CN117568399A - Galt gene knockout mouse model based on CRISPR-Cas9 system, construction method and application - Google Patents

Abstract

The invention relates to a Galt gene knockout mouse model, construction method and application based on the CRISPR-Cas9 system, and relates to the technical field of animal genetic engineering. It includes the following steps: Step 1: Design sgRNA targeting the Galt gene; Step 2: In vitro transcription The prepared sgRNA and Cas9mRNA are microinjected into mouse fertilized eggs together, and then transplanted into surrogate mothers to produce F0 generations. The F0 generations are identified by PCR, and the positive F0 generations are continuously bred for at least 2 generations, and Galt is obtained by screening. Gene knockout mouse model; the sgRNA1 sequence is shown in SEQ ID NO: 1. The present invention designed sgRNA for exon 9 of the mouse Galt gene, and used the CRISPR-Cas9 system to successfully construct a Galt gene knockout mouse model. The Galt gene undergoes a frameshift mutation during transcription, and the GALT protein cannot be translated correctly. , and then simulate human classic galactosemia, laying the foundation for biomedical research such as exploring the occurrence and development mechanism of human classic galactosemia, discovering new drug targets, and preclinical pharmacodynamic evaluation.

Description

Galt gene knockout mouse model and construction method based on CRISPR-Cas9 system application

Technical field

The present invention relates to the technical field of animal genetic engineering, and specifically relates to a Galt gene knockout mouse model based on the CRISPR-Cas9 system, its construction method and application.

Background technique

The CRISPR-Cas9 system is a gene editing tool modified based on the bacterial defense mechanism found in nature to resist phage infection and plasmid transfer. It can be used for genome editing, transcriptional interference, epigenetic regulation and genome imaging. The CRISPR-Cas9 system is not only low-cost, simple to operate, and highly efficient in editing, but it can also perform precise gene editing through homology-directed repair (HDR) after causing DNA double-strand breaks (DSB), as well as through non-homologous end joining ( NHEJ) performs random gene editing. Based on this, researchers have used the CRISPR-Cas9 system to construct many cell or animal disease models to study gene function and gene regulation, such as gene knockout human intestinal tissue-derived enteroid cell lines, TSC2-KO NIH-3T3 cell lines, muscular dystrophy Pig model of Alzheimer's disease, mouse model of Alzheimer's disease, etc., and certain research progress has been made from them.

Galactosemia (Galactosemia, GAL, OMIM#230440) is an autosomal recessive metabolic disease caused by the inactivation or dysfunction of key enzymes in the galactose Leloir metabolic pathway, including types I, II and III, respectively. It is caused by abnormalities in galactose-1-phosphate-uridyl transferase, galactokinase, and uridine diphosphate-galactose-4'-epimerase. Type I galactosemia, (Type IGalactosamia, GAL I, OMIM#230400), also known as Classic Galactosemia, has a prevalence rate of 1/30000 to 1/60000. Among the three types of galactosemia The highest incidence rate. Galactose is vital to the human body and has a wide range of functions. It is a key energy source for babies before weaning and is particularly important for early development.

GAL I presents as a potentially fatal disease in the neonatal period, leading to chronic debilitating complications and, if not treated promptly, neonatal death. The clinical symptoms of GAL I can be divided into acute symptoms and chronic complications. Acute symptoms are due to the fetus ingesting lactose in milk after birth. Abnormal lactose metabolism leads to liver damage such as hepatomegaly and cirrhosis. Subsequently, symptoms such as cataracts and renal tubular insufficiency appear, which is a serious threat to life. Lactose and galactose dietary restriction treatment can effectively relieve the acute symptoms of GAL I, but it cannot solve the chronic complications of GAL I. Chronic complications are mainly neurological and reproductive system lesions, manifested as speech disorders, movement disorders, and ovarian insufficiency. At present, limiting the patient's dietary intake of lactose and galactose is the main treatment measure, and there are no other treatments. For this reason, researchers have tried to develop drugs such as specific antibodies and molecular protective agents to treat GAL I, but they have not found an ideal treatment. In view of this, the present invention provides a Galt gene knockout mouse model, construction method and application based on the CRISPR-Cas9 system.

Contents of the invention

The technical problem to be solved by the present invention is to provide a Galt gene knockout mouse model, construction method and application based on the CRISPR-Cas9 system. The purpose is to use the CRISPR-Cas9 system to construct Galt gene knockout mice, aiming to establish a mouse model that simulates human GAL I disease, and to explore the occurrence and development mechanism of GAL I, discover new drug targets, and evaluate preclinical pharmacodynamics. Lay the foundation for biomedical research.

In order to solve the above technical problems, the first aspect of the present invention provides a method for constructing a Galt gene knockout mouse model based on the CRISPR-Cas9 system, which includes the following steps:

Step 1: Design sgRNA targeting the Galt gene based on the CRISPR-Cas9 system;

Step 2: sgRNA and Cas9 in vitro transcribed mRNA are microinjected into mouse fertilized eggs, and then the fertilized eggs are transplanted into surrogate mothers to produce F0 generations. The F0 generations are identified by PCR, and the positive F0 generations are Continuously breed for at least 2 generations and screen to obtain a Galt gene knockout mouse model; wherein the sgRNA sequence is shown in SEQ ID NO: 1.

The present invention adopts the Galt gene and uses the mouse Galt-201 (ENSMUST00000084695.12) transcript as the edited transcript. The Galt-201 transcript (ENSMUST00000084695.12) has a full length of 3.47kb, which contains 11 exons (Exons). 10 introns.

The beneficial effects of the present invention are: the present invention designs sgRNA for Galt exon 9, the pathogenic site of the classic galactosemia GALT gene, and uses the CRISPR-Cas9 system to co-inject sgRNA and Cas9 mRNA into fertilized eggs to successfully construct The Galt gene knockout mouse model was developed. The Galt gene undergoes a frameshift mutation during transcription, and the GALT protein cannot be translated correctly. The Galt gene knockout mouse model was successfully constructed through the above steps, and homozygotes were obtained through breeding.

The Galt gene knockout mouse model was obtained through the above identification. The specific identification methods include using qPCR, WB and other experiments to detect the expression level of the GALT gene in Galt gene knockout mice, and at the same time measuring the body weight of Galt gene knockout mice and wild-type mice. Recording was carried out, and the relevant phenotypes of the model mice were further analyzed through pathological section experiments, and the following conclusions were drawn:

(1) The expression levels of Galt mRNA and GALT protein were detected through qPCR experiments and WB experiments. The experimental results showed that the expression levels of Galt mRNA and GALT protein in Galt gene knockout mice were significantly lower than those in wild-type mice, and There is a statistical difference.

(2) In the first 15 weeks of mouse growth, the weight of wild-type mice is slightly higher than that of Galt knockout mice.

(3) Pathological and histomorphological analysis of major tissues and organs was performed through HE staining of paraffin sections. The results showed that compared with wild-type mice, the heart, spleen, and kidney tissues of Gal gene knockout mice had no abnormalities. , while the livers of Gal gene knockout mice experienced varying degrees of edema and lung damage.

On the basis of the above technical solution, the present invention can also make the following improvements.

Furthermore, bases 829 to 837 of the cDNA sequence of the Galt gene of the positive F0 generation mouse were deleted and 1 base T was inserted, while other nucleotide sequences of the Galt gene remained unchanged.

The above-mentioned deleted DNA sequence is located in exon 9 of the Galt gene, causing it to undergo a frameshift mutation during transcription, and the GALT protein cannot be translated correctly.

Further, the step 2 includes the following specific steps:

Step 21: Use mouse ovulation induction and in vitro fertilization to obtain mouse fertilized eggs;

Step 22: After in vitro transcription, sgRNA and Cas9 mRNA are microinjected into the cytoplasm of the mouse fertilized eggs to obtain injected fertilized eggs;

Step 23: The injected fertilized eggs are transplanted into the fallopian tubes of the surrogate mother mice. After development is completed, the F0 generation mice are obtained. PCR identification is performed on the F0 generation. The positive F0 generation is mated with wild-type mice to obtain the F1 generation hybrid. The zygotes and F1 generation heterozygotes were crossed to screen the homozygous generation of Galt gene knockout as a Galt gene knockout mouse model.

Further, the specific primers used for the PCR identification include: Galt-M-ES-1, Galt-M-EA-1, Galt-M-IS-1 and Galt-M-IA-1; the Galt- The M-ES-1 sequence is shown in SEQ ID NO:3, the Galt-M-EA-1 sequence is shown in SEQ ID NO:4, and the Galt-M-IS-1 sequence is shown in SEQ ID NO:5 As shown, the Galt-M-IA-1 sequence is shown in SEQ ID NO: 6.

Further, the mass ratio of the sgRNA to Cas9 mRNA is (0.9-1.1).

Furthermore, the donor of the mouse single fertilized eggs is a C57BL/6J mouse; the surrogate mother mouse is an ICR mouse.

The second aspect provides a Galt gene knockout mouse model based on the CRISPR-Cas9 system. The Galt gene knockout mouse model based on the CRISPR-Cas9 system is prepared by any of the preparation methods described above.

The third aspect provides a kit for constructing a Galt gene knockout mouse model based on the CRISPR-Cas9 system, the kit including the above-mentioned sgRNA and the Cas9 mRNA.

The fourth aspect provides the application of the above-mentioned Galt gene knockout mouse model or the above-mentioned kit in the development and/or screening and treatment of type I galactosemia.

Further, the substance is a drug.

Description of the drawings

Figure 1 is a structural diagram of the Galt-201 transcript in the Ensembl database of the present invention;

Figure 2 is a map of the mouse Galt gene targeting site of the present invention;

Figure 3 shows the offspring of F1 generation Galt ^-/- and Galt ^+/- gene knockout mice of the present invention;

Figure 4 is the Sanger sequencing results of the offspring of F1 generation Galt ^-/- and Galt ^+/- gene knockout mice of the present invention;

Figure 5 is the non-denaturing electrophoresis result of total RNA in the liver of Galt gene knockout mice of the present invention;

Figure 6 is the detection of Glat mRNA expression level in the liver of Galt gene knockout mice of the present invention;

Figure 7 is the electrophoresis result of protein level detection in Galt gene knockout mice of the present invention, wherein A is a graph of GALT protein expression levels in Galt gene knockout mice, and B is a statistical result graph;

Figure 8 shows HE staining of pathological sections of heart, liver, spleen, lung and kidney tissue of WT mice and Galt gene knockout mice of the present invention.

Detailed ways

The principles and features of the present invention are described below. The examples cited are only used to explain the present invention and are not intended to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.

Example

1. Experimental animals, main reagents and commonly used solutions

1.1 Experimental animals

SPF (Specific Pathogen Free) grade C57BL/6J and ICR mice were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. and raised in the Animal Laboratory of the School of Intelligent Medicine and Biotechnology, Guilin Medical College. The design and protocol of the present invention were approved by the Animal Care and Use Committee of Guilin Medical College, and the animal ethics approval number is GLMC201803033. The mice were euthanized by intraperitoneal injection of an overdose of 200 mg/kg sodium pentobarbital, and then samples of various tissues of the mice were quickly collected for subsequent experiments. The WT mice mentioned below are pure wild-type C57BL/6J mice.

1.2 Description of main reagents and consumables

Table 1 Main reagents and consumables

1.3 Common solution preparation

1) Preparation of 10×TBS solution and 1×TBST buffer

Table 2 Preparation of 10×TBS solution and 1×TBST buffer

The preparation of 10×TBS solution requires adjusting the pH to 7.6 with 12 N HCl, and adding distilled water to a final volume of 1L.

2) Preparation of transfer mold solution, preparation of 5×SDS-PAGE electrophoresis solution and preparation of 50×TAE solution

Table 3 Preparation of transfer mold solution, preparation of 5×SDS-PAGE electrophoresis solution and preparation of 50×TAE solution

3) Preparation of LB liquid culture medium and preparation of LB solid culture medium

Table 4 Preparation of LB liquid culture medium

serial number Reagent name Dosage 1 Yeast extract 0.5g/100ml 2 trypsin 1g/100ml 3 NaCl 1g/100ml 4 ddH ₂ O Adjust volume to 100ml

Preparation of LB solid medium: Compared with LB liquid medium, the preparation of LB solid medium also adds 1.5g/100ml agar powder, and the rest is the same as the preparation of LB liquid medium.

2. Experimental methods

2.1 Design of sgRNA sequence targeting mouse Galt gene

The mouse Galt (Gene ID: 14430) gene is located on chromosome 4 and has a total of 10 transcripts. Based on the comparison of the genome and transcriptome information of human GALT and mouse Galt in NCBI and Ensembl databases, the mouse Galt-201 (ENSMUST00000084695.12) transcript was selected as the edited transcript after analyzing its gene structure. The full length of the Galt-201 transcript (ENSMUST00000084695.12) is 3.47kb, which contains 11 exons (Exons) and 10 introns (Introns) (Figure 1). The Gal gene knockout mouse of the present invention has 9 bases deleted in Galt exon 9, and a T base is inserted. The Galt gene knockout mouse has a base deletion in exon 9, resulting in A frameshift mutation occurs during transcription, and the GALT protein cannot be translated correctly.

Specifically, the present invention designed an sgRNA sequence for the mouse Galt gene based on the CRISPR-Cas9 gene editing system (see Table 5 for sequence information), causing the mouse Galt gene to mutate at the Galt c.847+1G site (as shown in Figure 2) , causing translation errors of GALT protein. Galt-sgRNA (SEQ ID NO. 1) and the Cas9 mRNA are used to microinject into mouse fertilized eggs, and then the fertilized eggs are transplanted into surrogate mothers to produce F0 generations, and PCR identification is performed on the F0 generations. , the positive F0 generation was continuously bred for at least 2 generations, and the Galt gene knockout mouse model was screened.

Table 5 Designed sgRNA sequence information

sequence name Sequence(5'-3') serial number Galt-sgRNA AGCCTTACCATGCCAGCCCA SEQ ID NO.1

2.2 Preparation of Galt gene knockout mouse model

2.2.1 Obtaining mouse fertilized eggs

(1) Superovulation. C57BL/6 female mice aged 3-3.5W were selected and injected with pregnant mare serum gonadotropin (PMSG), 10UI/mouse, at 5 pm on the first day of the experiment; at 5 pm on the third day (with the At the same time of the day), inject human chorionic gonadotropin (HCG), 10UI/mouse, into the female mice that had been injected with PMSG.

(2) Preparation of embryo culture dishes. After the hormone injection is completed on the third day, use 35mm petri dishes on a clean workbench to make TYH sperm capacitance dishes, HTF fertilization dishes, and KSOM embryo culture dishes. After all petri dishes are made, place them in a 37.5°C, 5% _CO2 incubator overnight to equilibrate.

(3) Sperm capacitation. At around 9 a.m. on the fourth day of the experiment, suitable C57 BL/6 male mice aged 3 to 6 months were selected and killed by cervical dislocation. They were dissected with surgical instruments, the abdominal cavity was opened, the epididymis was found and removed. Use absorbent paper to remove the excess blood and fat on it, put it into the mineral oil in the TYH sperm capacitation dish, use ophthalmic scissors to make a small opening on the epididymis, and use microscopic forceps to remove the semen from the small opening under a stereomicroscope. Squeeze out and transfer the semen into a 200 µL droplet of TYH. Then put the TYH sperm capacitation dish into a 37.5°C, 5% CO2 incubator to capacitate for 50 minutes.

(4) In vitro fertilization. After killing the female mouse, use ophthalmic scissors to expose the skin on the back, cut off the ovary and fallopian tube segments, use absorbent paper to remove excess fat and blood, put them into a fertilization dish, find the enlarged fallopian tube section under a stereomicroscope, and use a 1mL syringe to remove the ovarian and fallopian tube segments. The egg mass is scratched into the HTF droplet. After the sperm capacitation is completed, use a sample gun to draw 2.5 μL of sperm along the edge of the droplet and add it to each 200 μL fertilization drop. Place the HTF fertilization dish into the incubator to continue culturing.

2.2.2 Microinjection of mouse fertilized eggs

(1) Cleaning and selection of fertilized eggs. After the sperm and egg cells are incubated for about 8 hours, the embryos are initially screened in the HTF fertilization dish to remove unfertilized eggs, stillbirths, malformed embryos, multinucleated embryos and other embryos that cannot be used for experiments, and select embryos that are plump and round, with intact zona pellucida and clear pronuclei. . Fertilized egg cells with a moderate distance between the two nuclear regions are transferred from 200 μL HTF droplets to 50 μL HTF droplets for initial cleaning. Then these embryos are transferred to the M2 embryo cleaning dish and washed 6-10 times with an oral pipette. Remove the cumulus cells and other impurities attached to the fertilized eggs, and return the cleaned embryos to the incubator for later use.

(2) Preparation of microinjection solution. Take out the sgRNA and Cas9 mRNA from the -80°C refrigerator and place them on ice to dissolve. In a clean bench, use an enzyme-free pipette tip to absorb sgRNA and Cas9 mRNA in a certain proportion and mix them thoroughly in an enzyme-free 1.5 EP tube, so that the final concentration of sgRNA and Cas9 mRNA in the mixed solution is 200ng/uL. Place the mixed solution in a centrifuge and centrifuge at 2000 rpm for 2 minutes. After centrifugation, use a Microloader micro-loading needle to draw 1 uL of the mixed solution and transfer it to the microinjection needle.

(3) Transfer of fertilized eggs. Transfer 50-100 cleaned fertilized eggs to the M2 culture droplets in the injection dish, arrange them evenly in a straight line, and place the injection dish in the incubator and incubate for 5-10 minutes.

(4) Instrument debugging before microinjection. Before starting microinjection, debug the microinjection operating system.

(5)Microinjection. After the instrument is debugged, adjust the fixing needle to use negative pressure to fix the fertilized egg, and avoid the polar body when fixing. After the fertilized eggs are fixed, the mixed solution is injected into the cytoplasm of the fertilized eggs through a microinjection needle. After the injection is completed, use an oral suction tube to move the injected fertilized eggs to the egg collection dish and wash them 5-10 times. Then move the washed fertilized eggs to the KOSM embryo culture dish, and place the KOSM embryo culture dish in the culture chamber. Continue culturing in the box.

2.2.3 Embryo transfer

(1) Ligation of ICR male mice. Select 3.5-4 weeks old ICR male mice and place them in cages after ligation.

(2) Preparation of ICR receptor female mice. At around 5 pm on the day of in vitro fertilization, select ICR female mice in estrus that are 6-12 weeks old and weigh 25-35g. They will be caged with ICR-ligated male mice, and the ICR female mice with visible plugs will be selected as recipients.

(3) Selection of fertilized eggs. The fertilized eggs that normally develop to the 2-cell stage after microinjection are selected and counted, and 15 of them are put into a group of microdroplets in the KSOM embryo culture dish for later use.

(4)Embryo transfer. Weigh and anesthetize the plugged ICR receptor female mouse, open an opening on the back and find the ampulla of the fallopian tube. Use microscissors to cut the fallopian tube at the front of the ampulla. Use a three-stage oral suction tube to move the embryo in, and use air bubbles to enter the fallopian tube to expand. As a sign of successful transplantation, the muscle tissue and skin of the mouse were sutured, and the mice were placed on a hot stage until they woke up, and were given post-operative care until they became pregnant and gave birth.

2.2.4 Genotype identification of newborn mice

(1) Amplification of the pseudomutation site region in F0 generation mice. ICR recipient female mice give birth about 21 days after embryo transfer, and the newborn mice are the F0 generation. Seven days after the mouse was born, the mouse tail was cut, and the mouse tail DNA was extracted using the Tiangen Blood/Cell/Tissue Genomic DNA Extraction Kit (DP304-02), and PCR amplification of the pseudo-mutation site region was performed. PCR product sequencing and genotype analysis. The primer sequences of the PCR reaction, Galt-M-ES-1 and Galt-M-EA-1, are shown in Table 6, the reaction system is shown in Table 7, and the PCR amplification reaction procedure is shown in Table 8.

(2) PCR product gel recovery. 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, 120V, 30 min. After electrophoresis, cut out a single band that meets the target size and put it into an EP tube. Follow the instructions of Tiangen General DNA Product Purification Kit (DP204-02) for gel recovery. The concentration of the recovered PCR product must reach 40ng/μL. meet the requirements for the next step of the experiment.

(3) Connection transformation. Mix 1 μL of gel recovery product and 1 μL of pMD 19-T Vector in a PCR tube, make up to 5 μL of ddH ₂ O, and add 2.5 μL of Solution I enzyme. All reactions are prepared on ice. Place the mixture in a PCR machine at 16°C for 1 hour for ligation. After the connection is completed, take out the E. coli from the -80°C refrigerator and place it on ice to dissolve it. Take 30 μL of E. coli and 7.5 μL of the ligation product and put it in a 1.5 mL EP tube. Mix well and keep in ice bath for 30 minutes. The metal bath is 42°C for 90 seconds. Ice bath 2min. Add 150 μL of LB liquid culture medium without antibiotics to the EP tube, place it in a 37°C shaker, 220 rpm, and incubate for 40 minutes to recover the bacteria. After shaking the bacteria, spread the bacterial solution evenly into the LB solid culture medium containing ampicillin, and place the culture medium in a 37°C incubator for 16-18 hours until a single colony grows.

(4) Pick a single colony. Use a small pipette tip to pick a single colony into a PCR tube containing 10 μL ddH ₂ O on the ultra-clean workbench, and pick 10-15 single colonies from each LB solid medium.

(5) Bacterial liquid PCR. Use the picked single colony as a template to perform bacterial liquid PCR. The bacterial liquid PCR system is shown in Table 9, and the PCR reaction procedure is shown in 10.

(6) Sequencing. After PCR of the bacterial liquid, perform 1% agarose gel electrophoresis. Move 8ul of the remaining bacterial liquid that meets the target size and has a single band into a 2ml centrifuge tube, add 1.5mL of LB liquid culture medium containing ampicillin, and place it on a shaker Incubate overnight at 37°C, 150rpm, and sequence the bacterial solution the next day.

Table 6 Primer sequences for PCR amplification of the pseudomutation site region

Table 7 PCR amplification reaction system of pseudomutation site region

Table 8 PCR amplification reaction procedure of pseudomutation site region 1

Table 9 Bacterial liquid PCR amplification reaction system

serial number reaction components Volume(μL) 1 Single colony liquid 2 2 M13F(10pmol/μL) 0.5 3 M13R(10pmol/μL) 0.5 4 Premix ^TaqTM 5 5 ddH ₂ O 2

Table 10 Bacterial liquid PCR amplification reaction procedure

2.2.5 Breeding of Galt knockout mice

The F0 generation Galt gene editor and WT mice were co-caged for breeding, and the resulting mice were the F1 generation. The tails of the F1 generation mice were cut, and the DNA was extracted for PCR amplification and Sanger sequencing to identify the genotype. The F1 generation identified as Galt ^+/- gene knockout mice were mated between groups to breed homozygotes, and their offspring were subjected to Sanger sequencing to screen for homozygous genotypes.

2.3 Detection of Galt gene knockout mice at the molecular level

2.3.1 Detection of mRNA expression levels in Galt gene knockout mice

(1) Extraction of total RNA. After euthanizing WT mice and Galt ^-/- gene knockout mice, their livers were removed and total RNA was extracted using the Trizol method on ice (at least 3 mice from each group were taken). Place the liver in a 2mL EP tube and weigh it. Add 1mL of pre-cooled Trizol reagent for every 100mg of tissue. Add 2-3 magnetic beads to the EP tube and grind it with an animal tissue grinder. Place the ground tissue into Let stand on ice for 10 minutes, then centrifuge at 12000g, 4°C for 15 minutes, and transfer the supernatant to another EP tube. Add 1/5 Trizol volume of chloroform to the EP tube, mix vigorously by inverting (do not use a vortex shaker to avoid RNA fragment breakage), let stand at room temperature for 3 minutes, and stratification will be visible (repeat this step several times to fully separate the liquid) layer), centrifuge at 12000g, 4°C for 15 minutes. After centrifugation, the solution is divided into three layers, the upper layer is aqueous phase, the middle layer is protein, and the lower layer is organic phase, and RNA is located in the upper aqueous phase. Use a pipette to carefully draw the upper aqueous phase into a new EP tube. Add 1/2 Trizol volume of pre-cooled isopropyl alcohol to the EP tube, mix gently with a pipette, let stand at room temperature for 10 minutes, centrifuge at 12000g, 4°C for 15 minutes, carefully discard the supernatant after centrifugation, and the precipitate is extracted Total RNA. Add 1 times the Trizol volume of 75% ethanol to the EP tube for washing, then centrifuge at 7500g, 4°C for 5 minutes, discard the supernatant, and let stand at room temperature for 5-15 minutes to ensure that the water and ethanol are completely evaporated.

(2) Total RNA quality detection. After the RNA precipitate is completely dry, add 30 μL of enzyme-free water to the EP tube to dissolve the precipitate, and measure the concentration. The A260/A280 ratio of the pure RNA sample is about 2.0, and then use 1% agarose gel electrophoresis to check whether the extracted RNA is intact.

(3) Genomic DNA removal and reverse transcription. The extracted total RNA was reverse transcribed. Follow the instructions of TaKaRa's reverse transcription kit (RR047A) and prepare a reaction system for removing genomic DNA from total RNA in an enzyme-free PCR tube. The reaction system is shown in Table 11. Place the PCR tube in a PCR machine at 42°C. React for 2 minutes. Then prepare a reverse transcription reaction system on ice. The reaction system is shown in Table 12. The reaction program is 37℃15min; 85℃5s; 4℃∞.

(4)qPCR. Follow TaKaRa TB Ex TaqTMⅡ kit (RR820L, TaKaRa) instructions, prepare qPCR reaction system (see Table 13). This experiment uses a two-step PCR amplification program: 95°C for 30s; 95°C for 5s, 60°C for 30s for 40repeats; 95°C for 15s, 60°C for 30s, and 95°C for 15s (dissociation). Using mouse GAPDH as the internal reference gene, the 2^-ΔΔCT method was used to calculate the relative expression level of the Galt gene. The primers for the qPCR reaction are shown in Table 14.

Table 11 Reaction system for removing genomic DNA

reaction components Volume (μL) 5×gDNA Eraser Buffer 2.0 gDNA Eraser 1.0 Total RNA 1μg Total RNA RNase Free dH ₂ O up to 10μL

Table 12 Reverse transcription reaction system

reaction components Volume (μL) Remove genomic DNA 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 13 qPCR reaction solution preparation

reaction components Volume (μL) 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 Sterilized water 8.5 Total 25

Table 14Galt gene qPCR amplification primer sequence

2.3.2 Detection of protein expression levels in Galt gene knockout mice

(1) Extraction of total protein. After euthanasia of 3 WT mice and 3 Galt gene knockout mice, their livers were placed in a 2mL EP tube and weighed. 100-200μL cell lysis solution (Beyotime, P0013) was added to every 20mg of tissue. PMSF (Beyotime, ST506) needs to be added before use to make the final concentration of PMSF 1mM. Add 2-3 magnetic beads to the EP tube, grind with an animal tissue grinder, place the fully ground tissue suspension on ice for lysing for 30 minutes, then centrifuge at 12000 rpm, 4°C for 15 minutes, and move the supernatant to another place. In a new EP tube, total tissue protein is stored in the supernatant.

(2) Determination of total protein concentration by BCA method: The concentration of the extracted tissue total protein was measured according to the instructions of Beyuntian Biotechnology Company's BCA protein concentration determination kit (P0012S). Finally, draw a standard curve based on the values measured by the microplate reader, and calculate the total protein concentration based on the standard curve.

(3) Protein denaturation. According to the concentration measured by the BCA method, take out a part of the protein sample, mix the sample with 5×SDS-PAGE loading buffer and quantify it to 5ug/μl. Place the mixed sample in a metal bath and heat it at 100°C for 10 minutes. During the heating process, Invert and mix evenly to heat evenly. After cooling to room temperature, centrifuge at 12,000 rpm and 4°C for 10 min, and store at -20°C for later use.

(4) Preparation of SDS-PAGE gel. Prepare 10% separation gel according to the instructions of Beyotime Biotechnology Company's SDS-PAGE gel preparation kit (P0012A). Add the separation gel to the glass plate and press it with ddH ₂ O. Leave it at room temperature until the separation gel solidifies. , remove ddH ₂ O, prepare a stacking gel and fill the glass plate, insert the comb into the stacking gel, and let it stand at room temperature until the stacking gel solidifies.

(5) SDS-PAGE electrophoresis. After assembling the prepared gel, place it into the electrophoresis tank and fill it with new 1× SDS-PAGE electrophoresis buffer. Take out the protein sample from -20℃, centrifuge at 12000rpm and 4℃ for 10min. When loading the sample, 2 μL of protein Marker (Thermo Fisher, 26616) was loaded into the first well, and then the protein samples were loaded sequentially, with 6 μL of protein sample in each well, that is, the total amount of protein loaded was 30 μg. After the spotting is completed, start electrophoresis, electrophoresis at 80V constant voltage until the sample runs to the separation gel, switch the voltage, and stop electrophoresis at 120V constant voltage until the sample runs to the bottom of the separation gel.

(6) Transfer film. After electrophoresis, pour the transfer solution into a white tray, separate the gel from the glass plate and immerse it in the transfer solution. According to the Marker strips on the gel, use a gel cutting board to cut out the target protein and internal reference protein respectively. Glue block, immerse the cut glue into the transfer liquid and set aside. Then cut two pieces of PVDF membrane similar in size to the glue and soak them in anhydrous methanol for about 1 minute. Assemble the "sandwich" in the order of blackboard, sponge pad, filter paper, glue, PVDF membrane, filter paper, sponge pad, and white board. Be careful that there are no air bubbles between each layer. Put the "sandwich" into the film transfer apparatus and transfer the film at a constant current of 250mA for 90 minutes.

(7) Closed. After the transfer, take out the PVDF membrane, wash it once with 1×TBST buffer, and destain it on a shaking table for 5 minutes. Then use 1×TBST buffer to prepare 5% skim milk for blocking for 2 h.

(8) Primary antibody incubation. After blocking, wash 4 times with 1×TBST buffer, 10 min each time. Recombinant Anti-GALT antibody (Abcom, ab178406) and GAPDH Polyclonal antibody (Proteintech, 10494-1-AP) were diluted with Western primary antibody diluent (Beyotime, P0023A) at a ratio of 1:2000 and 1:5000 respectively. Incubate the PVDF membrane with the diluted primary antibody and keep it in the refrigerator at 4°C overnight.

(9) Secondary antibody incubation. Take out the PVDF membrane that was incubated overnight from the refrigerator, recover the primary antibody, and wash the PVDF membrane 4 times with 1× TBST buffer for 10 min each time. Incubate with horseradish peroxidase-labeled goat anti-rabbit secondary antibody diluted 1:5000 in 5% skim milk at room temperature for 2 h. After incubation with the secondary antibody, wash the PVDF membrane 4 times with 1×TBST buffer for 10 min each time.

(10) Glow. Mix the reagent A and reagent B in the ultra-sensitive ECL chemiluminescence kit (P0018S) of Beyotime Biotechnology Company in a ratio of 1:1, drop the luminescent mixture evenly on the PVDF membrane, and observe using the Tanon gel imaging system result.

2.4 Phenotypic analysis of Galt gene knockout mice

2.4.1 Observation on the growth of Galt gene knockout mice

The body weights of Galt gene knockout mice and WT mice were recorded to observe whether the gene knockout mice showed obvious symptoms such as growth retardation.

2.4.2 Pathological section analysis of Galt gene knockout mice

(1) Material collection and fixation. After euthanizing WT mice and Galt gene knockout mice of similar age, the liver, kidney, ovary and other tissues were removed and fixed in 4% paraformaldehyde solution for 48 hours, then rinsed with tap water for 30 minutes.

(2) Dehydration and transparency. Dehydrate the fixed tissue through graded alcohol: 75% ethanol for 1 hour, 85% ethanol for 1 hour, 95% ethanol (I) for 1 hour, 95% ethanol (II) for 1 hour, absolute ethanol (I) for 1 hour; absolute ethanol (II) )1h. After dehydration, clear treatment with xylene solution twice, 30 minutes each time.

(3) Wax dipping, embedding and sectioning. Soak the transparently treated tissue in paraffin (soft wax) at 50-52°C for 1 hour, paraffin (I, hard wax) at 58-60°C for 1 hour, and paraffin (II) at 58-60°C for 1 hour. After the wax immersion is completed, the tissue is embedded in paraffin (hard wax) at 58-60°C. Use a slicer from the American Thermo Fisher Company for slicing. After slicing, place the slices at 65°C for 3-4 hours.

(5) HE staining: Stain the sections according to the instructions of the hematoxylin and eosin (HE) staining kit (G1120) of Beijing Solebao Technology Co., Ltd. Dewax in xylene twice, 5 minutes each time. Gradient alcohol rehydration (sequence: 100%, 95%, 85%, 75%), 3 minutes per gradient. Soak in distilled water for 2 minutes. Stain with hematoxylin staining solution for 2-5 minutes (the specific time depends on the situation), wash away the floating color with distilled water, differentiate with differentiation solution for 10-60 seconds, and soak twice with tap water for 3-5 minutes each time. Eosin staining solution 30s-2min, pour off the excess staining solution and quickly dehydrate (over gradient alcohol: 75%, 85%, 95% and absolute ethanol (Ⅰ) soak for 2-3s each, soak in absolute ethanol (Ⅱ) Wash for 1 minute). Clear with xylene twice, 1 minute each time, seal with neutral gum, and observe under a microscope.

2.5 Statistical analysis

All data result analysis in this invention uses GraphPad Prism 9 software for statistical analysis, t-test (Student's t-test) is used to compare two groups of data, and one-way analysis of variance (One-way Anova) is used to compare three groups and For comparisons between more than three groups of data, all experimental results are processed in the form of mean ± standard deviation (X ± SD), and * indicates P < 0.05.

3. Results

3.1 Construction of Galt gene knockout mice based on CRISPR-Cas9 technology

3.1.1 Generation of F0 generation mice

Use the sgRNA and Cas9 mRNA in 2.1 above for microinjection, and prepare the Galt gene knockout mouse model according to the description in 2.2 above. Four Galt ^+/- gene knockout mice were obtained, all of which were two female mice and two male mice. mice, these 4 mice were bred as the F0 generation.

3.1.2 Generation of F1 generation mice

F0 generation mice of Galt ^+/- gene knockout heterozygous mice were caged with WT mice to breed F1 generation mice. The tails of the F1 generation mice produced by Galt gene knockout mice were cut, DNA was extracted, and the F1 generation genotype was detected through Sanger sequencing and TA cloning to obtain Galt ^+/- gene knockout mice.

3.1.3 Breeding of homozygous mice

The F1 generation Galt ^+/- gene knockout mice were homozygously bred in cages within the group. After they gave birth to pups, DNA samples from the pups were taken for PCR amplification and Sanger sequencing to determine the genotype. Figure 3 shows the first generation of pups produced within the F1 generation Galt ^+/- knockout mouse group. The sequencing results (see Figure 4) show that the sequencing results of mouse No. 1 have no set of peaks. Comparison with the WT mouse sequence shows that mouse No. 1 is a Galt ^-/- gene knockout mouse (homozygous). The mouse had 9 bases deleted in the target region and inserted a T base; the sequencing results of mouse No. 2 showed a set of peaks and was a Galt ^+/- gene knockout mouse (heterozygote).

3.2 Detection of Galt gene knockout mice at the molecular level

The livers of WT mice and Galt ^-/- gene knockout mice were taken respectively, and total RNA was extracted using the Trizol method. The concentration of the extracted total RNA is shown in Table 15, and the electrophoresis results are shown in Figure 5. Select RNA with better quality for qPCR, and perform statistical analysis on the obtained data. The results are shown in Figure 6. The expression level of Galt mRNA in Galt ^-/- gene knockout mice was significantly lower than that in WT mice, and the difference was statistically significant.

WB experimental detection found that in liver tissue, the expression level of GALT protein in Galt ^-/- gene knockout mice was significantly lower than that in WT mice, and the difference was statistically significant (see Figures 7A and 7B).

Table 15 Total RNA concentration in mouse liver (ng/μL)

3.3 Phenotypic analysis of Galt gene knockout mice

3.3.1Growth of Galt gene knockout mice

In order to explore whether Galt gene knockout mice will develop GAL I growth retardation characteristics. Under normal feeding conditions, the body weights of WT mice and Galt ^-/- gene knockout mice were weighed. The results showed that during the first 15 weeks of growth, wild-type mice weighed slightly more than Galt knockout mice.

3.3.2 Analysis of pathological sections of Galt gene knockout mice

In WT mice, GALT protein is expressed in major organs: heart, liver, spleen, lung, and kidney, and the expression level is relatively high, so the above results from Galt ^-/- gene knockout mice and WT mice were taken The organs were pathologically sectioned, stained with HE, and observed under a microscope to see whether their morphology was different from that of WT mice.

As shown in Figure 8, in the HE staining of heart tissue sections from WT mice and Galt ^-/- gene knockout mice, the cardiomyocytes in the longitudinal section are arranged parallel to each other. Although there are bifurcations, they are anastomotic to each other and form a network; the cardiomyocytes are Oval, usually single-nucleated, occasionally double-nucleated, lightly stained, located in the center. The cross-sections of myocardial fibers appear as round or irregular small pieces. In some cross-sections, round and lightly stained nuclei can be seen, in some cross-sections there are no nuclei, and the center of the cross-section is often lightly stained. The shapes are all normal and there are no lesions.

As shown in Figure 8, structures such as red pulp, white pulp, trabeculae, and splenic corpuscles can be observed in the pathological sections of the spleen of WT mice and Galt ^-/- gene knockout mice, and there are no abnormalities in the morphological structure of the spleen.

As shown in Figure 8, in the renal tissue pathological sections of WT mice and Galt ^-/- gene knockout mice, normal cortex, medulla, glomeruli, distal convoluted tubules, and proximal convoluted tubules can be observed, and no Renal fibrosis, kidney damage, glomerular occlusion and other diseases.

As shown in Figure 8, compared with WT mice, liver tissue pathological sections of Galt ^-/- gene knockout mice can be observed in liver tissue pathological sections with enlarged hepatocytes, loose and empty cytoplasm, reticular or transparent, and large Most cell nuclei are centered, indicating that the liver tissue of Galt ^-/- gene knockout mice exhibits pathological changes of severe edema.

As shown in Figure 8, compared with WT mice, the alveolar walls in the lung tissue pathological sections of Galt ^-/- gene knockout mice were thickened, the shape of the alveoli changed, and some alveoli were broken or fused, accompanied by neutral Lesions with granulocytic infiltration.

4 Conclusion

The present invention aims at the GALT gene, the causative gene of classic galactosemia, and successfully constructs Galt ^-/- gene knockout mice. Galt ^-/- gene knockout mice have 9 bases deleted in the exon (bases 829 to 837 in the cDNA sequence) and a T base inserted, causing the Galt gene to shift during transcription. Code mutation, GALT protein cannot be translated correctly. The present invention uses qPCR and WB experiments to detect the Galt mRNA expression level and GALT protein expression level of Galt ^-/- gene knockout mice and WT mice respectively, and further analyzes the relevant phenotypes of the model mice through pathological section experiments. Concluded as follow:

(1) sgRNA was designed for the classic galactosemia-causing gene GALT, and the CRISPR-Cas9 system was used to co-inject sgRNA and Cas9 mRNA into fertilized eggs, and Galt ^-/- gene knockout mice were successfully constructed.

(2) The expression levels of Galt mRNA and GALT protein were detected through qPCR experiments and WB experiments respectively. The experimental results showed that the expression levels of Galt mRNA and GALT protein in Galt ^-/- gene knockout mice were significantly lower than those in WT mice. and has statistical significance.

(3) Compared with WT mice, Galt ^-/- gene knockout male mice have slightly lower body weight than WT mice. The morphology and function of tissues and organs were analyzed through pathological sections and HE staining. Compared with WT mice, it was found that the livers of Galt ^{-/- gene} knockout mice had severe edema. The alveolar walls of the lungs are thickened, the shape of the alveoli changes, some alveoli break or fuse, and are accompanied by neutrophil infiltration.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.

Claims (10)

1. The method for constructing a Galt gene knockout mouse model based on the CRISPR-Cas9 system is characterized by including the following steps: Step 1: Design sgRNA targeting the Galt gene based on the CRISPR-Cas9 system; Step 2: The sgRNA and the Cas9 mRNA transcribed in vitro are microinjected into mouse fertilized eggs, and then the fertilized eggs are transplanted into the surrogate mother mice to produce F0 generations, and the F0 generations are PCR identification, the positive F0 generation was continuously bred for at least 2 generations, and the Galt gene knockout mouse model was screened; Wherein, the sgRNA1 sequence is shown in SEQ ID NO: 1. 2. The construction method of the Galt gene knockout mouse model based on the CRISPR-Cas9 system according to claim 1, characterized in that the cDNA sequence of the Galt gene of the positive F0 generation mouse is based on bases 829 to 837 was knocked out and one base T was inserted, while the other nucleotide sequences of the Galt gene remained unchanged. 3. The method for constructing a Galt gene knockout mouse model based on the CRISPR-Cas9 system according to claim 1 or 2, characterized in that the step 2 includes the following specific steps: Step 21: Use mouse ovulation induction and in vitro fertilization to obtain mouse fertilized eggs; Step 22, microinject the sgRNA and Cas9 in vitro transcribed mRNA into the cytoplasm of the mouse fertilized egg to obtain the injected fertilized egg; Step 23: The injected fertilized eggs are transplanted into the fallopian tubes of the surrogate mother mice. After development is completed, the F0 generation mice are obtained. PCR identification is performed on the F0 generation. The positive F0 generation is mated with wild-type mice to obtain the F1 generation hybrid. The zygotes and F1 generation heterozygotes were crossed to screen the homozygous generation of Galt gene knockout as a Galt gene knockout mouse model. 4. The construction method of the Galt gene knockout mouse model based on the CRISPR-Cas9 system according to claim 3, characterized in that the specific primers used for the PCR identification include: Galt-M-ES-1, Galt -M-EA-1, Galt-M-IS-1 and Galt-M-IA-1; the Galt-M-ES-1 sequence is shown in SEQ ID NO: 3, the Galt-M-EA- The 1 sequence is shown in SEQ ID NO:4, the Galt-M-IS-1 sequence is shown in SEQ ID NO:5, and the Galt-M-IA-1 sequence is shown in SEQ ID NO:6. 5. The method for constructing a Galt gene knockout mouse model based on the CRISPR-Cas9 system according to claim 3, characterized in that the mRNA mass ratio of the sgRNA and the Cas9 after in vitro transcription is 1:(0.9- 1.1). 6. The construction method of the Galt gene knockout mouse model based on the CRISPR-Cas9 system according to claim 3, characterized in that the donor of the fertilized eggs is a C57BL/6J mouse; the surrogate mother mouse is an ICR mice. 7. A Galt gene knockout mouse model based on the 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 a Galt gene knockout mouse model based on the CRISPR-Cas9 system as claimed in claim 7, characterized in that the kit includes the sgRNA and the Cas9 after in vitro transcription of mRNA. 9. Application of a Galt gene knockout mouse model according to claim 7 or a kit according to claim 8, characterized in that it is used to develop and/or screen substances for treating type I galactosemia. 10. The application according to claim 9, characterized in that the substance is a drug.