CN112005968A - Construction method and application of galactosyltransferase GalT gene point mutation mouse model - Google Patents
Construction method and application of galactosyltransferase GalT gene point mutation mouse model Download PDFInfo
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- CN112005968A CN112005968A CN202010911812.7A CN202010911812A CN112005968A CN 112005968 A CN112005968 A CN 112005968A CN 202010911812 A CN202010911812 A CN 202010911812A CN 112005968 A CN112005968 A CN 112005968A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knockout animals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
Abstract
The invention provides a construction method and application of a galactosyltransferase GalT gene point mutation mouse model, which comprises the following steps: (1) constructing a target: searching a CDS region of a galactosyltransferase B4GalT gene, determining an exon part and determining a gene mutation site; (2) obtaining fertilized eggs, and performing microinjection: obtaining cas9 mRNA and gRNA by in vitro transcription, obtaining oligo donor DNA by synthesis, and performing microinjection on cas9 mRNA, gRNA and oligo donor DNA into fertilized eggs of a C57BL/6J mouse to obtain an F0 generation mouse; (3) breeding the positive F0 mouse and C57BL/6J to obtain positive F1 heterozygote with B4GalT gene site-directed mutation; (4) positive homozygote is obtained by PCR sequencing confirmation after the positive heterozygote mouse is mated, which indicates that the mouse model is successfully constructed. The model of the invention has simple manufacture and good repeatability, can be used for the research of medicaments for human glycosylation disorder diseases, and has important practical significance for the search of treatment methods.
Description
Technical Field
The invention belongs to the field of mouse models, and particularly relates to a construction method and application of a galactosyltransferase GalT gene point mutation mouse model.
Technical Field
Congenital glycosylation disease (CDG), originally known as carbohydrate-Deficient Glycoprotein Syndrome (CDGs), is a disease with abnormal N-glycosylation and clinical features involving the development of many organ systems, especially certain regions of the brain, and the function of the gastrointestinal, hepatic, visual and immune systems, with almost all patients having insufficient sialylation of serum glycoproteins. The current research on glycosylation diseases is mostly focused on clinical cases, but extensive systemic studies cannot be performed due to the fact that many cases are typed and the number of samples is small. The site-directed mutagenesis mouse can be propagated in batches, and is beneficial to multi-sample research.
Galactosyltransferases not only play an important role in cell motility, embryonic development, development of the nervous system, immune system and inflammatory responses, but also play an essential role in the sialylation process of glycoprotein N-glycosylation. The mutant galactosyltransferase can be used as a theoretical basis for developing congenital glycosylation disease model mice.
Disclosure of Invention
Based on the state of the prior art, the invention aims to provide a construction method and application of a galactosyltransferase GalT gene point mutation mouse model. By utilizing the techniques of homologous recombination, random insertion, RNAi and the like, strategies of knockout, site-specific mutation, gene inactivation and the like are applied to modify the galactose transferase gene so as to inactivate the galactose transferase gene.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for constructing a galactosyltransferase GalT gene point mutation mouse model comprises the following steps: designing a gene knockout strategy aiming at a B4GalT gene of a mouse, and selecting a knockout region mutation domain; obtaining F0 generation mice by adopting a gene editing method, breeding the positive F0 generation mice with C57BL/6J to obtain positive F1 generation heterozygotes with B4GalT gene site-directed mutation, and obtaining positive homozygotes by PCR sequencing confirmation after mating the positive heterozygote mice.
Further, a method for constructing a galactosyltransferase GalT gene point mutation mouse model, which comprises the following specific steps:
(1) constructing a target: searching a CDS region of a galactosyltransferase B4GalT gene, determining an exon part and determining a gene mutation site;
(2) obtaining fertilized eggs, and performing microinjection: obtaining cas9 mRNA and gRNA by in vitro transcription, obtaining oligo donor DNA by synthesis, and performing microinjection on cas9 mRNA, gRNA and oligo donor DNA into fertilized eggs of a C57BL/6J mouse to obtain an F0 generation mouse;
(3) breeding the positive F0 mouse and C57BL/6J to obtain positive F1 heterozygote with B4GalT gene site-directed mutation;
(4) positive homozygote is obtained by PCR sequencing confirmation after the positive heterozygote mouse is mated, which indicates that the mouse model is successfully constructed.
Furthermore, the 286 th amino acid of the 4 th exon B4GalT1 gene of the 4 th chromosome of the mouse.
Furthermore, the mutation site is that the 286 th amino acid tyrosine is mutated into leucine.
Furthermore, the DNA sequence of the gRNA is shown in SEQ ID NO. 1.
Further, the sequence of the oligo donor DNA is shown in SEQ ID NO 2.
Furthermore, the identification primer of the F0 generation mouse is shown as SEQ ID NO. 3.
Further, positive F1 mice were used for stock protection and establishment, and mating was not allowed in principle between different stock protection and establishment.
The invention also protects the application of the mouse model obtained by the construction method of the galactosyltransferase GalT gene point mutation mouse model in medicaments for treating glycosylation related diseases.
Further, the invention provides the application of the mouse model in diagnosis and treatment of galactosyltransferase and glycosylation disorder diseases.
The invention also protects the application of the mouse model obtained by the construction method of the galactosyltransferase GalT gene point mutation mouse model in the preparation of lactose-free milk products.
The invention protects the kit for obtaining the galactosyltransferase GalT gene point mutation mouse model.
Further, the kit contains gRNA shown in SEQ ID NO. 1.
Furthermore, it also contains oligo donor DNA shown in SEQ ID NO. 2.
The invention also protects the application of the mouse model in the research of the action of galactosyltransferase.
Advantageous effects
The construction method of the galactosyltransferase GalT gene point mutation mouse model and the provided mouse model fill a gap in the research field of the pathogenesis of the glycosylated airport disease, and the model is simple to manufacture, good in repeatability and low in manufacturing cost; the B4GalT1 galactosyltransferase-free activity mouse model can be used for researching the action of galactosyltransferase in mammals and has a certain value for researching lactose-free milk products; therefore, the method can be popularized and applied at home and abroad.
Drawings
Figures 1A and 1B are schematic diagrams of the strategy construction using CRISP-Cas 9; 1C is a peak spectrogram of mouse genotype identified by PCR;
FIG. 2 is a comparison of the N-chain oligosaccharide groups in sera of mice of different genotypes.
Detailed Description
The invention is further described with reference to the following figures and examples.
Example 1 suggest a site-directed mutagenesis of the B4GalT1-Y286 gene in a mouse model
Cas9 sample mixing system
gRNA design
As shown in the construction strategy diagram of FIG. 1, the gene substitution of exon4 (exon4) was determined. The sequence information of the upstream and downstream of the mutation site is as follows:
GTCATTACTTGGGGTAAAAACTTACTAGCCATGGGCCCTCTGTCTGTGCTTCCTCTCCAGCCTGCCATATGTTCAGTATTTTGGAGGTGTCTCTGCTCTCAGTAAACAACAGTTTCTTGC；
the sequence information of the oligo donor DNA is shown in SEQ ID NO: 2:
GTCATTACTTGGGGTAAAAACTTACTAGCCATGGGCCCTCTGTCTGTGCTTCCTCTCCAGCCTGCCATATGTTCAGCTGTTTGGAGGTGTCTCTGCTCTCAGTAAACAACAGTTTCTTGC。
designing a gRNA sequence which is shown as SEQ ID NO: 1: 5'-CTGCCATATGTTCAGTATTTTGG-3' are provided.
2. Preparation of Cas9 Mixed sample System
The Cas9 sample mixing system is a mixture and is derived from ThermoFisher, and comprises gRNAs, Cas9 and buffers, wherein the concentration of the gRNAs is 3 mug/muL of Cas9 protein, and the concentration of the gRNAs is 1 mug/muL.
Second, microinjection and transplantation
The method for preparing F0 mouse includes the following steps: supervola of mice; injecting fertilized eggs and transplanting; obtaining F0 mouse; wherein the mouse strains: c57 BL/6J.
The specific method comprises the following steps:
(1) selecting experimental mice: selecting mice with age of 5-6 weeks and weight of 18-22 g.
(2) Superovulation and fertilized egg injection of mice: the mice are supervolved and injected, namely female mice with the age of 4 weeks are injected with pregnant mare serum gonadotropin (PSMG) which is derived from Solarbio; injecting human chorionic gonadotropin (hCG) after 48 hours, wherein the hCG is derived from Solarbio, taking eggs after 14 hours, then performing in vitro fertilization, and taking fertilized eggs; and (3) mixing and injecting the Cas9 mixed sample system into fertilized eggs, and transplanting the fertilized eggs into a pseudopregnant female mouse to obtain an F0 mouse.
(3) Transplanting: after the injection is finished, transplanting the fertilized eggs into the uterus of a pseudopregnant female mouse; the transplanted receptor 2 is raised in one cage, and the patient can be continuously observed within one week after the operation, has pain response and is timely injected with analgesic.
Identification and breeding of mice of three and F0 generations
Microinjected F0 mice were identified by PCR and electrophoresis. PCR amplification primers, PCR reaction system, and procedure are shown in the following table.
The amplification primers were as follows:
| Primer | Sequence 5'-->3' | Primer Type |
| I | AGGCAGGCAGATCTCAAGTCAGC | Forward |
| II | AAAGCCCACATAGGAAGCCAAAGT | Reverse |
the PCR system was as follows:
PCR procedure:
| step (ii) of | Temperature/. degree.C | Time of day | Note that |
| 1 | 94 | 5min | - |
| 2 | 94 | 40sec | - |
| 3 | 63 | 40sec | - |
| 4 | 72 | 1min | Repeating 2-4 steps for 34 |
| 5 | 72 | 1min | - |
| 6 | 12 | - | Preservation of |
The sequencing primer is shown as SEQ ID NO. 3: 5'-AGGCAGGCAGATCTCAAGTCAGC-3' are provided.
Through PCR amplification and electrophoretic identification, 6F 0 generation mice are determined to be mutant heterozygotes.
Fourth, the verification and identification of the mice
Mating the positive heterozygotes to obtain offspring, cutting tails and identifying to obtain 6 positive homozygotes which are 14,22,58,65,66 and 69 respectively, and concretely comprises the following table:
| numbering | Genotype(s) | Sex | Date of birth |
| 14 | Ho | ♀ | 2016/6/18 |
| 22 | Ho | ♀ | 2016/7/7 |
| 58 | Ho | ♀ | 2016/10/29 |
| 65 | Ho | ♂ | 2016/12/1 |
| 66 | Ho | ♂ | 2016/12/1 |
| 69 | Ho | ♀ | 2016/12/1 |
The amplification primers were as follows:
and (3) PCR system:
| PCR Components | Source brand | Volume (μ L) |
| ddH2O | / | 15.7 |
| 10X la taq PCR Buffer | Thermo Fisher | 2 |
| 2.5mM dNTP | Thermo Fisher | 0.8 |
| Primer I(20pmol/μL) | Kinseruit | 0.2 |
| Primer II(20pmol/μL) | Kinseruit | 0.2 |
| Taq DNA polymerase | Thermo Fisher | 0.1 |
| Mouse tail genome | Thermo Fisher | 1 |
| Total | / | 20 |
PCR procedure:
| procedure for measuring the movement of a moving object | Temperature/. degree.C | Time of day | Remarks for note |
| 1 | 94 | 5min | - |
| 2 | 94 | 40s | - |
| 3 | 63 | 40s | - |
| 4 | 72 | 1min | Repeating the steps for 2-4 and 34 |
| 5 | 72 | 1min | - |
| 6 | 12 | - | Preservation of |
Sequencing primer:
| primer types | Sequence of(5’-3’) |
| Sequencing primer | AGGCAGGCAGATCTCAAGTCAGC |
The "peak pattern" of the sequencing results for the heterozygote mice is: mutated base sites, peak shape is bimodal; wild type and homozygous mice, because the PCR product is a single DNA molecule, the sequencing results for both genotypes are "peak-shape maps", both normal single peaks at the site of mutation, but the peak-shape signals for the two genotypes are different. Thus, at the site of the mutant base site, a heterozygote mouse with a double peak appears; the peak shape chart is normal unimodal, which may be wild type, or homozygote, and needs to be judged according to the sequence information of the mutation site; refer to fig. 1C.
Example 2 analysis of homozygote serum glycosylation
The operation method comprises the following steps:
(1) pretreatment of serum
50 μ L each of sera from homozygote (Ho) mice and wild type (Wt) mice were removed from a-20 ℃ refrigerator and thawed at room temperature.
(2) Enzymatic release of N-chain oligosaccharides
The prepared serum sample was digested with N-glycylamidase (PNGase F) purified in advance, and the reaction system was as follows: after the serum was dissolved in 28. mu.L of deionized water, 23. mu.L of 500mM phosphate buffer pH 7.5 and 12.5. mu.L of a denaturant (2% SDS aqueous solution, 3%. beta. -mercaptoethanol) were added in this order, and the mixture was thoroughly mixed and placed on a metal bath, and the mixture was heated at 95 ℃ for 10min to denature the protein, and then the sample was placed on ice, after cooling to room temperature, 19. mu.L of 10% polyethylene glycol octylphenyl ether (Triton-100) solution was added and mixed, and then incubated with 100. mu.L of PNGase F (1.6mg/mL) overnight at 37 ℃.
(3) Purification of N-chain oligosaccharides
The reacted sample was purified using a Supelclean ENVI-CarbTM pre-packed column by first activating a carbon column, washing the carbon column with 3mL of 80% Acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA), 3mL of distilled water, 3mL of 80% ACN containing 0.1% TFA, and 3mL of distilled water; the reacted solution was centrifuged at 12100 g at 4 ℃ for 10min, the supernatant was transferred to a carbon column by a pipette, after it completely flowed out, the column was washed with 3.0mL of distilled water, and finally 20% ACN and 40% ACN (both containing 0.1% TFA) eluted fractions (each 1.5mL) were collected, respectively, and the collected sample was placed in a vacuum centrifugal concentration dryer and spin-dried.
(4) Ultra-high performance liquid chromatography (UPLC) detection of serum N-chain oligosaccharides
The spun-dried sample was labeled with 2AB solution. 2, preparing an AB solution: containing 35 mmol. L-12-Aminobenzoic acid, 0.1 mol. L-1Sodium cyanoborohydride, and the solvent is dimethyl sulfoxide (DMSO) and glacial acetic acid (volume ratio is 7: 3). And (3) uniformly mixing 10 mu L of 2AB solution in the spin-dried sample, and reacting for 4 hours at 65 ℃ in a metal bath. Adding 20 μ L purified water into labeled mouse liver N-chain oligosaccharide, mixing well with vortex oscillator, centrifuging for 2min at 12100 g, mixing supernatant 15 μ L with 35 μ L acetonitrile, centrifuging for 3min at 12100 g, and placing supernatant 40 μ L into sample injection bottle with sample injection amount of 30 μ L. The column used for the detection was BEH Glycan column (1.7 μm, 2.1X 150mm, Waters); mobile phase A is 50 mmol.L at pH 4.5-1Ammonium formate solution; the mobile phase B is chromatographic pure acetonitrile; the excitation wavelength is 330 nm; the detection wavelength is 420 nm; the column temperature was set at 60 ℃.
Serum outcome analysis
From FIG. 2, it can be seen that sialylation and galactosylation of serum N-glycans of mice were almost completely eliminated, and the model mouse was suitable for the study of glycosylation disorder diseases.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be protected.
Sequence listing
<110> Nanjing university of agriculture
Construction method and application of <120> galactosyltransferase GalT gene point mutation mouse model
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctgccatatg ttcagtattt tgg 23
<210> 2
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtcattactt ggggtaaaaa cttactagcc atgggccctc tgtctgtgct tcctctccag 60
cctgccatat gttcagctgt ttggaggtgt ctctgctctc agtaaacaac agtttcttgc 120
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aggcaggcag atctcaagtc agc 23
Claims (8)
1. A method for constructing a galactosyltransferase GalT gene point mutation mouse model is characterized by comprising the following main steps:
(1) constructing a target: searching a CDS region of a galactosyltransferase B4GalT gene, determining an exon part and determining a gene mutation site;
(2) obtaining fertilized eggs, and performing microinjection: obtaining cas9 mRNA and gRNA by in vitro transcription, obtaining oligo donor DNA by synthesis, and performing microinjection on cas9 mRNA, gRNA and oligo donor DNA into fertilized eggs of a C57BL/6J mouse to obtain an F0 generation mouse;
(3) breeding the positive F0 mouse and C57BL/6J to obtain positive F1 heterozygote with B4GalT gene site-directed mutation;
(4) positive homozygote is obtained by PCR sequencing confirmation after the positive heterozygote mouse is mated, which indicates that the mouse model is successfully constructed.
2. The method for constructing a galactosyltransferase GalT gene point mutation mouse model according to claim 1, wherein the mutation site is 286 th amino acids in exon 4B 4GalT1 gene of mouse chromosome 4.
3. The method for constructing a galactosyltransferase GalT gene point mutation mouse model according to claim 1, wherein the DNA sequence of gRNA is shown in SEQ ID NO. 1.
4. The method for constructing a galactosyltransferase GalT gene point mutation mouse model according to claim 1, wherein the sequence of the oligo donor DNA is shown in SEQ ID NO. 2.
5. The method for constructing a galactosyltransferase GalT gene point mutation mouse model according to claim 1, wherein the primer for identifying F0 mouse is shown in SEQ ID NO. 3.
6. The use of the mouse model obtained by the method for constructing a galactosyltransferase GalT gene point mutation mouse model according to claim 1 in screening drugs for glycosylation-related diseases.
7. The use of the mouse model obtained by the method for constructing a galactosyltransferase GalT gene point mutation mouse model according to claim 1 in the preparation of lactose-free milk products.
8. Obtaining a kit of a galactosyltransferase GalT gene point mutation mouse model.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114214360A (en) * | 2021-12-27 | 2022-03-22 | 西安英创生物技术有限公司 | Congenital myasthenia gravis mouse model, and construction method and application thereof |
| CN114317603A (en) * | 2022-01-12 | 2022-04-12 | 北京航空航天大学 | Construction method and application of Foxi3 gene site-directed mutagenesis mouse model |
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Cited By (3)
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| CN114214360A (en) * | 2021-12-27 | 2022-03-22 | 西安英创生物技术有限公司 | Congenital myasthenia gravis mouse model, and construction method and application thereof |
| CN114317603A (en) * | 2022-01-12 | 2022-04-12 | 北京航空航天大学 | Construction method and application of Foxi3 gene site-directed mutagenesis mouse model |
| CN114317603B (en) * | 2022-01-12 | 2023-10-13 | 北京航空航天大学 | Construction method and application of Foxi3 gene site-directed mutagenesis mouse model |
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