CN117568399A - Galt gene knockout mouse model based on CRISPR-Cas9 system, construction method and application - Google Patents
Galt gene knockout mouse model based on CRISPR-Cas9 system, construction method and application Download PDFInfo
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- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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Abstract
The invention relates to a Galt gene knockout mouse model based on a CRISPR-Cas9 system, a construction method and application thereof, and relates to the technical field of animal genetic engineering, comprising the following steps: step 1: designing sgRNA targeting Galt genes; step 2: microinjection is carried out on sgRNA prepared by in vitro transcription and Cas9mRNA together into fertilized eggs of mice, then the fertilized eggs are transplanted into a surrogate mother mouse body to generate F0 generation, PCR identification is carried out on the F0 generation, positive F0 generation is continuously propagated for at least 2 generations, and a Galt gene knockout mouse model is obtained by screening; the sgRNA1 sequence is shown as SEQ ID NO. 1. The invention designs sgRNA aiming at the No. 9 exon of the mouse Galt gene, successfully constructs a Galt gene knockout mouse model by utilizing a CRISPR-Cas9 system, and ensures that the Galt gene is subjected to frame shift mutation during transcription and the GALT protein cannot be translated correctly, thereby simulating the human classical galactosylation and laying a foundation for exploring the occurrence and development mechanism of the human classical galactosylation, discovering new targets of medicines, evaluating preclinical pharmacodynamics and other biomedical researches.
Description
Technical Field
The invention relates to the technical field of animal genetic engineering, in particular to a Galt gene knockout mouse model based on a CRISPR-Cas9 system, a construction method and application.
Background
The CRISPR-Cas9 system is a gene editing tool engineered according to the bacterial defense mechanisms in nature against phage infection and plasmid transfer that can act on genome editing, transcriptional intervention, epigenetic regulation, and genome imaging. The CRISPR-Cas9 system is low in price, simple to operate and high in editing efficiency, and can carry out accurate gene editing through Homologous Directional Repair (HDR) and random gene editing through non-homologous end joining (NHEJ) after DNA Double Strand Break (DSB) is caused. Based on this, researchers have constructed many cell or animal disease models using CRISPR-Cas9 system to study gene functions and gene regulation, such as gene knockout human intestinal tissue-derived enteroid cell line, TSC2-KO NIH-3T3 cell line, muscular dystrophy pig model, alzheimer's disease mouse model, etc., and have made a certain research progress therefrom.
Galactosyls (galctosemia, GAL, OMIM # 230440) are autosomal recessive inherited metabolic disorders caused by inactivation or dysfunction of key enzymes in the galactose lepair metabolic pathway, including type I, type II and type III, caused by abnormalities in galactose-1 phospho-uridine transferase, galactose kinase and uridine diphosphate-galactose-4' -epimerase, respectively. Type I galactosylation (Type IGalactosamia, GAL I, OMIM # 230400) is also known as classical galactosylation (Classic Galactosemia), the prevalence rate is 1/30000-1/60000, and the prevalence rate is highest among three galactosylations. Galactose is vital to human body, has wide functions, is a key energy source before weaning of infants, and is particularly important for early development.
GAL I presents a potentially fatal disease during neonatal periods, which can lead to chronic debilitating complications and, if not treated in time, neonatal death. Clinical symptoms of GAL I can be divided into acute symptoms and chronic complications. The acute symptoms are caused by the fact that lactose in milk is taken after birth of a fetus, liver injury such as liver enlargement and liver cirrhosis is caused by lactose metabolism abnormality, and then symptoms such as cataract and renal insufficiency appear, and life is seriously threatened. Lactose and galactose diet restriction treatment is effective in alleviating the acute symptoms of GAL I, however, the chronic complications of GAL I are not addressed. Chronic complications are mainly lesions of the nervous system and reproductive system, manifested by language disorders, movement disorders, ovarian dysfunction, etc. Currently, limiting the dietary intake of lactose and galactose by patients is a major therapeutic measure, and no other therapeutic approach exists. For this reason, researchers have attempted to treat GAL I by developing specific antibodies and molecular protectants, etc., but have not found ideal treatments. In view of this, the invention provides Galt gene knockout mouse models based on CRISPR-Cas9 systems, construction methods and applications.
Disclosure of Invention
The invention aims to provide a Galt gene knockout mouse model based on a CRISPR-Cas9 system, a construction method and application thereof. The purpose is to construct Galt gene knockout mice by using a CRISPR-Cas9 system, to establish a mouse model for simulating human GAL I diseases, and to lay a foundation for exploring GAL I generation and development mechanisms, discovering new targets of medicines, evaluating preclinical pharmacodynamics and other biomedical researches.
In order to solve the technical problems, the first aspect of the invention provides a construction method of a Galt gene knockout mouse model based on a CRISPR-Cas9 system, which comprises the following steps:
step 1, designing sgRNA targeting Galt genes based on a CRISPR-Cas9 system;
step 2, microinjection of sgRNA and mRNA of Cas9 after in vitro transcription into a fertilized ovum of a mouse, then transplanting the fertilized ovum into a surrogate mother mouse body to generate an F0 generation, carrying out PCR identification on the F0 generation, continuously breeding positive F0 generation for at least 2 generations, and screening to obtain a Galt gene knockout mouse model; wherein, the sgRNA sequence is shown as SEQ ID NO: 1.
The present invention adopts Galt gene and uses mouse Galt-201 (ENSMUST 00000084695.12) transcript as edit transcript, the Galt-201 transcript (ENSMUST 00000084695.12) has 3.47kb whole length, which contains 11 Exons (Exons) and 10 Introns (Introns).
The beneficial effects of the invention are as follows: the invention designs sgRNA aiming at a pathogenic site Galt 9 exon of a classical galactosylated GALT gene, utilizes a CRISPR-Cas9 system to co-inject the sgRNA and the Cas9mRNA into fertilized eggs, successfully constructs a Galt gene knockout mouse model, causes frame shift mutation when the Galt gene is transcribed, ensures that the GALT protein cannot be translated correctly, successfully constructs the Galt gene knockout mouse model through the steps, and obtains homozygotes through breeding.
The above identification is carried out to obtain a Galt gene knockout mouse model, the specific identification method comprises the steps of adopting qPCR, WB and other experiments to detect the expression level of the GALT gene of the Galt gene knockout mouse, recording the weights of the Galt gene knockout mouse and a wild type mouse, further analyzing the relevant phenotype of the model mouse through pathological section experiments, and obtaining the following conclusion:
(1) The expression level of Galt mRNA and the expression level of GALT protein are detected by qPCR experiments and WB experiments, and the experimental results show that: galt mRNA and GALT protein expression levels were significantly lower in Galt knockout mice than in wild-type mice, and were statistically different.
(2) The weight of wild-type mice was slightly higher than that of Galt knockout mice 15 weeks before mice were grown.
(3) The pathological and histomorphological analysis of the major tissue organs by paraffin section HE staining revealed that heart, spleen and kidney tissues of Gal knockout mice were not abnormal, but liver of Gal knockout mice was edematous to different extents and lung injury was observed as compared with wild type mice.
On the basis of the technical scheme, the invention can be improved as follows.
Further, bases 829 to 837 on the cDNA sequence of the positive F0-generation mouse Galt gene were knocked out, and 1 base T was inserted, and the other nucleotide sequences of the Galt gene were kept unchanged.
The deleted DNA sequence is located on exon 9 of Galt gene, resulting in frame shift mutation during transcription and improper translation of GALT protein.
Further, the step 2 includes the following specific steps:
step 21, the mouse ovulation promotion and in vitro fertilization are carried out to obtain a mouse fertilized egg;
step 22, microinjecting sgRNA and Cas9mRNA into cytoplasm of the mouse fertilized egg together after in vitro transcription to obtain injected fertilized egg;
step 23, transplanting the fertilized eggs after injection into oviduct of a surrogate mother mouse, obtaining F0 generation mice after development is completed, carrying out PCR identification on the F0 generation, mating positive F0 generation with a wild type mice to obtain F1 generation heterozygotes, hybridizing the F1 generation heterozygotes, and screening homozygous offspring of Galt gene knockout as a Galt gene knockout mouse model.
Further, specific primers for the PCR identification include: galt-M-ES-1, galt-M-EA-1, galt-M-IS-1 and Galt-M-IA-1; the sequence of Galt-M-ES-1 IS shown as SEQ ID NO. 3, the sequence of Galt-M-EA-1 IS shown as SEQ ID NO. 4, the sequence of Galt-M-IS-1 IS shown as SEQ ID NO. 5, and the sequence of Galt-M-IA-1 IS shown as SEQ ID NO. 6.
Further, the mass ratio of sgRNA to Cas9mRNA is (0.9-1.1).
Further, the donor of the single fertilized ovum of the mouse is a C57BL/6J mouse; the surrogate mother mouse is an ICR mouse.
In a second aspect, a Galt gene knockout mouse model based on a CRISPR-Cas9 system is provided, which is prepared by the preparation method of any one of the above-described methods.
A third aspect provides a kit for constructing a Galt gene knockout mouse model based on a CRISPR-Cas9 system, the kit comprising the sgRNA and the Cas9mRNA as described above.
In a fourth aspect, there is provided the use of the above Galt knockout mouse model or the above kit for developing and/or screening a therapeutic type I galactosylation.
Further, the substance is a drug.
Drawings
FIG. 1 is a block diagram of Galt-201 transcripts in Ensembl database of the present invention;
FIG. 2 is a map of the targeting sites of the mouse Galt gene of the invention;
FIG. 3 shows the F1 Galt generation according to the invention -/- And Galt +/- Offspring of the knockout mice;
FIG. 4 shows the F1 Galt generation according to the invention -/- And Galt +/- Gene knockout mouse offspring Sanger sequencing results;
FIG. 5 shows the results of total RNA non-denaturing electrophoresis in liver of Galt knockout mice of the present invention;
FIG. 6 shows the measurement of the expression level of Glat mRNA in liver of Galt knockout mice of the present invention;
FIG. 7 shows the results of electrophoresis for detecting the protein level of the Galt gene knockout mouse, wherein A is a graph of the GALT protein expression level in the Galt gene knockout mouse, and B is a graph of the statistical result;
FIG. 8 shows HE staining of heart, liver, spleen, lung and kidney histopathological sections of WT mice and Galt knockout mice of the invention.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Examples
1. Experimental animal, main reagent and common solution
1.1 laboratory animals
C57BL/6J and ICR mice of grade SPF (Specific pathogen Free) were purchased from Hunan Style Lekka laboratory animals Co., ltd and kept in the Guilin medical college of intelligent medicine and biotechnology college of animal laboratories. The design and the scheme of the invention are approved by the animal nursing and use committee of Guilin medical college, and the animal ethical examination batch number is GLMC201803033. Mice were euthanized by intraperitoneal injection of an excess of 200mg/kg sodium pentobarbital, followed by rapid collection of individual tissue samples from the mice for subsequent experiments. WT mice in the following are purely wild C57BL/6J mice.
1.2 description of Main reagents and consumables
TABLE 1 Main reagents and consumables
1.3 preparation of a commonly used solution
1) Preparation of 10 XTBS solution and preparation of 1 XTST buffer
TABLE 2 preparation of 10 XTBS solution and preparation of 1 XTBST buffer
Wherein the 10 XTBS solution was formulated requiring pH adjustment to 7.6 with 12N HCl, distilled water was added to a final volume of 1L.
2) Preparation of transfer solution, preparation of 5 XSDS-PAGE electrophoresis solution and preparation of 50 XTAE solution
TABLE 3 preparation of transfer solution, preparation of 5 XSDS-PAGE electrophoresis solution and preparation of 50 XTAE solution
3) Preparation of LB liquid culture medium and preparation of LB solid culture medium
Table 4 preparation of LB liquid Medium
| Sequence number | Reagent name | Dosage of |
| 1 | Yeast extract | 0.5g/100ml |
| 2 | Pancreatic proteins | 1g/100ml |
| 3 | NaCl | 1g/100ml |
| 4 | ddH 2 O | Constant volume to 100ml |
Preparation of LB solid medium: compared with the LB liquid medium, the preparation of the LB solid medium is also added with 1.5g/100ml of agar powder, and the rest is the same as the preparation of the LB liquid medium.
2. Experimental method
2.1 design of the sgRNA sequence of the target mouse Galt Gene
The mouse Galt (Gene ID: 14430) Gene is located on chromosome 4 and has a total of 10 transcripts. The human GALT in the NCBI and Ensembl databases was selected as the editing transcript from the mouse GALT-201 (ensmset 00000084695.12) transcripts after analysis of their genetic structure based on comparison with the mouse GALT genomic and transcriptome information. Galt-201 transcript (ENSMUST 00000084695.12) was 3.47kb in length, and contained 11 Exons (Exons) and 10 Introns (Introns) (FIG. 1). The Gal gene knockout mouse of the invention knocks out 9 bases in the exon of Galt 9 and inserts a T base, and the Galt gene knockout mouse generates frame shift mutation during transcription due to the base deletion on the exon 9, so that the GALT protein cannot be translated correctly.
Specifically, the invention designs a sgRNA sequence (sequence information is shown in table 5) aiming at a mouse Galt gene based on a CRISPR-Cas9 gene editing system, so that the mouse Galt gene is mutated at a Galt c.847+1G site (shown in figure 2), and the translation error of a GALT protein is caused. And (3) microinjection is carried out on Galt-sgRNA (SEQ ID NO. 1) and the Cas9mRNA together into fertilized eggs of a mouse, then the fertilized eggs are transplanted into a surrogate mother mouse body to generate F0 generation, PCR identification is carried out on the F0 generation, positive F0 generation is continuously propagated for at least 2 generations, and a Galt gene knockout mouse model is obtained through screening.
Table 5 shows sgRNA sequence information
| Sequence name | Sequence (5 '-3') | Sequence number |
| Galt-sgRNA | AGCCTTACCATGCCAGCCCA | SEQ ID NO.1 |
2.2 preparation of Galt Gene knockout mouse model
2.2.1 obtaining fertilized eggs of mice
(1) Superovulation. C57BL/6 female mice with the age of 3-3.5W are selected, and Pregnant Mare Serum Gonadotropin (PMSG) and 10 UI/female mice are injected into the female mice at 5 pm on the first day of the experiment; female mice that had been injected with PMSG were injected with Human Chorionic Gonadotrophin (HCG), 10 UI/day at 5 pm on the third day (same time as the first day).
(2) Preparation of embryo culture dishes. After the hormone injection on day three, TYH sperm capacitation dishes, HTF fertilization dishes, KSOM embryo culture dishes were made in a 35mm dish in an ultra clean bench. After all dishes are manufactured, the dishes are placed at 37.5 ℃ and 5% CO 2 The incubator was equilibrated overnight.
(3) Sperm capacitation. About 9 in the morning on the fourth day of the experiment, selecting an adaptive C57BL/6 male mouse of 3-6 months, killing the male mouse by a cervical dislocation method, dissecting the male mouse by using a surgical instrument, opening the abdominal cavity, finding the epididymis and taking down the epididymis. The excess blood and fat were removed with absorbent paper, placed in mineral oil in TYH sperm capacitation dishes, cut with an ophthalmic scissors to open a small opening on the epididymis, squeeze semen from the small opening under a stereoscopic microscope using micromanipulation forceps, and move semen into 200 μl TYH droplets. TYH sperm capacitation dishes were then placed in a 5% CO2 incubator at 37.5℃for 50min.
(4) In vitro fertilization. After the female mice were sacrificed, the back skin was exposed with an ophthalmic scissors, the ovaries and fallopian tube segments were cut off with absorbent paper to remove excess fat and blood thereon, placed in a fertilization dish, and the enlarged parts of the fallopian tubes were found under a stereoscopic microscope, and the egg masses were scraped into HTF droplets with a 1mL syringe. After sperm capacitation was completed, 2.5 μl of sperm was aspirated along the droplet edge with a loading gun and added to each 200 μl fertilization drop, and the HTF fertilization dishes were placed in an incubator for continued culture.
2.2.2 microinjection of fertilized mouse eggs
(1) Cleaning and selecting fertilized eggs. After sperm and egg cells are incubated for about 8 hours, embryo primary screening is carried out in an HTF fertilization dish, unfertilized eggs, dead embryos, malformed embryos, multi-core embryos and other embryos which cannot be subjected to experiments are removed, full and round fertilized egg cells with complete zona pellucida, clear prokaryotes and moderate two-nucleus area size are selected and transferred from 200 mu L HTF liquid drops to 50 mu L HTF liquid drops for primary cleaning, then the embryos are transferred into an M2 embryo cleaning dish, cleaning is carried out for 6-10 times by using an oral suction tube, cumulus cells and other impurities attached to the fertilized eggs are removed, and the cleaned embryos are put back into an incubator for standby.
(2) And (3) preparing a microinjection liquid. The sgrnas, cas9mRNA were removed from the-80 ℃ refrigerator and placed on ice for solubilization. And (3) respectively sucking the sgRNA and the Cas9mRNA in the ultra-clean bench by using an enzyme-free gun head according to a certain proportion, and fully and uniformly mixing the sgRNA and the Cas9mRNA in an enzyme-free 1.5EP tube, so that the final concentration of the sgRNA and the Cas9mRNA in a mixed solution is 200ng/uL. The mixture was placed in a centrifuge and centrifuged at 2000rpm for 2min, after the centrifugation was completed, 1uL of the mixture was aspirated by using a Microloader microsampling needle and transferred into a microinjection needle.
(3) Transfer of fertilized eggs. Transferring 50-100 washed fertilized eggs into M2 culture droplets of an injection dish, uniformly aligning the fertilized eggs, and placing the injection dish into an incubator for incubation for 5-10min.
(4) Instrument tuning prior to microinjection. Before starting microinjection, the microinjection operating system is debugged.
(5) Microinjection. After the instrument is debugged, the fertilized eggs are fixed by adjusting the fixing needle through negative pressure, and the polar body is avoided during the fixation. After the fertilized egg is fixed, the mixed solution is injected into the cytoplasm of the fertilized egg through a microinjection needle. After the injection is finished, the injected fertilized eggs are moved into an egg picking dish by an oral suction tube for cleaning for 5-10 times, the cleaned fertilized eggs are moved into a KOSM embryo culture dish, and the KOSM embryo culture dish is placed into an incubator for continuous culture.
2.2.3 embryo transfer
(1) Ligation of ICR male mice. ICR male mice of 3.5-4 weeks of age were selected and kept in cages after ligation.
(2) Preparation of ICR recipient females. In the day of in vitro fertilization, about 5 pm, selecting estrus ICR female mice with a week age of 6-12 weeks and a weight of 25-35g, and caging the ICR female mice with ICR ligature male mice to select ICR female mice with a thrombus as a receptor.
(3) And (5) fertilized egg selection. Fertilized eggs which develop normally to the 2-cell stage after microinjection are selected and counted, and 15 fertilized eggs are put into droplets of a KSOM embryo culture dish as a group for standby.
(4) Embryo transfer. Weighing and anesthetizing an ICR acceptor female mouse with a thrombus, opening the back, finding the ampulla of the oviduct, shearing the oviduct part at the front end of the ampulla by using a micro-shearing port, transferring the embryo into an oral suction tube by using a three-section method, taking the expanded part of the oviduct into which air bubbles enter as a success transplanting sign, suturing muscle tissues and skin of the mouse, placing the mouse on a hot bench for awakening, nursing after operation and carrying out pregnancy and farrowing.
2.2.4 genotyping of neonatal mice
(1) Amplification of the F0 mice pseudo-mutation site region. ICR recipient females born approximately 21 days after embryo transfer, and neonatal mice were F0 generation. After 7 days of birth, the mice were sheared, rat tail DNA was extracted with a tenna blood/cell/tissue genomic DNA extraction kit (DP 304-02), and PCR amplification was performed on the region of the pseudomutation site, and the PCR products were sequenced for genotyping. Primer sequences Galt-M-ES-1 and Galt-M-EA-1 of the PCR reaction are shown in Table 6, reaction systems are shown in Table 7, and PCR amplification reaction procedures are shown in Table 8.
(2) And (5) recycling the PCR product glue. The mouse DNA confirmed to have undergone gene editing was subjected to PCR again, and the PCR product was subjected to 1% agarose gel electrophoresis at 120V for 30min. After electrophoresis, cutting out a single strip which meets the target size and is in an EP tube, and recovering the gel according to the operation instruction of a common DNA product purification kit (DP 204-02), wherein the concentration of the recovered PCR product needs to reach 40 ng/. Mu.L to meet the requirement of the next experiment.
(3) Ligation transformation. 1. Mu.L of the gel recovery product and 1. Mu.L of pMD 19-T Vector were mixed in a PCR tube and ddH was added 2 O was made up to 5. Mu.L, 2.5. Mu.L of Solution I enzyme was added and all reactions were formulated on ice. The mixture was placed in a PCR apparatus at 16℃for 1h for ligation. After the connection is finished, taking out the escherichia coli from a refrigerator at the temperature of minus 80 ℃ and placing the escherichia coli on ice for dissolution, taking 30 mu L of the escherichia coli and 7.5 mu L of a connection product, placing the connection product on an EP pipe with the volume of 1.5mL for uniform mixing, and carrying out ice bath for 30min and metal bath at the temperature of 42 ℃ for 90s; ice bath for 2min. 150. Mu.L of LB liquid medium without antibiotics is added into an EP tube, put into a shaking table at 37 ℃ and incubated at 220rpmCulturing for 40min to revive thallus. After the fungus shaking is finished, evenly coating the fungus liquid in LB solid culture medium containing ampicillin, and placing the culture medium in a constant temperature oven at 37 ℃ for culturing for 16-18h until single colony grows.
(4) Single colonies were picked. Single colonies were picked with a small tip in an ultra clean bench to 10. Mu.L ddH 2 In the PCR tube of O, 10-15 single colonies were picked from each LB solid medium.
(5) Bacterial liquid PCR. Bacterial liquid PCR was performed using the picked single colony as a template, the bacterial liquid PCR system is shown in Table 9, and the PCR reaction procedure is shown in Table 10.
(6) Sequencing. After bacterial liquid PCR, 1% agarose gel electrophoresis was performed, 8ul of the remaining bacterial liquid which meets the target size and is single in band was transferred to a 2mL centrifuge tube, and 1.5mL of LB liquid medium containing ampicillin was added, and the bacterial liquid was placed in a shaker at 37℃and 150rpm, cultured overnight, and the bacterial liquid was sequenced the next day.
TABLE 6 PCR amplification primer sequences for regions of pseudo-mutation sites
TABLE 7 PCR amplification reaction System for pseudo-mutation site region
TABLE 8 PCR amplification reaction procedure 1 for the region of the pseudo-mutation site
TABLE 9 bacterial liquid PCR amplification reaction system
| Sequence number | Reaction components | Volume (mu L) |
| 1 | Single colony bacterial liquid | 2 |
| 2 | M13F(10pmol/μL) | 0.5 |
| 3 | M13R(10pmol/μL) | 0.5 |
| 4 | Premix Taq TM | 5 |
| 5 | ddH 2 O | 2 |
TABLE 10 bacterial liquid PCR amplification reaction procedure
2.2.5 breeding of Galt Gene knockout mice
Editing F0 generation Galt gene, breeding with WT mice in cages, cutting F1 generation to obtain F1 generation miceThe mouse tail, extracting DNA, PCR amplification and sanger sequencing to identify genotype. Will be identified as Galt +/- The F1 generation of the gene knockout mice are subjected to inter-group mating and homozygous breeding, and offspring produced by the mice are subjected to sanger sequencing, so that homozygous genotypes are screened.
2.3 detection of the molecular level of Galt Gene knockout mice
2.3.1 detection of mRNA expression level in Galt Gene knockout mice
(1) Extraction of total RNA. WT mice, galt -/- After euthanasia of the knockout mice, livers were taken and total RNA was extracted on ice by Trizol (at least 3 mice per group). The liver was weighed in a 2mL EP tube, 1mL of pre-chilled Trizol reagent was added to each 100mg of tissue, 2-3 beads were added to the EP tube, ground with an animal tissue grinder, the ground tissue was placed on ice for 10min, followed by 12000g,4℃and centrifugation for 15min, and the supernatant was transferred to another EP tube. 1/5Trizol volume of chloroform was added to the EP tube, mixed by forced inversion (vortex shaker was not used to avoid fragmentation of RNA fragments), left to stand at room temperature for 3min, and allowed to separate (this procedure was repeated several times to allow adequate separation of the liquid), 12000g, and centrifuged at 4℃for 15min. After centrifugation, the solution was separated into 3 layers, the upper layer being the aqueous phase, the middle layer being the protein, the lower layer being the organic phase, and the RNA being in the upper aqueous phase, the upper aqueous phase was carefully aspirated into the new EP tube with a pipette. Adding 1/2Trizol volume of precooled isopropanol into an EP tube, gently mixing by a pipette, standing at room temperature for 10min,12000g, centrifuging at 4 ℃ for 15min, carefully discarding the supernatant after centrifugation, and precipitating to obtain the total RNA extracted. To the EP tube, 75% ethanol was added in an amount of 1 time the volume of Trizol for washing, followed by 7500g, centrifugation at 4℃for 5min, the supernatant was discarded, and standing at room temperature for 5-15min, to ensure complete evaporation of water and ethanol.
(2) And (5) detecting total RNA quality. After the RNA pellet was completely dried, 30. Mu.L of enzyme-free water was added to the EP tube to dissolve the pellet, and the concentration was determined to be about 2.0 for a pure RNA sample A260/A280, followed by 1% agarose gel electrophoresis to determine whether the extracted RNA was intact.
(3) Removal of genomic DNA and reverse transcription. The total RNA extracted was reverse transcribed. The reaction system for removing genomic DNA from total RNA was prepared in an enzyme-free PCR tube according to TaKaRa reverse transcription kit (RR 047A) and shown in Table 11, and the PCR tube was placed in a PCR instrument and reacted at 42℃for 2min. Then preparing a reverse transcription reaction system on ice, wherein the reaction system is shown in table 12, and the reaction procedure is that the temperature is 37 ℃ for 15min; 5s at 85 ℃;4 ℃ is infinity.
(4) qPCR. According to TaKaRa company TBThe Ex TaqTM II kit (RR 820L, taKaRa) was used to prepare qPCR reaction systems (see Table 13). The experiment uses a two-step PCR amplification procedure: 95 ℃ for 30s;95 ℃ for 5s,60 ℃ 30s for 40repeats;95℃15s,60℃30s,95℃15s (dissociation). The relative expression level of Galt gene was calculated by 2-DeltaCT method using mouse GAPDH as an internal reference gene, and the primers for qPCR reaction are shown in Table 14.
TABLE 11 genomic DNA removal reaction System
| Reaction components | Volume (mu L) |
| 5×gDNA Eraser Buffer | 2.0 |
| gDNA Eraser | 1.0 |
| Total RNA | Total RNA 1. Mu.g |
| RNase Free dH 2 O | up to 10μL |
TABLE 12 reverse transcription reaction system
| Reaction components | Volume (mu L) |
| Genomic DNA removal reaction solution | 10.0 |
| PrimeScript RT Enzyme Mix I | 1.0 |
| RT Primer Mix | 1.0 |
| 5×PrimeScript Buffer 2(for Real Time) | 4.0 |
| RNase Free dH 2 O | 4.0 |
| Total | 20 |
TABLE 13qPCR reaction liquid formulation
| Reaction components | Volume (mu L) |
| TB Green Premix Ex Taq II(2×) | 12.5 |
| PCR Forward Primer(10μM) | 1.0 |
| PCR Reverse Primer(10μM) | 1.0 |
| RT reaction solution (cDNA solution) | 2 |
| Sterilizing water | 8.5 |
| Total | 25 |
TABLE 14 qPCR amplification primer sequences for Galt Gene
2.3.2 detection of protein expression level in Galt Gene knockout mice
(1) And (5) extracting total protein. After euthanizing 3 WT mice and 3Galt knockout mice, the livers were weighed in 2mL EP tubes, 100-200. Mu.L of cell lysate (Biyun, P0013) was added per 20mg of tissue, and PMSF (Biyun, ST 506) was added to the lysate before use to give a final concentration of 1mM PMSF. 2-3 magnetic beads were added to the EP tube, ground with an animal tissue grinder, the ground tissue suspension was subjected to ice lysis for 30min, followed by 12000rpm,4℃and centrifugation for 15min, and the supernatant was transferred to another fresh EP tube, and total tissue proteins were present in the supernatant.
(2) BCA assay to determine total protein concentration: the concentration of extracted total tissue proteins was determined according to BCA protein concentration assay kit (P0012S) instructions from the yunnan biotechnology company. And finally, drawing a standard curve according to the numerical value measured by the enzyme labeling instrument, and calculating the total protein concentration according to the standard curve.
(3) Protein denaturation. Taking out a part of protein sample according to the concentration measured by BCA method, mixing the sample with 5 XSDS-PAGE loading buffer, quantifying to 5ug/μl, placing the mixed sample in a metal bath, heating at 100deg.C for 10min, reversing and mixing at any time during heating process, heating to uniformity, cooling to room temperature, centrifuging at 12000rpm at 4deg.C for 10min, and storing at-20deg.C for use.
(4) Gel preparation of SDS-PAGE gels. 10% of the separation gel was prepared according to the specification of SDS-PAGE gel preparation kit (P0012A) from Biyun biotechnology Co., ltd, and the separation gel was added to a glass plate and ddH was used 2 O is pressed into glue, and is kept stand at room temperature, and ddH is removed after the separated glue is solidified 2 O, preparing concentrated glue, filling a glass plate, inserting a comb into the concentrated glue, standing at room temperature, and solidifying the concentrated glue.
(5) SDS-PAGE electrophoresis. After the prepared gel is assembled, the gel is placed in an electrophoresis tank, and the electrophoresis tank is filled with a new 1 XSDS-PAGE electrophoresis buffer. Protein samples were removed from-20℃and centrifuged at 12000rpm and 4℃for 10min. In loading, 2. Mu.L of protein Marker (Thermo Fisher, 26616) was applied to the first well, followed by sequential loading of protein samples, with 6. Mu.L of protein sample per well, i.e.a total protein loading of 30. Mu.g. After the sample application is finished, electrophoresis is started, the electrophoresis is stopped when the sample runs to the bottom of the separation gel under the constant voltage of 80V and the constant voltage of 120V is converted.
(6) And (5) transferring films. After electrophoresis, pouring the transfer film liquid into a white tray, separating the glue from the glass plate, immersing the glue into the transfer film liquid, cutting off glue blocks containing target proteins and reference proteins by using a glue cutting plate according to the Marker strip on the glue, and immersing the cut glue into the transfer film liquid for later use. Then cutting two PVDF films with the same size as the glue, and soaking in absolute methanol for about 1 min. The "sandwich" was assembled in the order blackboard, foam-rubber cushion, filter paper, glue, PVDF film, filter paper, foam-rubber cushion, whiteboard, note that no air bubbles could be present between each layer. The sandwich was placed in a transfer film apparatus, 250mA constant flow film for 90min.
(7) And (5) sealing. After the transfer, the PVDF membrane was removed, washed 1 time with 1 XTBST buffer and decolorized in a shaker for 5min. 5% skim milk was then prepared with 1 XTBST buffer and blocked for 2h.
(8) And (5) incubating the primary antibody. After the end of the blocking, the cells were washed 4 times with 1 XTBE buffer for 10min each. Recombinant Anti-GALT antibodies (Abcom, ab 178406), GAPDH Polyclonal antibody (Proteintech, 10494-1-AP) were diluted with Western primary antibody dilutions (Biyun Tian, P0023A) at a ratio of 1:2000, 1:5000, respectively. The PVDF membrane was incubated with diluted primary antibody overnight in a refrigerator at 4 ℃.
(9) And (5) incubating the secondary antibody. The overnight incubated PVDF membrane was removed from the refrigerator, and the primary antibody was recovered, and the PVDF membrane was washed with 1 XTBST buffer 4 times for 10min each. The goat anti-rabbit secondary antibody labeled with horseradish peroxidase diluted with 5% skim milk at a ratio of 1:5000 was incubated for 2h at room temperature. After the secondary antibody incubation, the PVDF membrane was washed with 1 XTBST buffer 4 times for 10min each.
(10) And (5) emitting light. The reagent A and the reagent B in the hypersensitive ECL chemiluminescence kit (P0018S) of Biyun biotechnology company are uniformly mixed according to the proportion of 1:1, a luminescent mixed solution is uniformly dripped on a PVDF film, and a Tanon gel imaging system is utilized for observing results.
2.4 phenotyping of Galt knockout mice
2.4.1 observation of growth of Galt Gene knockout mice
The body weights of Galt knockout mice and WT mice were recorded to see whether the knockout mice exhibited obvious symptoms such as growth retardation.
2.4.2 analysis of pathological section of Galt Gene knockout mice
(1) Drawing materials and fixing. After euthanizing the WT mice and Galt gene knockout mice with similar ages, taking the liver, kidney, ovary and other tissues, putting the tissues into 4% paraformaldehyde solution for fixation for 48 hours, and flushing the tissues for 30 minutes by tap water.
(2) Dehydration and transparency. Dehydrating the fixed tissue by using gradient alcohol: 75% ethanol 1h,85% ethanol 1h,95% ethanol (I) 1h,95% ethanol (II) 1h, absolute ethanol (I) 1h; absolute ethanol (II) for 1h. After dehydration, the mixture was subjected to transparency treatment with a xylene solution twice for 30 minutes each.
(3) Wax dipping, embedding and slicing. Soaking the transparent tissue in paraffin (soft wax) at 50-52deg.C for 1 hr, and paraffin (I, hard wax) at 58-60deg.C for 1 hr; soaking in paraffin (II) at 58-60deg.C for 1 hr. After the wax dipping is finished, the tissues are embedded by paraffin (paraffin) at 58-60 ℃. Slicing by using a microtome of Thermo Fisher company of America, and standing at 65deg.C for 3-4 hr.
(5) HE staining: the sections were stained according to the Hematoxylin Eosin (HE) staining kit (G1120) instructions from beijing solebao technologies. Dewaxing was performed twice in xylene for 5min each. Gradient alcohol rehydration (order: 100%, 95%, 85%, 75%) for 3min per gradient. Soaking in distilled water for 2min. Hematoxylin staining solution is used for staining for 2-5min (the specific time is determined according to the situation), floating color is washed off by distilled water, differentiation solution is differentiated for 10-60s, and tap water is used for soaking and washing twice for 3-5min each time. Eosin staining solution for 30s-2min, pouring off excessive staining solution, and rapidly dehydrating (gradient alcohol: 75%, 85%, 95% and absolute alcohol (I) for 2-3s, absolute alcohol (II) for 1 min). Xylene was transparent twice, 1min each, sealed with neutral gum and observed under a mirror.
2.5 statistical analysis
All data result analyses were statistical analysis using GraphPad Prism 9 software, comparison between two sets of data was performed using t-test (Student's t-test), comparison between three and more sets of data was performed using One-way Anova, all experimental results were processed in mean ± standard deviation (X ± SD) form, P <0.05 was expressed in X.
3. Results
3.1 construction of Galt Gene knockout mice based on CRISPR-Cas9 technology
Production of 3.1.1F0 generation mice
Microinjection was performed using the sgrnas and Cas9mRNA of 2.1 above, and a Galt knockout mouse model was prepared according to the description of 2.2 above, yielding 4 galts +/- The gene knockout mice are two female mice and two male mice, and the 4 mice are bred as F0 generation.
Production of 3.1.2F1 generation mice
Galt +/- F0 generation mice of the gene knockout heterozygous mice are respectively bred with WT mice in cages. Cutting the tail of F1 generation mice produced by Galt gene knockout mice, extracting DNA, detecting the genotype of F1 generation by Sanger sequencing and TA cloning to obtain Galt +/- A knockout mouse.
3.1.3 breeding of homozygous mice
Respectively taking F1 generation Galt +/- The knockout mice are bred into homozygotes by using a combined cage, after the mice are bred, DNA samples of the mice are taken for PCR amplification and Sanger sequencing to determine the genotype. FIG. 3 shows the F1 Galt generation +/- The primary young mice produced are bred in the gene knockout mice group. The sequencing result (see FIG. 4) shows that the sequencing result of the mouse No.1 has no peak, and the mouse No.1 is Galt when the result is compared with the sequence of the WT mouse -/- A knockout mouse (homozygote) which knocks out 9 bases in the targeting region and inserts one T base; the sequencing result of mouse No. 2 shows that there is a cover peak, which is Galt +/- Knockout mice (heterozygotes).
3.2 detection of the molecular level of the Galt Gene knockout mouse
WT mice and Galt were taken separately -/- The liver of the knockout mice was subjected to Trizol method to extract total RNA, the concentration of the total RNA extracted is shown in Table 15, and the result of electrophoresis is shown in FIG. 5. The qPCR was performed with the better quality RNA, and the data obtained was statistically analyzed, and the results are shown in FIG. 6. Galt -/- Galt mRNA expression levels were significantly lower in knockout mice than in WT mice, with statistical differences.
WB assay detectionGalt found in liver tissue -/- GALT protein expression levels were significantly lower in the knockout mice than in the WT mice, with statistical differences (see fig. 7A and 7B).
TABLE 15 Total RNA concentration in mouse liver (ng/. Mu.L)
3.3 phenotyping of Galt knockout mice
3.3.1 growth of Galt knockout mice
To investigate whether Galt knockout mice would exhibit GAL I growth retardation characteristics. Under the condition of normal feeding, the WT mice and Galt are subjected to the following conditions -/- The knockout mice were weighed. As a result, it was found that the weight of the wild-type mice was slightly higher than that of the Galt knockout mice 15 weeks before the growth of the mice.
3.3.2 pathological section analysis of Galt Gene knockout mice
In WT mice, GALT protein is found in the major viscera: galt is taken because it is expressed in heart, liver, spleen, lung and kidney and its expression level is high -/- The above organs of the knockout mice and the WT mice were subjected to pathological section, HE staining, and whether or not the morphology thereof was different from that of the WT mice was observed under a microscope.
As shown in FIG. 8, WT mice and Galt -/- In the HE staining of heart tissue sections of gene knockout mice, myocardial cells of longitudinal sections are arranged in parallel with each other, and although there are bifurcation, they are anastomosed to form a net; cardiomyocytes are oval, generally mononuclear, sometimes binuclear, and shallowly stained in the center. The myocardial fiber has small blocks with circular or irregular cross sections, and has round cell nuclei with shallow staining, no nuclei and shallow staining in the center. The shape is normal, and no pathological changes exist.
As shown in FIG. 8, in WT mice and Galt -/- Red marrow, white marrow, trabecula, spleen corpuscle and other structures can be observed in spleen tissue pathological sections of the gene knockout mice, and the morphological structure of the spleen is not abnormal.
As shown in FIG. 8, in WT mice and Galt -/- In kidney tissue pathological sections of the gene knockout mice, normal cortical, medullary, glomerulus, distal tubular and proximal tubular can be observed, and lesions such as renal fibrosis, renal injury, glomerular occlusion and the like are not generated.
As shown in FIG. 8, galt compared with WT mice -/- The hepatomegaly, the cytokinesis and the empty cytoplasmic loosening can be observed in the liver tissue pathological section of the gene knockout mouse, and the cytokinesis is netty or transparent, and most of nuclei are centered, which indicates Galt -/- Liver tissue of knockout mice exhibited pathological changes of severe edema.
As shown in FIG. 8, the cells were observed to be in Galt compared with the WT mice -/- Alveolar wall thickening, alveolar shape change, partial alveolar rupture or fusion in lung tissue pathological sections of the knockout mice, and lesions accompanied by neutrophil infiltration.
Conclusion 4
The invention successfully constructs Galt aiming at the pathogenic gene GALT gene of classical galactosylation -/- A knockout mouse. Galt -/- The knockout mouse knocks out 9 bases (829 to 837 bases on cDNA sequence) on exon, and inserts a T base, which causes frame shift mutation of Galt gene during transcription, and GALT protein cannot be translated correctly. The invention utilizes qPCR and WB experiments to respectively detect Galt -/- The Galt mRNA expression level, galt protein expression level of the knockout mice and WT mice were further analyzed for the relevant phenotype of the model mice by pathology section experiments, leading to the following conclusions:
(1) The method comprises the steps of designing sgRNA aiming at a classical galactosylation pathogenic gene GALT gene, and utilizing a CRISPR-Cas9 system to co-inject the sgRNA and Cas9mRNA into fertilized eggs to successfully construct Galt -/- A knockout mouse.
(2) The expression level of Galt mRNA and the expression level of GALT protein are respectively detected by qPCR experiment and WB experiment, and the experimental result shows that -/- Galt mRNA and GALT protein expression levels of the knockout mice are significantly lower than those of the WT mice, and the expression levels of the Galt mRNA and GALT protein are statistically significant.
(3) Galt compared to WT mice -/- The weight of the knockout male mice is slightly lower than that of the WT mice. Analysis of morphology and function of tissues and organs by pathological section and HE staining, galt was found compared with WT mice -/- Severe edema, galt, occurred in the liver of the knockout mice -/- Lung alveolar wall thickening, alveolar shape changes, partial alveolar rupture or fusion, with neutrophil infiltration lesions in the knock-out mice.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. The construction method of the Galt gene knockout mouse model based on the CRISPR-Cas9 system is characterized by comprising the following steps:
step 1, designing sgRNA targeting Galt genes based on a CRISPR-Cas9 system;
step 2, microinjection is carried out on the sgRNA and mRNA of the Cas9 after in vitro transcription into a fertilized egg of a mouse, then the fertilized egg is transplanted into a surrogate mother mouse body to generate an F0 generation, PCR identification is carried out on the F0 generation, positive F0 generation is continuously propagated for at least 2 generations, and a Galt gene knockout mouse model is obtained through screening;
wherein, the sgRNA1 sequence is shown in SEQ ID NO: 1.
2. The method for constructing a Galt gene knockout mouse model based on CRISPR-Cas9 system according to claim 1, wherein bases 829 to 837 on the cDNA sequence of the positive F0-generation mouse Galt gene are knocked out and 1 base T is inserted, and other nucleotide sequences of the Galt gene remain unchanged.
3. The method for constructing a Galt gene knockout mouse model based on CRISPR-Cas9 system according to claim 1 or 2, wherein the step 2 comprises the following specific steps:
step 21, the mouse ovulation promotion and in vitro fertilization are carried out to obtain a mouse fertilized egg;
step 22, microinjecting sgRNA and mRNA of the Cas9 subjected to in vitro transcription into cytoplasm of the mouse fertilized egg together to obtain injected fertilized egg;
step 23, transplanting the fertilized eggs after injection into oviduct of a surrogate mother mouse, obtaining F0 generation mice after development is completed, carrying out PCR identification on the F0 generation, mating positive F0 generation with a wild type mice to obtain F1 generation heterozygotes, hybridizing the F1 generation heterozygotes, and screening homozygous offspring of Galt gene knockout as a Galt gene knockout mouse model.
4. The method for constructing a Galt gene knockout mouse model based on CRISPR-Cas9 system according to claim 3, wherein the specific primers for the PCR identification comprise: galt-M-ES-1, galt-M-EA-1, galt-M-IS-1 and Galt-M-IA-1; the sequence of Galt-M-ES-1 IS shown as SEQ ID NO. 3, the sequence of Galt-M-EA-1 IS shown as SEQ ID NO. 4, the sequence of Galt-M-IS-1 IS shown as SEQ ID NO. 5, and the sequence of Galt-M-IA-1 IS shown as SEQ ID NO. 6.
5. The method for constructing a Galt gene knockout mouse model based on a CRISPR-Cas9 system according to claim 3, wherein the mass ratio of the sgRNA to the mRNA of Cas9 after in vitro transcription is 1 (0.9-1.1).
6. The method for constructing a Galt gene knockout mouse model based on CRISPR-Cas9 system according to claim 3, wherein the donor of fertilized egg is C57BL/6J mouse; the surrogate mother mouse is an ICR mouse.
7. A Galt gene knockout mouse model based on a CRISPR-Cas9 system, characterized in that the Galt gene knockout mouse model is prepared by the construction method according to any one of claims 1 to 6.
8. A kit for constructing the CRISPR-Cas9 system-based Galt gene knockout mouse model of claim 7, wherein the kit comprises the sgRNA and the mRNA of Cas9 after in vitro transcription.
9. Use of a Galt knockout mouse model according to claim 7 or a kit according to claim 8 for developing and/or screening substances for the treatment of type I galactosyls.
10. The use according to claim 9, wherein the substance is a medicament.
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