CN106282231B - Construction method and application of mucopolysaccharide storage disease type II animal model - Google Patents

Abstract

The present invention provides a method for constructing a mucopolysaccharidosis type II animal model, including designing a target for mouse IDS gene, synthesizing an oligonucleotide chain, and annealing the synthesized oligonucleotide chain to a pUC57-T7-sgRNA plasmid Connect to obtain sgRNA expression vector; obtain mouse fertilized eggs; mix the transcribed Cas9 mRNA and sgRNA evenly to obtain RNA mixture, microinject the RNA mixture into the cytoplasm of mouse fertilized eggs, and then implant the surviving fertilized eggs into females In vivo, the F0 generation mice were obtained by breeding; the genomic DNA of the F0 generation mice was extracted, used as a template, and the PCR reaction was carried out. The product was digested with enzymes, and mutant mice were selected to determine the Founder mice; Mice were bred to obtain F1 generation mice. The invention also provides the animal model constructed by the method and the use of the model in the study of mucopolysaccharidosis type II.

Description

Construction method and application of mucopolysaccharidosis type II animal model

technical field

The invention belongs to the technical field of medical biology, and in particular, the invention relates to a method for constructing an animal model.

Background technique

Mouse disease models play a key role in the study of human disease pathogenesis and drug screening, and are especially valuable in evaluating drug treatment effects and selection of treatment methods.

Gene knockout is a new type of molecular biology technology developed since the late 1980s. It is a technology that inactivates or deletes specific genes in the body through a certain pathway. Gene knockout in the usual sense mainly uses the principle of DNA homologous recombination to replace the target gene segment with the designed homologous segment, so as to achieve the purpose of gene knockout. This is a complex molecular biology technique, also known as "gene targeting" technique, so far thousands of mouse models of gene mutation have been constructed using this technique. However, this traditional gene knockout method is complicated, time-consuming and expensive. In recent years, everyone has been looking for a new method that is faster, more economical and more applicable.

Mucopolysaccharidosis type II (MPS II, MIM309900), also known as Hunter syndrome, is an X-linked recessive genetic disorder caused by mutations in the lysosomal enzyme idose-2-sulfate Enzyme (iduronate-2-sulfatase, IDS) deficiency, so that mucopolysaccharide (mucop01ysaccharides, MPS) deposition in the body, a large number of urinary excretion of dermatin sulfate (dermatin sulfate B, DS), heparin sulfate (heparitinsulfate, HS). The patient's appearance at birth is generally normal, but as more and more mucopolysaccharides are stored in the body, the course of MPS II progressively worsens, with typical symptoms and a poor prognosis. The IDS gene is located at Xq27.3-Xq28, and MPS II is highly correlated with the mutation of the IDS gene. Due to its extremely low incidence, it is also known as an "orphan disease". At present, the research and treatment of this disease is still difficult in the world.

SUMMARY OF THE INVENTION

In view of the complex process, harsh conditions and huge cost in the current animal model construction, and the lack of researchable animal models in the current research on mucopolysaccharidosis type II, the present invention provides a mucopolysaccharidosis type II The construction method of the animal model, the animal model obtained by the method, and the use of the animal model in the study of mucopolysaccharidosis type II.

In a first aspect, the present invention provides a method for constructing a mucopolysaccharidosis type II animal model, characterized in that it comprises the following steps:

(1) Design a target for the mouse IDS gene, synthesize an oligonucleotide chain, and connect the synthesized oligonucleotide chain to the pUC57-T7-sgRNA plasmid by annealing to obtain an sgRNA expression vector;

(2) obtaining mouse fertilized eggs;

(3) Mixing the transcribed Cas9 mRNA and sgRNA uniformly to obtain an RNA mixture, microinjecting the RNA mixture into the cytoplasm of mouse fertilized eggs, then implanting the surviving fertilized eggs into female mice, and breeding to obtain F0 generation mice;

(4) Extracting the genomic DNA of the F0 generation mouse as a template, using primers containing the target of sgRNA to carry out a PCR reaction, the product is digested with enzymes, and mutant mice are selected to determine the Founder mouse;

(5) F1 generation mice were obtained by mating Founder mice with wild-type male mice.

In the aforementioned construction method, the following two pairs of oligonucleotide chains are synthesized in step (1):

ms Ids E5-1-sgRNA1: TAGGACAAAGCAGATTGGCG and AAACCGCCAATCTGCTTTGT;

ms Ids E5-2-sgRNA2: TAGGATGTGGCAGATGTGCCTGA and AAACTCAGGCACATCTGCCACAT.

In the aforementioned construction method, in step (2), a 4-5 week-old female mouse is used as a donor, and PMSG and hCG are injected intraperitoneally for superovulation, followed by mating with male mice, and mouse fertilized eggs are obtained the next day. .

Aforesaid construction method, in step (4), the primer that comprises sgRNA action target is:

ms Ids E5C9 forward primer: AGTTCTGGTCTGGAGACACAATT;

ms Ids E5C9 reverse primer: AGGCATCCTGGTAGGTAGGTTAT.

In the aforementioned construction method, in step (4), the PCR reaction system was 50 μL, and the PCR reaction conditions were: 95°C pre-denaturation for 5 min, followed by 95°C denaturation for 30s, 55°C annealing for 30s, 72°C extension for 30s, 30 cycles, A subsequent extension at 72°C for 10 min.

In the aforementioned construction method, in step (4), the T7 endonuclease is used for enzymatic cleavage.

In the aforementioned construction method, in step (4), after selecting the mutant mouse, firstly, the mutation is determined by TA cloning and sequencing, and then the Founder mouse is determined.

The aforementioned construction method also includes the identification of the genotype of the mucopolysaccharidosis type II animal model, including the steps of: extracting the genomic DNA of the F1 generation mice; designing primers containing sgRNA targets, and using the extracted F1 generation mice. The genomic DNA was used as a template, PCR reaction was performed, and the product of PCR reaction was sequenced.

Aforesaid construction method, the described primer that comprises sgRNA action target site is:

ms Ids E5C9 forward primer: AGTTCTGGTCTGGAGACACAATT;

ms Ids E5C9 reverse primer: AGGCATCCTGGTAGGTAGGTTAT.

In the aforementioned construction method, the reaction system of the PCR reaction is 50 μL, and the reaction conditions are: 95°C pre-denaturation for 5 min, followed by 95°C denaturation for 30s, 55°C annealing for 30s, 72°C extension for 30s, 30 cycles, followed by 72°C extension for 10min .

The aforementioned construction method also includes the phenotypic analysis of the mucopolysaccharidosis type II animal model, including the steps of: extracting the heart, liver, spleen and kidney four tissues of F1 generation mice and male wild-type mice respectively, and making them into Tissue sections were stained with alcian blue and compared with those from male wild-type mice.

In a second aspect, the present invention provides a mucopolysaccharidosis type II animal model constructed by the construction method of the first aspect.

In the aforementioned mucopolysaccharidosis type II animal model, nucleotides 18956-18975 bp of the idose-2-sulfatase gene of the X chromosome are deleted, and its sequence is AACTCCACGCCAATCTGCTT.

In the third aspect, the present invention provides the mucopolysaccharidosis type II animal model constructed by the construction method of the first aspect or the mucopolysaccharidosis type II animal model of the second aspect in the study of mucopolysaccharidosis type II animal model use.

The technical scheme of the present invention has at least the following beneficial effects:

(1) The mucopolysaccharidosis type II mouse animal model established by the present invention based on the CRISPR/Cas9 gene knockout technology provides an extremely important tool for further in-depth research on the pathogenic mechanism and therapeutic effect evaluation of mucopolysaccharidosis type II. animal model.

(2) The period for obtaining the mucopolysaccharidosis type II mouse animal model by the method of the present invention is short, only 2 months.

(3) The IDS gene knockout model mice obtained by the present invention, whose genotype is marked as X- ^ids X ^ids , are mice with normal phenotype, which can be normally cultured in the laboratory. Mice (genotype X ^ids Y) are mated, and their genes can be stably inherited. The inventors have successfully established an IDS gene knockout model mouse strain that can stably inherit genes. It is mated with wild-type C57BL/6 male mice (genotype X ^ids Y) to obtain F1 generation mice. In theory, 25% of the mice in the F1 generation have the genotype of X- ^ids Y, that is, mucopolysaccharide. Storage disease type II mouse animal model (genotype X- ^ids Y), and theoretically 25% of the mice have the genotype X- ^ids X ^ids , that is, IDS knockout model mice (genotype X ids) ^-ids X ^ids ). In this case, once the IDS knockout model mice (genotype X- ^ids X ^ids ) are obtained, the mucopolysaccharidosis type II mice can be obtained by simple natural mating process in the laboratory model (genotype X- ^ids Y) without the need to redo complex gene knockout experiments. Therefore, the method of the present invention obtains an IDS gene knockout model mouse that can simultaneously produce the IDS gene knockout model mouse and the mucopolysaccharidosis type II mouse animal model, which is the most ingenious part of the technical solution of the present invention. where.

Description of drawings

Figure 1 is a schematic diagram of the mechanism of RNA-mediated Cas9 system-directed genome modification;

Figure 2 is an analysis of CRISPR/Cas9-mediated IDS gene modification;

Figure 3 is a schematic diagram of the DNA sequence structure at the target;

Figure 4(A) and Figure 4(B) are the genotype identification and sequencing results of the mucopolysaccharidosis type II mouse animal model;

Figure 5 shows the results of alcian blue staining of tissue sections of mucopolysaccharidosis type II mouse animal model.

Detailed ways

In order to fully understand the purpose, features and effects of the present invention, the present invention will be described in detail by the following specific embodiments, but the present invention is not limited thereto. The experimental methods that do not specify specific conditions in the following specific embodiments can be selected according to conventional methods and conditions, or according to the product description. The substances involved in the following specific embodiments are conventionally commercially available.

The CRISPR/Cas9 gene knockout technology involved in the present invention is the latest generation method that can be used to construct gene knockout mice in a targeted manner after zinc finger nuclease, TALEN and other technologies, and shows great potential in directional modification of genes. The present invention provides a mucopolysaccharidosis type II mouse animal model and a construction method based on the CRISPR/Cas9 gene knockout technology, and provides further research on the pathogenic mechanism and therapeutic effect evaluation of mucopolysaccharidosis type II. extremely important animal model.

I. Construction of mucopolysaccharidosis type II mouse model

1. Construction of CRISPR Targeted Modification Gene Vector

The inventors designed two targets for the IDS gene (for the Mouse BAC-146N21 Chromosome Xcontains iduronate-2-sulfatase gene (the idose-2-sulfatase gene contained in the mouse BAC-146N21X chromosome) (Accession: AC002315) 18963-18981 bp nucleotide region and 18985-19005 bp nucleotide region of exon 5 (nucleotides 18934-19134 bp), where ms Ids E5-1-sgRNA1 targets the 18963-18981 bp nucleotide region, ms Ids E5- 2-sgRNA2 targeting the 18985-19005 bp nucleotide region), two pairs of oligonucleotide chains (ms Ids E5-1-sgRNA1: TAGGACAAAGCAGATTGGCG and AAACCGCCAATCTGCTTTGT; ms Ids E5-2-sgRNA2: TAGGATGTGGCAGATGTGCCTGA and AAACTCAGGCACATCTGCCACAT) were synthesized for preparation sgRNA (small guide RNA, small guide RNA), its mechanism of action is shown in Figure 1. The synthesized oligonucleotides were annealed (at 95°C for 5 min and then naturally cooled to room temperature), and ligated into the pUC57-T7-sgRNA expression vector (Addgene, NO.51132) recovered by BsaI digestion to construct the sgRNA expression vector. Verify the correctness of the ligated fragment by sequencing, select the correct clone, and extract the plasmid after expanding the culture to prepare the template for in vitro transcription.

The Cas9 (D10A) expression plasmid (Pst1374-NLS-Cas9-ZF) was linearized by AgeI digestion, extracted and purified by phenol-chloroform, and dissolved in nuclease-free water as a template for in vitro transcription. Synthesis of Cas9 mRNA was accomplished in vitro by the kit T7UltraKit (Ambion, AM1345) using T7 RNA polymerase. The sgRNA expression vector was cut and linearized with DraI, extracted and purified by phenol-chloroform, and dissolved in nuclease-free water as a template for in vitro transcription. The in vitro synthesis of sgRNA was accomplished in vitro by the MEGAshortscript Kit (Ambion, AM1354) using T7 RNA polymerase.

2. Superovulation of Embryos in Donor Mice

Donor female mice were treated with PMSG (serum gonadotropin), injected with hCG (human chorionic gonadotropin) 46 hours later, and mated with male mice. The fertilized eggs were collected the next day for microinjection.

3. Microinjection and embryo transfer

The transcribed Cas9 mRNA and sgRNA were mixed and the concentration was adjusted to 20ng/μl and 12.5ng/μl of each sgRNA. The RNA mixture was injected into the cytoplasm of mouse fertilized eggs by microinjection, and 143 surviving fertilized eggs were transplanted to 5. Pseudopregnant C57BL/6 female mice were implanted with 28 fertilized eggs per pseudopregnant female. About 3 weeks later, the mice were born, 19 F0 mice were obtained, and 17 survived.

4.Founder mouse identification

The embryo-transferred mice will be born about 19 days after the operation, and 20 days after the mice are born, their tails will be cut off to extract DNA and conduct PCR identification. A pair of primers containing sgRNA targets (ms Ids E5C9For: AGTTCTGGTCTGGAGACACAATT and ms Ids E5C9Rev: AGGCATCCTGGTAGGTAGGTTAT) were designed, and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The PCR reaction system was 50 μL (PCR reaction related reagents were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). Reaction conditions: 95°C for 5 min; (95°C for 30s, 55°C for 30s, 72°C for 30s), 30 cycles; 72°C for 10 min; store at 4°C. The amplified fragment size is 466bp. The PCR products were purified with a PCR purification kit (Beijing Tiangen). 100 ng of the purified PCR product was denatured and renatured in NEB Buffer 2, incubated with T7 endonuclease (NEB, M0302L) at 37°C for 40 min, and then separated by 1.5% agarose gel electrophoresis. T7 endonuclease can recognize incompletely paired double-stranded DNA and cut it. If CRISPR/Cas9 mutates the target, it will be recognized by the enzyme and cause double-stranded DNA breaks. Therefore, there are bands other than PCR product bands in agarose gel electrophoresis, indicating that the annealed product can be recognized and cleaved by T7 endonuclease, indicating that there may be a mutation in the target DNA sequence (Figure 2). In order to further verify the existence of the mutation and the specific situation of the mutation, the mutation was further detected by TA cloning and sequencing (Figure 3).

5. F1 obtained by mating of Founder mice with wild-type mice

When the female Founder mice are 4 weeks old, they can be mated with wild-type male mice, and the mice are identified by PCR 20 days after birth. If a positive mouse is born, it means that the transgene has been integrated into the germ cells, marking the successful establishment of the line. Obtain IDS knockout model mice, whose genotype is marked as X- ^ids X ^ids . Select F0 generation 23-1 female mice (genotype X- ^ids X ^ids ) and wild-type C57BL/6 male mice (genotype X ^ids Y) to obtain F1 generation mice.

II. Genotyping of mucopolysaccharidosis type II mouse animal model

Twenty days after the birth of F1 generation mice, the tail tips of F1 generation male mice and wild-type C57BL/6 male mice were cut, and the mouse genomic DNA was extracted to amplify the target gene. A pair of primers containing sgRNA targets (ms Ids E5C9For: AGTTCTGGTCTGGAGACACAATT and ms Ids E5C9Rev: AGGCATCCTGGTAGGTAGGTTAT) were designed, and the primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The PCR reaction system was 50 μL (PCR reaction related reagents were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). Reaction conditions: 95°C for 5 min; (95°C for 30s, 55°C for 30s, 72°C for 30s), 30 cycles; 72°C for 10 min; store at 4°C. The amplified fragment size is 466bp. The PCR product was purified with a PCR purification kit (Beijing Tiangen), and the purified PCR product was directly sent for sequencing. The mouse animal model, its genotype is X- ^ids Y, the sequencing results show that it lacks 20 bases of nucleotides 18956-18975bp of the Mouse BAC-146N21 Chromosome X containsiduronate-2-sulfatase gene (Accession: AC002315), the sequence is AACTCCACGCCAATCTGCTT, This result is consistent with the design, that is, a mouse animal model of mucopolysaccharidosis type II with correct genotype identification (genotype X- ^ids Y) is obtained.

III. Phenotypic analysis of mucopolysaccharidosis type II mouse animal model

Mice were sacrificed by cervical dislocation, and four types of tissues including heart, liver, spleen and kidney of mucopolysaccharidosis type II mouse model (genotype X- ^ids Y) and male wild mice were extracted, and the above four tissues were prepared. Paraffin tissue sections of the tissue, after the sections are made, the sections can be stained with alcian blue. Alcian blue is a cationic dye that is the most specific for showing acidic mucus substances. Compared with the Alcian blue staining of wild-type mouse tissue sections (Figure 5), it can be found that the heart, liver, spleen, and kidney tissues of the mucopolysaccharidosis type II mouse animal model (genotype X- ^ids Y) There is obvious mucopolysaccharide aggregation phenomenon, which proves that the mucopolysaccharidosis type II mouse animal model was successfully constructed.

Finally, it should be noted that: the above enumeration is only the specific embodiment of the present invention, of course those skilled in the art can make changes and modifications to the present invention, if these modifications and modifications belong to the scope of the claims of the present invention and its equivalent technology within the scope of protection of the present invention.

Claims (10)

1. A method for constructing a mucopolysaccharide storage disease type II animal model is characterized by comprising the following steps:

(1) aiming at a mouse IDS gene design target spot, synthesizing an oligonucleotide chain, and connecting the synthesized oligonucleotide chain with pUC57-T7-sgRNA plasmid through annealing to obtain a sgRNA expression vector;

(2) obtaining mouse fertilized eggs;

(3) uniformly mixing the transcribed Cas9mRNA and the sgRNA to obtain an RNA mixture, microinjecting the RNA mixture to cytoplasm of mouse fertilized eggs, then implanting the living fertilized eggs into female mice, and propagating to obtain F0 generation mice;

(4) extracting genome DNA of an F0 mouse, using the genome DNA as a template, performing PCR reaction by using a primer containing a sgRNA action target spot, performing enzyme digestion on a product, and selecting a mutant mouse, thereby determining a fountain mouse;

(5) mating the fountain mouse with a wild type male mouse to obtain an F1 generation mouse;

wherein, in the step (1), targets are designed aiming at 18963-18981bp nucleotide region and 18985-19005bp nucleotide region of the exon 5 of the iduronate-2-sulfatase gene contained in the X chromosome of the mouse;

wherein, the nucleotide 18956-18975bp deletion of the iduronate-2-sulfatase gene of the X chromosome of the mucopolysaccharide storage disease II type animal model has the sequence of AACTCCACGCCAATCTGCTT;

wherein, the following two pairs of oligonucleotide chains are synthesized in the step (1):

ms Ids E5-1-sgRNA 1: TAGGACAAAGCAGATTGGCG and AAACCGCCAATCTGCTTTGT;

ms Ids E5-2-sgRNA 2: TAGGATGTGGCAGATGTGCCTGA, and AAACTCAGGCACATCTGCCACAT.

2. The constructing method according to claim 1, wherein in the step (2), female mice of 4 to 5 weeks old are used as donors, and PMSG and hCG are intraperitoneally injected for superovulation, followed by mating with male mice, and fertilized eggs of the mice are obtained the next day.

3. The method for constructing the recombinant vector of claim 1, wherein in the step (4), the primers comprising sgRNA target functions are:

ms Ids E5C9 forward primer: AGTTCTGGTCTGGAGACACAATT, respectively;

ms Ids E5C9 reverse primer: AGGCATCCTGGTAGGTAGGTTAT are provided.

4. The method of claim 1, wherein in the step (4), the PCR reaction system is 50. mu.L, and the conditions of the PCR reaction are as follows: pre-denaturation at 95 ℃ for 5min, followed by denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, followed by extension at 72 ℃ for 10 min.

5. The method of claim 1, wherein in step (4), the digestion is performed with the T7 endonuclease.

6. The method according to claim 1, wherein in step (4), after selecting the mutant mice, the mutation is determined by further detection through TA cloning and sequencing, and then the fountain mice are determined.

7. The method of constructing a model according to any one of claims 1 to 6, further comprising genotyping a mucopolysaccharidosis type II animal model comprising the steps of: extracting genome DNA of F1 mouse; designing a primer containing a sgRNA target, performing PCR reaction by using the extracted genomic DNA of the F1 mouse as a template, and sequencing the product of the PCR reaction.

8. The construction method according to claim 7, wherein the primer containing the sgRNA target of action is:

ms Ids E5C9 forward primer: AGTTCTGGTCTGGAGACACAATT, respectively;

ms Ids E5C9 reverse primer: AGGCATCCTGGTAGGTAGGTTAT are provided.

9. The method according to claim 7, wherein the reaction system of the PCR reaction is 50. mu.L, and the reaction conditions are: pre-denaturation at 95 ℃ for 5min, followed by denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, followed by extension at 72 ℃ for 10 min.

10. The method of constructing a model according to any one of claims 1 to 7, further comprising a phenotypic analysis of a mucopolysaccharidosis type II animal model comprising the steps of: four tissues of heart, liver, spleen and kidney of the F1 mouse and the male wild mouse are respectively extracted to prepare tissue sections, the tissue sections are stained with alcian blue, and the tissue sections of the F1 mouse are compared with the tissue sections of the male wild mouse.

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