Method for constructing leptin gene modified obese golden hamster model and application thereof

Abstract

The application discloses a construction method of a Leptin gene modified obese golden hamster model and application thereof, and the sgRNA sample prepared by targeting the hamster Leptin gene is microinjected into a single two-cell stage embryo blastocyst, and then is transferred into a real pregnant surrogate to develop in vivo after culture recovery, so that the hamster Leptin gene can be accurately targeted and modified, and a genetically modified hamster is obtained. The two-cell stage embryo gene editing which overcomes the hamster embryonic development arrest period can overcome the embryonic development arrest caused by in vitro operation, and the litter effect is stable. The golden hamster can be directly infected with SARS-CoV-2 and show similar pathological characteristics to humans, and the ob / ob golden hamster model will fill the blank of a severe model animal of a novel coronavirus infection, and plays an important role in the fields of infection mechanism research, treatment drug screening and new pathogenic virus strain evaluation of SARS-CoV-2.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering animal model construction technology, specifically relating to a method for constructing a Leptin gene-modified obese golden hamster (ob / ob) model and its application. Background Technology

[0002] Obesity is a disease characterized by excessive accumulation of body fat, leading to physiological and pathological changes detrimental to health. Obese patients often experience chronic low-grade systemic inflammation. Patients infected with SARS-CoV-2 with a body mass index (BMI) of 28.0 ± 2.5 exhibit more severe symptoms, and existing studies have demonstrated that SARS-CoV-2 is more pathogenic in obese mice. However, due to the significantly different metabolic characteristics of humans, mice are not the optimal animal model for studying the pathogenesis of obesity-related SARS-CoV-2 infection. Golden hamsters share high homology with humans at key binding sites between the ACE2 receptor and SARS-CoV-2. Multiple studies have consistently shown that SARS-CoV-2 infection in golden hamsters leads to robust viral replication and substantial lung lesions, similar to the course observed in patients infected with SARS-CoV-2. Furthermore, compared to mice, golden hamsters have metabolic characteristics similar to humans, making them a more suitable animal model for studying lipid metabolism-related diseases. Therefore, golden hamsters are a more suitable animal model for simulating severe obesity-related SARS-CoV-2 infection. Summary of the Invention

[0003] In response to the technical problems raised in the background art, the present invention provides a method for establishing a Leptin gene-modified obese golden hamster (ob / ob) model and its application.

[0004] This invention utilizes CRISPR-Cas9 technology, under a red-light chamber with a microscope equipped with a red filter, to inject Cas9 protein and sgRNAs targeting exon 2 of the leptin gene into two-cell embryos to generate leptin-deficient golden hamsters, thus avoiding early embryonic developmental arrest. Constructed offspring were obtained through the transfer of albino recipient embryos in a true pregnancy and used to simulate a severe SARS-CoV-2 infection model associated with obesity.

[0005] The objective of this invention is achieved through the following technical methods:

[0006] In a first aspect, the present invention claims protection for a method for constructing a Leptin gene-modified obese golden hamster model, the method comprising: injecting a Cas9 functional element and sgRNA targeting exon 2 of the Leptin gene into a two-cell embryo under a red light source, transplanting the embryo into a recipient, thereby obtaining a Leptin gene-modified obese golden hamster (ob / ob) model.

[0007] Furthermore, the target site of the gene to be knocked out in golden hamster was determined, and an sgRNA targeting the hamster leptin gene was designed. The nucleotide sequence of the sgRNA is shown in SEQ ID NO:1.

[0008] gLeptin-Sg1 (sgRNA): GGTGCAAATATCTAACGACC (SEQ ID NO: 1).

[0009] Furthermore, the process for preparing the sgRNA is as follows: a double-stranded DNA fragment is formed by annealing with primers gLeptin-Sg1F and gLeptin-Sg1R; the double-stranded DNA fragment is inserted into the PUC57-CRISP9 vector linearized with BsaI; after transformation and screening, an sgRNA expression plasmid is obtained; using the verified sgRNA expression plasmid as a template, high-concentration sgRNA is synthesized and purified by in vitro transcription; the nucleotide sequence of gLeptin-Sg1F is shown in SEQ ID NO:2, and the nucleotide sequence of gLeptin-Sg1R is shown in SEQ ID NO:3.

[0010] gLeptin-Sg1F: TAGGTGCAAATATCTAACGACC (SEQ ID NO: 2).

[0011] gLeptin-Sg1R:AAACGGTCGTTAGATATTTGCA (SEQ ID NO:3).

[0012] Furthermore, the PUC57-CRISP9 vector is a PUC57-CRISP9-sgRNA-GFP plasmid, and the construction method of the PUC57-CRISP9-sgRNA-GFP plasmid includes: digesting the PUC57 plasmid with Not I and Xho I restriction endonucleases to obtain a 2608 bp fragment as the PUC57-CRISP9 vector backbone; inserting the U6-T7-GFP-tracrRNA sequence as shown in SEQ ID NO:9 into the PUC57-CRISP9 vector backbone to construct the PUC57-CRISP9-sgRNA-GFP plasmid.

[0013] Furthermore, the construction method specifically includes the following steps:

[0014] (1) Under red light, active sgRNA and Cas9 functional elements were co-injected into the cytoplasm or nucleus of two-cell embryos of golden hamsters, and the injected embryos were transplanted into recipient mother mice for gestation to obtain F0 generation hamsters, and PCR identification and sequencing were performed.

[0015] (2) F0 generation hamsters were crossed with wild-type hamsters to obtain F1 generation hamsters, and PCR identification and sequencing were performed;

[0016] (3) Cross F1 generation heterozygous hamsters to obtain F2 generation hamsters. After PCR identification and sequencing, stable homozygotes were obtained, which are the Leptin gene-modified obese golden hamster animal models.

[0017] Furthermore, the Cas9 functional element is Cas9 mRNA or Cas9 protein.

[0018] Furthermore, the specific primer pairs for PCR identification include: Leptin-TOF, Leptin-TOR, Leptin-TIF, and Leptin-TIR; the nucleotide sequence of Leptin-TOF is shown in SEQ ID NO:4, the nucleotide sequence of Leptin-TOR is shown in SEQ ID NO:5, the nucleotide sequence of Leptin-TIF is shown in SEQ ID NO:6, and the nucleotide sequence of Leptin-TIR is shown in SEQ ID NO:7.

[0019] Leptin-TOF: CCCAATAAGATTAGTCAGAAGTGAAGG (SEQ ID NO: 4)

[0020] Leptin-TOR:TGAGAATCACCTCCGCAGACT (SEQ ID NO:5)

[0021] Leptin-TIF: AGAGGAGGCTCAGAATAAGCAGAA (SEQ ID NO: 6)

[0022] Leptin-TIR:GAATGGCGTGTCTATCCTTAGTCAA (SEQ ID NO:7).

[0023] In the technical solution of this invention, all golden hamsters are kept in SPF-grade and equivalent breeding environments.

[0024] In the technical solution of this invention, all experiments were conducted at room temperature (28.5℃) under red light.

[0025] In the technical solution of this invention, all golden hamsters that provide embryos are induced to ovulate superovulate by intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) (15 IU / 100g) and then mated with male hamsters in a 1:1 cage.

[0026] In the technical solution of this invention, before and after the two-cell embryo transfer, the embryo is placed in an incubator in HEMC-11 culture medium. The culture conditions are: temperature 37.5℃, carbon dioxide content 10%, oxygen concentration 5%, and nitrogen concentration 85%.

[0027] In the technical solution of this invention, the target site of sgRNA is located at exon 2 of the Leptin gene or upstream and downstream of the exon. The Cas9 protein translated from Cas9 mRNA in vivo or the Cas9 protein in the injected sample binds to the target site under the guidance of sgRNA, thereby causing DNA double-strand breaks and generating non-homologous recombination repair.

[0028] As a specific embodiment of the present invention, the method for constructing the Leptin gene-modified obese golden hamster model of the present invention specifically includes the following steps:

[0029] (1) Structure of Leptin gene and design of target site for sgRNA: The target site of the gene to be knocked out in golden hamster was determined, and sgRNA targeting the Leptin gene in golden hamster was designed. The nucleotide sequence of the sgRNA is shown in SEQ ID NO:1.

[0030] Preparation of the sgRNA: A double-stranded DNA fragment was formed by annealing with primers gLeptin-Sg1F and gLeptin-Sg1R. This double-stranded DNA fragment encodes an sgRNA targeting the leptin gene (SEQ ID NO:1). The double-stranded DNA fragment was inserted into a BsaI-linearized PUC57-CRISP9 vector. After transformation and screening, an sgRNA expression plasmid was obtained. Using the verified sgRNA expression plasmid as a template, high-concentration sgRNA was synthesized and purified by in vitro transcription. The nucleotide sequences of gLeptin-Sg1F and gLeptin-Sg1R are shown in SEQ ID NO:2-3. The PUC57-CRISP9 vector is a PUC57-CRISP9-sgRNA-GFP plasmid. The construction method of the PUC57-CRISP9-sgRNA-GFP plasmid includes: digesting the PUC57 plasmid with Not I and Xho I restriction endonucleases to obtain a 2608 bp fragment as the PUC57-CRISP9 vector backbone; inserting the U6-T7-GFP-tracrRNA sequence, as shown in SEQ ID NO:9, into the PUC57-CRISP9 vector backbone to construct the PUC57-CRISP9-sgRNA-GFP plasmid.

[0031] (2) Donor preparation: Select 6-8 week old golden female mice. At 9:00 AM on the first day of estrus, inject pregnant mare serum gonadotropin (PMSG) (15 IU / 100g) intraperitoneally to induce superovulation. At 6:00 PM on the fourth day, mate with male mice in a 1:1 ratio. At 9:00 AM on the second day, examine vaginal secretions under a microscope for sperm. The presence of sperm indicates mating.

[0032] (3) Recipient preparation: Eight-week-old golden female mice were selected and mated with male mice in a 1:1 ratio at 6 pm on the fourth day of estrus. At 9 am on the second day, the vaginal secretions were examined under a microscope for sperm. The presence of sperm indicated that the recipient was a 0.5-day true pregnancy recipient.

[0033] (4) Embryo Acquisition: Hamster fertilized eggs are sensitive to pH, temperature and light. All experiments were performed at room temperature of 28.5℃ under red light. The donor female mice were anesthetized by intraperitoneal injection of 1.25% aphthine (1.8 ml / 100 g), and then euthanized by cervical dislocation. The fallopian tubes were excised from the abdomen and placed in culture droplets. Under a stereomicroscope, the ampulla of the fallopian tube was torn open with ophthalmic forceps to release the cell cluster. Two-cell embryos with uniform blastomeres were selected and transferred to HEMC-11 culture medium for temporary storage. The in vitro embryo acquisition process was completed within 30 minutes.

[0034] (5) Embryo injection: 20 ng / μl sgRNA and 50 ng / μl cas9 mRNA were injected into the cytoplasm of two-cell embryos through a micromanipulator. After injection, the embryos were cultured in a three-gas incubator with a temperature of 37.5℃, a carbon dioxide content of 10%, an oxygen concentration of 5%, and a nitrogen concentration of 85%.

[0035] (6) Embryo transfer: 1.25% aphrodisiac recipient female mice, after making an incision in the middle of the back skin, open the abdominal wall muscle layer between the abdominal ribs and iliac bone, use forceps to remove the fat pad and pull out one side of the ovary and fallopian tube, and aspirate 14-20 embryos for use. Under a stereomicroscope, use ophthalmic forceps to longitudinally tear the ampulla and fimbriae of the fallopian tube, and blow the embryo into the ampulla of the fallopian tube through the transfer tube.

[0036] (7) A method for constructing a Leptin gene knockout mutant golden hamster model, comprising: constructing a Leptin gene knockout mutant golden hamster model by using gLeptin-Sg1 (sgRNA). The Leptin gene mutation includes: ① deletion of the nucleotide sequence after amino acid 99 in the entire exon 2 sequence of the Leptin gene; the deletion of nucleotides after amino acid 99 in the entire exon 2 sequence of the Leptin gene leads to translation errors of the Leptin protein.

[0037] (8) Breeding and screening of the Leptin gene-modified obese golden hamster (ob / ob) model, which includes: ① injecting a mixture of Cas9 mRNA (or Cas9 protein) and sgRNA into golden hamster fertilized eggs and then transplanting them into surrogates to obtain F0 generation golden hamsters, and screening the positive golden hamsters among them as the F0 generation Leptin gene knockout mutant golden hamsters; ② screening the offspring obtained by crossing the F0 generation Leptin gene knockout mutant golden hamsters with wild-type golden hamsters, and taking the positive golden hamsters among them as the F1 generation heterozygous golden hamsters; ③ screening the offspring obtained by self-breeding the F1 generation heterozygous golden hamsters, and taking the positive golden hamsters among them as the F2 generation golden hamsters; the screening method for positive golden hamsters includes PCR identification and gene sequencing, and the positive homozygotes in the F2 generation golden hamsters are selected as the Leptin gene-modified diabetic golden hamster animal model.

[0038] In one embodiment, the specific primer pairs used for PCR identification and gene sequencing include: Leptin-TOF, Leptin-TOR, Leptin-TIF, and Leptin-TIR; the nucleotide sequences of Leptin-TOF, Leptin-TOR, Leptin-TIF, and Leptin-TIR are shown in SEQ ID NO:4-7.

[0039] This invention protects a method for modeling leptin-modified obese golden hamsters by genetically modifying fertilized eggs or two-cell embryos and then transplanting them into surrogate mother mice for development. When leptin-homozygous animals obtained using this method were mated with wild-type golden hamsters, no offspring were produced, indicating that the leptin-homozygous mutant is infertile like ob / ob mice. Letpin released from white adipose tissue (body fat) in the visceral epididymal adipose tissue (EAT) and inguinal subcutaneous adipose tissue (IAT) was detected. Western blot analysis showed that leptin protein was not detected in EAT and IAT, thus confirming the successful construction of the leptin-modified golden hamster model.

[0040] Secondly, the present invention seeks protection for the use of the leptin gene-modified hamster model constructed by the above method in at least one of the following (1)-(6):

[0041] (1) To study the infection mechanism of SARS-CoV-2 virus;

[0042] (2) To study the related functions and mechanisms of action of the leptin gene;

[0043] (3) Study the pathogenesis of obesity;

[0044] (4) Screening for drugs to treat SARS-CoV-2 virus infection;

[0045] (5) Develop vaccines to prevent SARS-CoV-2 virus infection;

[0046] (6) Evaluation of novel pathogenic strains of SARS-CoV-2.

[0047] The advantages of the Leptin gene-modified golden hamster model constructed in this invention are as follows:

[0048] (1) The method for constructing the golden hamster model of the present invention is highly efficient and has a stable reproductive transmission rate. Leptin-modified golden hamster offspring are obtained by gene modification of two-cell embryos through true pregnancy recipient transplantation. The heterozygous strain of this modified strain has normal fertility. Therefore, the present invention provides an important method for constructing a golden hamster model for applied research such as exploring the pathogenesis of obesity, novel coronavirus infection, and vaccine and drug development.

[0049] (2) Compared with wild-type hamsters, the ob / ob obese golden hamsters constructed in this invention have increased liver damage indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total protein (TP), which is helpful for obesity-related research.

[0050] (3) Ob / ob obese golden hamsters constructed based on the present invention died after SARS-CoV-2 infection, while WT golden hamsters survived after SARS-CoV-2 infection, which is helpful for the study of the mechanism and drugs of severe novel coronavirus infection. Attached Figure Description

[0051] Figure 1 This is a schematic diagram and comparison image of the ob / ob obese golden hamster construction according to an embodiment of the present invention.

[0052] Golden hamster leptin has two exons. An sgRNA target site was designed on the second exon (left image). Through microinjection and passage, a letpin-deficient (Leptin - / -) (ob / ob) golden hamster strain was obtained. This strain lacks four nucleotides (del4), leading to mistranslation of the letpin protein. The right image shows that at 8 weeks, the weight of leptin - / - hamsters was significantly greater than that of wild-type golden hamsters (wt).

[0053] Figure 2 This invention provides a statistical analysis of the fertility of male and female ob / ob obese golden hamsters, specifically Het (heterozygous) and Hom (homozygous).

[0054] Fertility tests showed that heterozygous Leptin + / - hamsters were fertile in both sexes, with no difference from the wild type. Homozygous ob / ob obese golden hamsters were male-sterile and female-infertile.

[0055] Figure 3 This is the Western blot result of ob / ob obese golden hamsters in an embodiment of the present invention.

[0056] Western blot analysis of ob / ob obese golden hamsters revealed the absence of leptin expression in the visceral epididymal adipose tissue (EAT) and inguinal subcutaneous adipose tissue (IAT). This result indicates that leptin gene knockout golden hamsters have been successfully obtained.

[0057] Figure 4 The growth curves of ob / ob obese golden hamsters and WT golden hamsters at 6-12 weeks are shown in the embodiments of the present invention.

[0058] Weight monitoring showed that significant differences were observed in male mice at 6 weeks of age, while in female mice, weight differences were not observed until after 9 weeks.

[0059] Figure 5 The overweight rate of ob / ob obese golden hamsters and WT golden hamsters in the embodiments of the present invention.

[0060] Letpin knockout hamsters showed minimal weight changes compared to wild-type (WT) hamsters. Male hamsters gained 25-38% weight between weeks 6 and 12, while females gained 10-12%. In contrast, OB mice gained 80-100% of their body weight. These results indicate that the presence of the leptin gene function is a species-specific variation.

[0061] Figure 6 These are representative biochemical indicators of ob / ob obese golden hamsters and WT golden hamsters at 8 weeks of age, as described in this embodiment of the invention.

[0062] Compared to 8-week-old wild-type hamsters, 8-week-old Letpin hamsters had higher levels of liver damage markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total protein (TP). Significantly elevated serum total cholesterol (CHOL), triglycerides (TRIG), and creatine kinase (CK) levels indicated dyslipidemia in Letpin hamsters.

[0063] Impaired cardiovascular function.

[0064] Figure 7 In this embodiment of the invention, ob / ob obese golden hamsters died after SARS-CoV-2 infection, while WT golden hamsters survived after SARS-CoV-2 infection.

[0065] SARS-CoV-2 infection can induce severe disease in letpin- / - hamsters. To establish an obesity-related infection model, letpin- / - and wild-type hamsters were intranasally infected with SARS-CoV-2. Body weight changes, clinical symptoms, and survival rates were monitored daily. Although the body weight curves overlapped between the two groups, we observed a significantly greater decrease in body weight in letpin- / - hamsters than in wild-type hamsters, with a mortality rate of approximately 40%.

[0066] Figure 8 The images show lung pathological changes in ob / ob obese golden hamsters and WT golden hamsters after H&E staining following SARS-CoV-2 infection, as illustrated in the embodiments of the present invention. Rectangles represent magnified areas.

[0067] Letpin- / - hamsters exhibited more severe pneumonia than wild-type hamsters at 3, 7, and 14 days post-infection. At 3 days post-infection, leptin-containing hamsters showed marked lesions in certain areas, characterized by thickened and dilated alveolar walls with neutrophil infiltration. (DPI, Days Post-Infection).

[0068] Figure 9 This is a structural diagram of the U6-T7-GFP-trcRNA sequence. Detailed Implementation

[0069] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make equivalent changes to the disclosed technical content to create equivalent embodiments. Any modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention fall within the protection scope of the present invention.

[0070] Example 1

[0071] This embodiment provides a method for constructing a hamster animal model modified with the leptin gene via microinjection. The specific steps of the construction method are as follows:

[0072] (1) Structure of Leptin gene (Gene ID: 101837349, Leptin protein: XM_005078071.4; XP_005078128.1, as shown in SEQ ID NO: 8) and design of sgRNA target site: The target site of the gene to be knocked out in golden hamster was determined, and sgRNA targeting the hamster Leptin gene was designed. The sgRNA is gLeptin-Sg1 (SEQ ID NO: 1: GGTGCAAATATCTAACGACC).

[0073] The designed sgRNA sequence was constructed into an expression vector using an in vitro ligation cloning method to obtain an sgRNA expression plasmid. Using the validated sgRNA expression plasmid as a template, high-concentration sgRNA was synthesized and purified through in vitro transcription.

[0074] The primers used in constructing the sgRNA expression plasmid include gLeptin-Sg1F and gLeptin-Sg1R.

[0075] gLeptin-Sg1F: TAGGTGCAAATATCTAACGACC (SEQ ID NO: 2;

[0076] gLeptin-Sg1R:AAACGGTCGTTAGATATTTGCA (SEQ ID NO:3).

[0077] The specific process of constructing the sgRNA expression plasmid and performing in vitro transcription is as follows: (a) Constructing the sgRNA expression plasmid

[0078] Construction of PUC57-CRISP9-sgRNA-GFP plasmid

[0079] The PUC57 (General Electric Company, GE) plasmid was purified by gel extraction using Not I and Xho I restriction endonucleases at 37°C for 2 hours to obtain a 2608 bp fragment, which was used as the backbone of the PUC57-CRISP9 vector.

[0080] The artificially synthesized sequence containing U6-T7-GFP-tracrRNA (containing Not I and Xho I restriction sites, nucleotide sequence as shown in SEQ ID NO:9, sequence structure as shown in...) was used. Figure 9 As shown, the vector backbone (2608 bp) was inserted into the PUC57-CRISP9 vector to construct PUC57-CRISP9-sgRNA-GFP, and the results were verified by sequencing.

[0081] Vector backbone preparation: The PUC57-CRISP9-sgRNA-GFP plasmid was linearized by digestion with BsaI restriction endonuclease. The target vector fragment of approximately 2965 bp was then separated and purified by gel electrophoresis for later use.

[0082] Oligo annealing: The two synthesized specific primers, gLeptin-Sg1F and gLeptin-Sg1R, were mixed. First, they were treated with T4 polynucleotide kinase (T4 PNK) to ensure 5' phosphorylation of the oligonucleotide chains, which is essential for subsequent ligation. Then, the two primers were annealed to form a double-stranded DNA fragment through a temperature cycling program (37°C, 30 min → 95°C, 5 min → slow cooling to 25°C).

[0083] Ligation and Transformation: The annealed double-stranded Oligo fragment (after dilution) was mixed with the purified linearized vector backbone and ligated using T4 DNA ligase to insert the sgRNA sequence into the vector. The ligation product was transformed into DH5α competent E. coli cells, and selection was performed using Amp+ (ampicillin resistance). Only colonies successfully transformed with the plasmid were allowed to grow. Positive Clone Identification: Single colonies were picked and cultured. After plasmid extraction, PCR amplification and sequencing were performed using universal primers M13F / M13R (M13R: CAG GAA ACA GCT ATG ACC; M13F: TGTAAA ACG ACG GCC AGT) to verify the correctness of the inserted sgRNA sequence.

[0084] (b) In vitro transcription to synthesize sgRNA

[0085] After obtaining a plasmid that has been correctly sequenced, it is used as a template to generate a large amount of the required sgRNA through in vitro transcription.

[0086] Transcription template amplification: Using the successfully validated plasmid as a template, PCR amplification was performed using M13F / M13R primers. The product was purified by gel excision and used as a template for in vitro transcription. The purpose of this step is to obtain a large number of pure sgRNA-encoded DNA fragments without the plasmid backbone.

[0087] In vitro transcription: Using the HiScribe T7 in vitro transcription kit, sgRNA was synthesized under the catalysis of T7 RNA polymerase, using the PCR product as a template. After the reaction was completed, TURBO DNase was added to degrade the DNA template, ensuring that the final product was pure RNA.

[0088] sgRNA purification and storage: The transcribed sgRNA was purified using ethanol precipitation to remove impurities such as salt ions, proteins, and unbound nucleotides from the reaction system. The purified sgRNA was reconstituted with RNase-free water, its concentration was determined, and it was diluted to a working concentration (e.g., 1000 ng / μl). Finally, it was stored at -80°C.

[0089] (2) Donor preparation: Select 6-8 week old golden female mice. At 9:00 AM on the first day of estrus, inject pregnant mare serum gonadotropin (PMSG) (15 IU / 100g) intraperitoneally to induce superovulation. At 6:00 PM on the fourth day, mate with male mice in a 1:1 ratio. At 9:00 AM on the second day, examine vaginal secretions under a microscope for sperm. The presence of sperm indicates mating.

[0090] (3) Recipient preparation: Eight-week-old golden female mice were selected and mated with male mice in a 1:1 ratio at 6 pm on the fourth day of estrus. At 9 am on the second day, the vaginal secretions were examined under a microscope for sperm. The presence of sperm indicated that the recipient was a 0.5-day true pregnancy recipient.

[0091] (4) Embryo Acquisition: Hamster fertilized eggs are sensitive to pH, temperature and light. All experiments were performed at room temperature of 28.5℃ under red light. The donor female mice were anesthetized by intraperitoneal injection of 1.25% aphthine (1.8 ml / 100 g), and then euthanized by cervical dislocation. The fallopian tubes were excised from the abdomen and placed in culture droplets. Under a stereomicroscope, the ampulla of the fallopian tube was torn open with ophthalmic forceps to release the cell cluster. Two-cell embryos with uniform blastomeres were selected and transferred to HEMC-11 culture medium for temporary storage. The in vitro embryo acquisition process was completed within 30 minutes.

[0092] (5) Embryo injection: 20 ng / μl sgRNA and 50 ng / μl cas9 mRNA were injected into the cytoplasm of two-cell embryos through a micromanipulator. After injection, the embryos were cultured in a three-gas incubator at 37.5℃, 1% carbon dioxide, 5% oxygen and 85% nitrogen.

[0093] (6) Embryo transfer: 1.25% aphrodisiac recipient female mice, after making an incision in the middle of the back skin, open the abdominal wall muscle layer between the abdominal ribs and iliac bone, use forceps to remove the fat pad and pull out one side of the ovary and fallopian tube, and aspirate 14-20 embryos for use. Under a stereomicroscope, use ophthalmic forceps to longitudinally tear the ampulla and fimbriae of the fallopian tube, and blow the embryo into the ampulla of the fallopian tube through the transfer tube.

[0094] (7) The identification primers are any one or a combination of two of the outer identification primer pair and the inner identification primer pair; F0 generation positive heterozygous hamsters are selected by sequencing and mated with wild-type hamsters to obtain F1 generation hamsters. F2 generation hamsters are obtained by hybridization among the F1 generation heterozygous hamsters selected by sequencing. The obtained F2 generation hamsters are identified by PCR and sequenced to obtain homozygotes in the F2 generation hamsters. The short chain screening and identification primer pairs include: Leptin-TOF, Leptin-TOR, Leptin-TIF and Leptin-TIR.

[0095] Leptin-TOF: CCCAATAAGATTAGTCAGAAGTGAAGG (SEQ ID NO: 4)

[0096] Leptin-TOR:TGAGAATCACCTCCGCAGACT (SEQ ID NO:5)

[0097] Leptin-TIF: AGAGGAGGCTCAGAATAAGCAGAA (SEQ ID NO: 6)

[0098] Leptin-TIR:GAATGGCGTGTCTATCCTTAGTCAA (SEQ ID NO:7).

[0099] Golden hamster leptin has two exons. sgRNA target sites (such as...) can be designed on the second exon. Figure 1 (Left image in the image). Sequencing results showed that a letpin-deficient (Leptin - / -) (ob / ob) golden hamster strain was obtained through microinjection and passage. This strain lacks 4 nucleotides (del4), leading to mistranslation of the letpin protein. Figure 1 The right image shows that the 8-week-old Leptin - / - hamsters had a significantly larger weight (wt) than wild-type golden hamsters.

[0100] (8) Comparative experiment: The experiment was divided into basic phenotype and SARS-CoV-2 infection.

[0101] ① Leptin- / - fertility was tested by mating male and female Leptin Hom (homozygous) golden hamsters with wild-type (WT), Het (heterozygous) hamsters with Het, and WT hamsters with WT. Hom hamsters were determined to be infertile, while other groups showed normal fertility. For example... Figure 2 As shown, heterozygous Leptin + / - individuals are fertile in both sexes, showing no difference from the wild type. Homozygous ob / ob obese golden hamsters are male-sterile and female-infertile.

[0102] ② Western blot analysis showed complete loss of leptin expression in the visceral epididymal adipose tissue (EAT) and inguinal subcutaneous adipose tissue (IAT) of Leptin- / - male golden hamsters (e.g., Figure 3 (As shown in the image), using Tubulin as a load control. This result demonstrates the successful acquisition of golden hamsters with the Leptin gene knocked out.

[0103] ③ Growth curves of Leptin- / - and WT male and female golden hamsters (N=9 per group) at 6-12 weeks. Weight monitoring showed that significant differences were observed in males at 6 weeks, while in females, weight differences were not observed until after 9 weeks (e.g. Figure 4 (As shown). A comparison of overweight rates between Leptin- / - male and female golden hamsters and wild-type controls showed that the weight of golden hamsters varied slightly compared to wild-type (WT) hamsters. Males gained 25-38% of their weight from 6-12 weeks, while females gained only 10-12% from 6-12 weeks (e.g., ...). Figure 5 (As shown). OB mice gained 80-100% of their body weight. This result indicates that the presence of leptin gene function varies across species.

[0104] ④ Compared to wild-type hamsters, letpin- / - showed increased liver damage markers, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total protein (TP). Significantly increased serum total cholesterol (CHOL) and triglycerides (TRIG), and decreased creatine kinase (CK) were also detected, indicating abnormal lipid levels and impaired cardiovascular function in letpins (e.g., Figure 6 (As shown).

[0105] ⑤ After SARS-CoV-2 infection, several Leptin- / - hamsters died after infection, while WT hamsters survived in the experiment (Leptin- / - n=13, WT n=14) (e.g. Figure 7 (As shown). SARS-CoV-2 infection can induce severe disease in letpin- / - hamsters. To establish an obesity-related infection model, letpin- / - and wild-type hamsters were intranasally infected with SARS-CoV-2. Weight changes, clinical symptoms, and survival rates were monitored daily. Although the weight curves of the two groups overlapped, we observed that the weight loss in letpin- / - hamsters was significantly greater than that in wild-type hamsters, and the mortality rate was approximately 40%.

[0106] HE staining revealed pathological analysis of lung tissue. Compared to WT hamsters, Leptin- / - hamsters exhibited more severe pneumonia at 3, 7, and 14 DPI. At 3 DPI, Leptin- / - hamsters showed significant lesions in certain areas, characterized by thickening and widening of the alveolar walls, and infiltration of neutrophils and lymphocytes. Additionally, mild hemorrhage, protein exudation, or hyaline membrane formation were observed, along with significant swelling and necrosis of numerous bronchial epithelial cells, accompanied by a small increase in inflammatory cells and perivascular inflammation. At 7 DPI, the lesions in Leptin- / - hamsters were more severe than in WT hamsters, characterized by significant proliferation of bronchial epithelial cells, inflammatory cell infiltration in most areas, and alveolar hemorrhage and extensive macrophage and lymphocyte infiltration in some areas. By 14 DPI, the lung lesions in Leptin- / - hamsters were further worse than in wild-type hamsters, as the affected areas did not recover or absorb properly, but instead showed consolidation and fibrosis, with significant proliferation of bronchial epithelial cells. Fibroblast proliferation and fibrosis were observed in parts of the alveolar walls and interstitial spaces, along with numerous infiltrative inflammatory cells. Pathological scoring confirmed that, during the period from 3 to 14 DPI, Leptin- / - hamsters exhibited more severe lung tissue damage (e.g., ...) compared to wild-type hamsters. Figure 8 (As shown). (Errors are expressed as mean ± SEM. Statistical significance was measured by Student's t-test and two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).

[0107] sequence list

[0108] Leptin protein sequence (XM_005078071.4; XP_005078128.1, SEQ ID NO:8)

[0109] MCWRPLCWFLWLWSYLSYVQAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSLPSRNVVQISNDLENLRDLLHLLASSKSCSLPQTSELQTRESLDGVLEASLYSTEVVALSRLQGSLQDILRQLDLSPEC.

[0110] Nucleotide sequence of U6-T7-GFP-trcRNA (SEQ ID NO:9):

[0111]

Claims

1. A method for constructing a Leptin gene-modified obese golden hamster model, characterized in that, The method includes: injecting Cas9 functional elements and sgRNA targeting exon 2 of the Leptin gene into two-cell embryos under a red light source, transplanting the embryos into recipients, and thus obtaining a Leptin gene-modified golden hamster model.

2. The construction method according to claim 1, characterized in that, The target site of the gene to be knocked out in golden hamster was determined, and an sgRNA targeting the hamster leptin gene was designed. The nucleotide sequence of the sgRNA is shown in SEQ ID NO:

1.

3. The construction method according to claim 1 or 2, characterized in that, The process for preparing the sgRNA is as follows: a double-stranded DNA fragment is formed by annealing with primers gLeptin-Sg1F and gLeptin-Sg1R; the double-stranded DNA fragment is inserted into the PUC57-CRISP9 vector linearized with BsaI; after transformation and screening, an sgRNA expression plasmid is obtained; using the verified sgRNA expression plasmid as a template, high-concentration sgRNA is synthesized and purified by in vitro transcription; the nucleotide sequence of gLeptin-Sg1F is shown in SEQ ID NO:2, and the nucleotide sequence of gLeptin-Sg1R is shown in SEQ ID NO:

3.

4. The construction method according to claim 3, characterized in that, The PUC57-CRISP9 vector is a PUC57-CRISP9-sgRNA-GFP plasmid. The construction method of the PUC57-CRISP9-sgRNA-GFP plasmid includes: digesting the PUC57 plasmid with Not I and Xho I restriction endonucleases to obtain a 2608 bp fragment as the PUC57-CRISP9 vector backbone; inserting the U6-T7-GFP-tracrRNA sequence, as shown in SEQ ID NO:9, into the PUC57-CRISP9 vector backbone to construct the PUC57-CRISP9-sgRNA-GFP plasmid.

5. The construction method according to any one of claims 1-4, characterized in that, The method specifically includes the following steps: (1) Under red light, active sgRNA and Cas9 functional elements were co-injected into the cytoplasm or nucleus of two-cell embryos of golden hamsters, and the injected embryos were transplanted into recipient mother mice for gestation to obtain F0 generation hamsters, and PCR identification and sequencing were performed. (2) F0 generation hamsters were crossed with wild-type hamsters to obtain F1 generation hamsters, and PCR identification and sequencing were performed; (3) Cross F1 generation heterozygous hamsters to obtain F2 generation hamsters. After PCR identification and sequencing, stable homozygotes were obtained, which are the Leptin gene-modified golden hamster animal models.

6. The construction method according to claim 1 or 5, characterized in that, The Cas9 functional element is Cas9 mRNA or Cas9 protein.

7. The construction method according to claim 5, characterized in that, The specific primer pairs for PCR identification include: Leptin-TOF, Leptin-TOR, Leptin-TIF, and Leptin-TIR; the nucleotide sequence of Leptin-TOF is shown in SEQ ID NO:4, the nucleotide sequence of Leptin-TOR is shown in SEQ ID NO:5, the nucleotide sequence of Leptin-TIF is shown in SEQ ID NO:6, and the nucleotide sequence of Leptin-TIR is shown in SEQ ID NO:

7.

8. The Leptin-modified hamster model constructed by any of the methods described in claims 1-7 is used in at least one of the following (1)-(6): (1) To study the infection mechanism of SARS-CoV-2 virus; (2) To study the related functions and mechanisms of action of the leptin gene; (3) Study the pathogenesis of obesity; (4) Screening for drugs to treat SARS-CoV-2 virus infection.

9. The application of the leptin gene-modified hamster model constructed by any of the methods described in claims 1-7 in the development of a vaccine to prevent SARS-CoV-2 virus infection.

10. The application of the leptin gene-modified hamster model constructed by any of the methods described in claims 1-7 in the evaluation of novel pathogenic strains of SARS-CoV-2.

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