 | Target sequence group, carrier and the method for point mutation animal model embryo are made using CRISPR-Cas9 system |
Non-Patent Citations (2)
Title |
---|
Mouse models of mitochondrial complex I dysfunction;Irwin M.H.等;《The International Journal of Biochemistry & Cell Biology》;20120810;第34-40页 * |
O75306,NDUS2_HUMAN;EBI UNIPROT;《EBI UNIPROT》;20191211;全文 * |
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