CN115197962A

CN115197962A - Construction method and application of Sirt3 gene knockout mouse animal model

Info

Publication number: CN115197962A
Application number: CN202210802311.4A
Authority: CN
Inventors: 赵静
Original assignee: Cyagen Biosciences Inc Suzhou
Current assignee: Cyagen Biosciences Inc Suzhou
Priority date: 2022-07-07
Filing date: 2022-07-07
Publication date: 2022-10-18

Abstract

The invention belongs to the technical field of transgenosis, and particularly relates to a construction method and application of a Sirt3 gene knockout mouse animal model, wherein the construction method comprises the steps of knocking out Exon 4 of the Sirt3 gene by using cas9 protein; furthermore, the sequence of the knockout region of Exon 4 is not 3 times, so that frameshift mutation is caused, and the purpose of knocking out the gene is achieved; exon 4 was knocked out using gRNA1 and gRNA 2; the sequence of gRNA1 is SEQ ID NO.1, and the sequence of gRNA2 is SEQ ID NO.2; the construction method provided by the invention has the advantages of simple steps, easy method, short period and high probability of obtaining positive mice, the Sirt3 gene has profound influence on nuclear gene expression, cancer, cardiovascular diseases, neuroprotection, aging and metabolic control, and the construction of the Sirt3 knockout mouse model can provide an important mouse tool model for the research of diseases such as metabolic diseases, cardiovascular diseases, neurodegenerative diseases and the like.

Description

Construction method and application of Sirt3 gene knockout mouse animal model

Technical Field

The invention belongs to the technical field of transgenosis, and particularly relates to a construction method and application of a Sirt3 gene knockout mouse animal model.

Background

Sirt3 is a member of the mammalian sirtuin family of proteins, which is an NAD + dependent protein deacetylase. Human sirtuins have a range of molecular functions and have become important proteins in the regulation of senescence, stress resistance and metabolism. In addition to protein deacetylation, studies have shown that human sirtuins may also function as intracellular regulatory proteins with mono ADP ribosyltransferase activity. Endogenous Sirt3 is a soluble protein located in the mitochondrial matrix and there is a large body of published literature that indicates a strong mechanistic link between mitochondrial function, senescence and carcinogenesis.

Sirt3 is mainly present in mitochondria and can play an important role in regulating mitochondrial function and metabolism by binding and deacetylating a number of important mitochondrial metabolism-related enzymes. Sirt3 has been shown to specifically modulate ROS production in ETC, inhibiting apoptosis by eliminating reactive oxygen species. Sirt3 is also involved in the metabolic reprogramming of the Warburg effect in cancer cells, potentiates the glycolytic production of ATP, and prevents the formation of cancer cells. It also controls the flow of mitochondrial oxidative pathways and ultimately the rate of Reactive Oxygen Species (ROS) production. SIRT3 can also affect multiple energy metabolic processes (e.g., tricarboxylic acid cycle, respiratory chain, fatty acid beta-oxidation, and ketogenesis) by targeting enzymes. Sirt 3-mediated deacetylation activates the enzymes responsible for reducing ROS, thus providing protection against oxidative stress-dependent phenomena or diseases such as cardiac hypertrophy, aging, cancer, cardiac dysfunction and neurodegeneration. Sirt3 also plays a role in multiple additional metabolic processes from acetate metabolism to BAT thermogenesis. Sirt3 has profound effects on nuclear gene expression, cancer, cardiovascular disease, neuroprotection, aging, and metabolic control. And the construction of the Sirt3 knockout mouse model can provide an important mouse tool model for the research of diseases such as metabolic, cardiovascular and neurodegenerative diseases. How to quickly and accurately construct a Sirt3 gene knockout mouse animal model is a problem which needs to be solved urgently at present.

Disclosure of Invention

In order to solve the technical problem, the invention discloses a construction method and application of a Sirt3 gene knockout mouse animal model.

The technical scheme of the invention is as follows:

a construction method of a Sirt3 gene knockout mouse animal model uses cas9 protein to knock out Exon 4 of Sirt3 gene. The invention utilizes CRISPR/Cas9 technology to knock out Sirt3 gene in a mouse body quickly and construct a Sirt 3-deficient mouse model. CRISPR/Cas9 is a technology guided by gRNA and editing a target gene using Cas9 nuclease, and its working principle is that crRNA (CRISPR-derived RNA) is combined with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex, which will guide Cas9 protein to cut double-stranded DNA at a sequence target site paired with crRNA, and by artificially designing both crRNA and tracrRNA, the sirna with a guiding effect is modified, thereby guiding Cas9 protein to cut the DNA at a fixed point, and generating a double-stranded DNA nick with a flat end, and further initiating a DNA damage repair mechanism, and sequences upstream and downstream of the break are mainly connected by Non-Homologous end joining (NHEJ) or Homologous recombination (Homologous recombination, grhr).

Further, according to the method for constructing the Sirt3 knockout mouse animal model, the knockout region sequence of Exon 4 is not 3 times, so that a frameshift mutation is caused, and the purpose of knocking out the gene is achieved.

Further, in the method for constructing the Sirt3 knockout mouse animal model, exon 4 is knocked out by using gRNA1 and gRNA 2;

the sequence of gRNA1 is SEQ ID No.1=5'-GACCTCTCAGCTGCACGTGGTGG-3';

the sequence of gRNA2 is SEQ ID No.2=5'-CTACCCCCAAGAATTGCAGAAGG-3'.

If the Cas9 protein needs to play a role, a 5'-NGG-3' motif at the downstream of a target site, namely a PAM sequence, a gene-specific sgRNA template sequence is positioned in front of the PAM sequence, more than 4T endings of the sgRNA sequence are avoided, and the optimal GC content is 30-70%. In addition, when the sgRNA is selected, the off-target effect of the sgRNA should be considered, and the off-target effect of the whole gene should be considered that the number of base mismatches at the off-target site is not more than 5 as much as possible. Based on the consideration, two gRNAs 1 and 2 aiming at Sirt3 genes are designed, the 5 'end and the 3' end of Exon 4 are respectively targeted, the off-target probability is low, and the knockout success rate is high.

Furthermore, the construction method of the Sirt3 gene knockout mouse animal model,

the method comprises the following steps:

1) Design of a knockout scheme: searching detailed information of the Sirt3 gene, selecting a gene knockout region which needs to cause frameshift mutation and possibly comprises a functional domain part of the gene, selecting Exon 4 containing a 101bp coding sequence of the Sirt3 gene as a knockout region, wherein the Exon 4 is a multiple other than 3, and can cause frameshift mutation after knockout, and directly knocking out 13.1 percent of coding regions of the Sirt3 gene;

2) gRNA sequence design:

according to the sequence of the mouse Sirt3 gene, two gRNA sequences aiming at the gene are designed and synthesized, and the sequence information is as follows:

the sequence of gRNA1 is SEQ ID No.1=5'-GACCTCTCAGCTGCACGTGGTGG-3';

the sequence of gRNA2 is SEQ ID No.2=5'-CTACCCCCAAGAATTGCAGAAGG-3';

3) Microinjection of Cas 9/gRNA: cas9 mRNA, artificially synthesized gRNA1 and gRNA2 are mixed uniformly and then injected into the cell nucleus of mouse fertilized eggs through microinjection, and an F0 mouse is obtained through delivery of a surrogate mouse;

4) PCR identification of Sirt3 knockout mice: and (3) carrying out PCR identification on the F0 mouse, identifying a correct positive mouse, mating with a wild mouse to obtain an F1 mouse, and identifying the homozygous mouse as a Sirt3 knockout mouse animal model.

The knockout strategy is low in off-target rate, and compared with the traditional method, the time period for obtaining the mouse model greatly saves time and cost.

Further, the method for constructing the Sirt3 knockout mouse animal model comprises the following steps in step 3):

selecting an SPF (specific pathogen free) female mouse with the age of 4-6 weeks as an ovum donor, injecting PMSG into the abdominal cavity of the mouse, injecting hCG after 48 hours, mating with a germchit male mouse with normal reproductive capacity immediately, collecting fertilized ova of the mouse, digesting and washing, and placing in an incubator at 37 ℃ for later use; cas9 mRNA is uniformly mixed with artificially synthesized gRNA1 and gRNA2, then the mixture is injected into mouse fertilized egg cell nucleus through a micro-injection mode, then the mixture is transplanted into the ampulla of an oviduct of a surrogate mother mouse, the surrogate mother mouse is weighed every other week, whether the surrogate mother mouse is pregnant or not is preliminarily judged, a newborn mouse is delivered 19-21 days after an operation, and after 5 days, a tail number is cut and PCR detection is carried out.

The method has the advantages of short time for obtaining the F0 mouse and high success rate of identification.

Further, in the method for constructing the Sirt3 knockout mouse animal model, the step 4) includes the following steps:

according to the Sirt3 gene knockout region, a pair of primers F1/R1 are respectively designed at the 5 'end of Exon 4 and the 3' end of Exon 4; carrying out PCR primer amplification on a born F0 mouse and sequencing, wherein the product amplified by the primer in a knockout mouse is 820bp, and the product amplified in a wild mouse is 1114bp; mating the positive mouse which is correctly identified with a wild mouse to obtain an F1 mouse, carrying out PCR amplification on the mouse 14 days after birth to identify the pure heterozygosity of the mouse, wherein the identification primer pair is F2/R1; the amplification product of the homozygous mouse is 820bp, the amplification product of the heterozygous mouse is 820bp/437bp/1114bp, and the amplification product of the wild mouse is 37bp/1114bp.

The identification method is accurate, has small probability of false positive and low identification cost, and can be used for batch and rapid identification.

Further, in the method for constructing the Sirt3 knockout mouse animal model, in the step 4):

sequence of F1 = SEQ ID No.3=5'-CAGTCAGTGACATCTTGGCTCTAC-3';

sequence of R1 = SEQ ID No.4=5'-CAGCCCAGCCTTATGTTCCTTTAC-3';

sequence of F2 = SEQ ID No.5=5'-ATGCACGGTCTGTCGAAGGTCC-3'.

The sequence is well designed and screened, so that the practicability and the specificity are good, and the identification cost is greatly reduced.

Further, in the method for constructing a Sirt3 knockout mouse animal model, in the step 4):

the sequenced primer sequence = SEQ ID No.6=5'-GCCAACCTGGTCTAAATAGTGAG-3'.

Further, according to the method for constructing the Sirt3 knockout mouse animal model, the PCR identification part of the mouse is the mouse tail, and the crude lysis method is selected for the mouse tail identification to extract DNA. The rat tail extracts DNA, and the operation is convenient.

Further, the construction method of the Sirt3 knockout mouse animal model is applied to the research of metabolic, cardiovascular and neurodegenerative diseases. Sirt3 has profound effects on nuclear gene expression, cancer, cardiovascular disease, neuroprotection, aging, and metabolic control, and the mouse model described above can greatly accelerate the progress of relevant research.

The invention has the following beneficial effects:

the invention designs two gRNAs of a specific target Sirt3 gene, and uses cas9 protein to knock out Exon 4 of the Sirt3 gene, wherein the sequence of the knocked region is not 3 times, so that a frame shift mutation is caused, and the purpose of knocking out the gene is achieved. The method for constructing the Sirt3 gene knockout mouse model by using the CRISPR/Cas9 system is simple and easy to implement, has short period and high probability of obtaining a positive mouse, can fully research the pathogenesis of Sirt3 in diseases such as cancer, cardiovascular diseases, neuroprotection, aging, metabolism and the like by using the mouse model, and provides service for further developing treatment modes aiming at the diseases.

Drawings

FIG. 1 is a schematic diagram of a Sirt3 knock-out mouse construction scheme according to the present invention; the dark region in the figure indicates the knockout region, the filled rectangle represents the exon of the gene, and the gRNA region represents the splicing region of the gRNA;

FIG. 2 is an identification strategy for Sirt3 knockout mice of the present invention; the dark region in the figure indicates the knockout region, the filled rectangle represents the exon of the gene, F1, F2, R1 represents the primer binding region for mouse identification;

FIG. 3 shows the result of identifying a Sirt3 knockout mouse of the present invention: PCR screening is carried out by utilizing a PCR primer PCR Primers1, and the amplification product after gene knockout is 820bp; 1114bp of wild type amplification product; the left figure is the DNA molecular weight marker, and the right figure is the identification product figure of PCR; PCR reactions were performed for 35 cycles in a 25. Mu.L system using P112-01 for Taq DNA polymerase and negative controls: water (without DNA template) and WT (400 ng mouse genomic DNA);

FIG. 4 shows the sequencing results of the PCR products: the Sirt3 gene was deleted 295bp as indicated by the arrow;

sequencing primer sequence = SEQ ID No.6=5'-GCCAACCTGGTCTAAATAGTGAG-3'.

Detailed Description

The method of the present invention is further illustrated below with reference to examples, in which experimental procedures not specifying specific conditions may be generally carried out under conventional conditions, such as those described in molecular cloning, a laboratory Manual written by J. Sambrook et al, or according to conditions recommended by the manufacturer. The present invention may be better understood and appreciated by those skilled in the art with reference to the following examples. However, the method of carrying out the present invention should not be limited to the specific method steps described in the examples of the present invention.

The CRISPR/Cas9 reagents were purchased from commercial kits.

Example 1

Design of a knockout scheme:

according to the requirements, the detailed information of the Sirt3 gene is searched, a gene knockout region is selected to cause frameshift mutation and include a functional domain part of the gene as much as possible, the knockout region is selected according to the actual situation, exon 4 containing a 101bp coding sequence of the Sirt3 gene is selected as a knockout region, the Exon 4 is a non-3 multiple, the frameshift mutation can be caused after knockout, and 13.1% of a coding region of the Sirt3 gene is directly knocked out, as shown in figure 1.

Example 2

gRNA sequence design

the sequence of gRNA1 is SEQ ID No.1=5'-GACCTCTCAGCTGCACGTGGTGG-3';

the sequence of gRNA2 is SEQ ID No.2=5'-CTACCCCCAAGAATTGCAGAAGG-3'.

Example 3

Microinjection of Cas9/sgRNA

Selecting an SPF female mouse (Cyagen) with the age of 4-6 weeks as an ovum donor, injecting PMSG (pregnant mare serum gonadotropin) into the abdominal cavity of the mouse, injecting hCG (human chorionic gonadotropin) after 48 hours, mating the mouse with a germchit with normal reproductive capacity, collecting fertilized eggs of the mouse, digesting and washing the fertilized eggs, and placing the fertilized eggs in an incubator at 37 ℃ for later use. Cas9 mRNA (20-200 ng/mu L) and artificially synthesized gRNA1 and gRNA2 (20-50 ng/mu L) are mixed uniformly and then injected into mouse fertilized egg cell nucleuses through a micro-injection method, then the mouse is transplanted into the ampulla of the oviduct of a surrogate mother mouse, the weight of the surrogate mother mouse is weighed every other week, whether the mouse is pregnant or not is preliminarily judged, the newborn mouse is delivered 19-21 days after an operation, and the number of the tail is cut after the newborn mouse is delivered 5 days, and PCR detection is carried out.

Example 4

PCR identification of Sirt3 knockout mice

The authentication strategy is shown in figure 2.

According to the Sirt3 gene knockout region, a pair of primers F1/R1 are respectively designed at the 5 'end of Exon 4 and the 3' end of Exon 4, PCR primer amplification and sequencing are carried out on the born F0 mouse, and the sequencing result is shown in the attached figure 4; the product amplified by the primer in a knockout mouse is 820bp, and the product amplified in a wild mouse is 1114bp; mating the positive mouse which is correctly identified with a wild mouse to obtain an F1 mouse, carrying out PCR amplification on the mouse 14 days after the birth to identify pure heterozygosity of the mouse, wherein the identification primer pair is F2/R1; the amplification product of the homozygous mouse is 820bp, the amplification product of the heterozygous mouse is 820bp/437bp/1114bp, and the amplification product of the wild mouse is 37bp/1114bp.

Sequence of F1 = SEQ ID No.3=5'-CAGTCAGTGACATCTTGGCTCTAC-3';

sequence of R1 = SEQ ID No.4=5'-CAGCCCAGCCTTATGTTCCTTTAC-3';

sequence of F2 = SEQ ID No.5=5'-ATGCACGGTCTGTCGAAGGTCC-3';

sequencing primer sequence = SEQ ID No.6=5'-GCCAACCTGGTCTAAATAGTGAG-3'.

The PCR identification part of the mouse is a mouse tail, and a crude cracking method is selected for mouse tail identification to extract DNA; the PCR reaction system is shown in Table 1 below:

TABLE 1 PCR reaction System

The PCR reaction procedure is as follows in table 2:

TABLE 2 PCR reaction procedure

Carrying out agarose gel electrophoresis on the PCR product, wherein the electrophoresis result is shown in figure 3, and the amplification product after gene knockout is 820bp; 1114bp of wild type amplification product. The left panel shows DNA molecular weight markers, and the right panel shows PCR-derived identification products. PCR reactions were amplified for 35 cycles in a 25. Mu.L system using P112-01 for Taq DNA polymerase and negative controls: water (without DNA template) and WT (400 ng mouse genomic DNA).

From the above examples it can be seen that: the invention designs two gRNAs of a specific target Sirt3 gene, and uses cas9 protein to knock out Exon 4 of the Sirt3 gene, wherein the sequence of the knocked region is not 3 times, so that the frame shift mutation is caused, and the purpose of knocking out the gene is achieved. The method for constructing the Sirt3 gene knockout mouse model by using the CRISPR/Cas9 system is simple and easy to implement, has short period and high probability of obtaining a positive mouse, and can fully research the pathogenesis of Sirt3 in diseases such as cancer, cardiovascular diseases, neuroprotection, aging, metabolism and the like and provide service for further developing a treatment mode aiming at the diseases.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.

Claims

1. A construction method of a Sirt3 gene knockout mouse animal model is characterized in that Exon 4 of Sirt3 gene is knocked out by using cas9 protein.

2. The method for constructing a Sirt3 knockout mouse animal model according to claim 1, wherein the sequence of the knockout region of Exon 4 is not a multiple of 3, resulting in a frameshift mutation, thereby achieving the purpose of knockout of the gene.

3. The method for constructing a Sirt3 knockout mouse animal model according to claim 1, wherein Exon 4 is knocked out by using gRNA1 and gRNA 2;

the sequence of gRNA1 is SEQ ID No.1=5'-GACCTCTCAGCTGCACGTGGTGG-3';

the sequence of gRNA2 is SEQ ID No.2=5'-CTACCCCCAAGAATTGCAGAAGG-3'.

4. The method for constructing an animal model of a Sirt3 knockout mouse according to claim 1, comprising the steps of:

1) Design of a knockout scheme: searching for detailed information of the Sirt3 gene, selecting a gene knockout region which needs to cause frameshift mutation and possibly comprises a functional domain part of the gene, selecting Exon 4 of the Sirt3 gene containing a 101bp coding sequence as a knockout region, wherein the Exon 4 is a non-3 multiple, and can cause frameshift mutation after knockout, so that 13.1 percent of coding regions of the Sirt3 gene can be directly knocked out;

2) gRNA sequence design:

the sequence of gRNA1 is SEQ ID No.1=5'-GACCTCTCAGCTGCACGTGGTGG-3';

the sequence of gRNA2 is SEQ ID NO.2=5'-CTACCCCCAAGAATTGCAGAAGG-3';

5. The method for constructing a Sirt3 knock-out mouse animal model according to claim 4, wherein the step 3) comprises the steps of:

selecting an SPF (specific pathogen free) female mouse with the age of 4-6 weeks as an ovum donor, injecting PMSG into the abdominal cavity of the mouse, injecting hCG after 48 hours, mating with a germchit male mouse with normal reproductive capacity immediately, collecting fertilized ova of the mouse, digesting and washing, and placing in an incubator at 37 ℃ for later use; cas9 mRNA, artificially synthesized gRNA1 and gRNA2 are mixed uniformly and then injected into mouse fertilized egg cell nucleus through a micro-injection method, then the mixture is transplanted into the ampulla of the oviduct of a surrogate mother mouse, the surrogate mother mouse is weighed every other week, whether the surrogate mother mouse is pregnant or not is preliminarily judged, a newborn mouse is delivered 19-21 days after an operation, and after 5 days, the newborn mouse is cut off and numbered and is subjected to PCR detection.

6. The method for constructing a Sirt3 knock-out mouse animal model according to claim 4, wherein the step 4) comprises the steps of:

according to the Sirt3 gene knockout region, a pair of primers F1/R1 are respectively designed at the 5 'end of Exon 4 and the 3' end of Exon 4; we carried out PCR primer amplification and sequencing on the born F0 mouse, the product amplified by the primer in the knockout mouse is 820bp, and the product amplified in the wild mouse is 1114bp; mating the positive mouse which is correctly identified with a wild mouse to obtain an F1 mouse, carrying out PCR amplification on the mouse 14 days after birth to identify the pure heterozygosity of the mouse, wherein the identification primer pair is F2/R1; the homozygous mouse amplification product is 820bp, the heterozygous mouse amplification product is 820bp/437bp/1114bp, and the wild type mouse amplification product is 37bp/1114bp.

7. The method for constructing a Sirt3 knock-out mouse animal model according to claim 6, wherein the step 4) comprises:

sequence of F1 = SEQ ID No.3=5'-CAGTCAGTGACATCTTGGCTCTAC-3';

the sequence of R1 = SEQ ID No.4=5'-CAGCCCAGCCTTATGTTCCTTTAC-3';

sequence of F2 = SEQ ID No.5=5'-ATGCACGGTCTGTCGAAGGTCC-3'.

8. The method for constructing a Sirt3 knock-out mouse animal model according to claim 6, wherein the step 4) comprises:

the sequenced primer sequence = SEQ ID No.6=5'-GCCAACCTGGTCTAAATAGTGAG-3'.

9. The method of claim 6, wherein the PCR site of the mouse is rat tail, and the DNA is extracted by crude lysis method for rat tail identification.

10. Use of the method of constructing a Sirt3 knockout mouse animal model according to any of claims 1-9 for the study of metabolic, cardiovascular, and neurodegenerative diseases.