Construction method and application of Dcaf17 gene modified golden hamster model

Abstract

The invention discloses a construction method of a Dcaf17 gene modified golden hamster model and application of the Dcaf17 gene modified golden hamster model. The method comprises the following steps: under a red light source, injecting a Cas9 functional element and sgRNA of a targeted Dcaf17 gene exon 1 through a two-cell embryo, and transplanting the embryo into a receptor body, so as to obtain the Dcaf17 gene modified golden hamster. The Dcaf17 gene modified golden hamster model is constructed by utilizing two-cell stage embryo gene editing, and the Dcaf17 gene modified golden hamster homozygous male rat has the phenotype characteristics of no fertilization ability, normal testis and the like, and is used for replacing ligation male rats to prepare false pregnancy receptor rats. The receptor can efficiently identify and utilize a false pregnancy receptor through microscopic examination of sperms by a microscope. Therefore, the invention solves the technical difficulty of receptor preparation in related experiments of assisted reproduction of the golden hamster, and can be used for phenotype research of the Dcaf17 gene and related applications of assisted reproduction of the golden hamster.

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 Dcaf17 gene-modified golden hamster model and its application. Background Technology

[0002] Mutations in Dcaf17 (DDB1 and CUL4 associated factor 17) lead to Woodhouse-Sakati syndrome (WSS), which causes male infertility and oligospermia / asthenospermia. The pathogenesis of this syndrome requires further investigation. Furthermore, in assisted reproductive experiments involving embryo transfer in golden hamsters, females require mating stimulation to maintain pregnancy. Vasectomized mice are used for mating, but males lack coagulation glands, making it impossible to determine mating status using sperm plugs, resulting in low efficiency in recipient preparation using vasectomized mice. Utilizing the basal phenotype of male golden hamster infertility and abnormal spermatogenesis caused by Dcaf17 deletion, sperm analysis of vaginal secretions after mating can determine whether mating has occurred. Moreover, the application of Dcaf17 mutant mice will effectively reduce surgical infections and physiological and psychological damage to golden hamsters, conforming to the 3R (optimization, replacement, and reduction) principle of laboratory animals. Dcaf17 mutant mice hold significant research and application potential in gene function analysis and assisted reproduction in golden hamsters. Summary of the Invention

[0003] In view of the technical problems raised in the background art, the purpose of this invention is to provide a method for constructing a Dcaf17 gene-modified golden hamster 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 construct golden hamsters with a Dcaf17 mutation by injecting Cas9 protein and sgRNAs targeting exon 1 of Dcaf17 into two-cell embryos, thus avoiding early embryonic developmental arrest. Offspring of these constructed embryos are obtained through the transfer of true pregnancy albino recipient embryos and used to establish a pseudopregnancy recipient model in assisted reproductive experiments.

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

[0006] In a first aspect, the present invention seeks to protect a method for constructing a Dcaf17 gene-modified golden hamster model, the method comprising: injecting a Cas9 functional element (Cas9 mRNA or Cas9 protein) and sgRNA targeting exon 1 of the Dcaf17 gene into a two-cell embryo under a red light source, transplanting the embryo into a recipient, thereby obtaining a Dcaf17 gene-modified golden hamster.

[0007] Furthermore, the target site of the gene to be knocked out in golden hamster was determined, and an sgRNA targeting the DCaf17 gene in golden hamster was designed. The nucleotide sequence of the sgRNA is shown in SEQ ID NO:1.

[0008] SEQ ID NO: 1: GGCTTCTTCGCACGCGACGCGGG.

[0009] Furthermore, the designed sgRNA was amplified in vitro to construct a plasmid expressing the sgRNA, and the sgRNA was transcribed in vitro. The primers for constructing the plasmid expressing the sgRNA included Dcaf17-Sg1F and gDcaf17-Sg1R. The nucleotide sequence of Dcaf17-Sg1F is shown in SEQ ID NO:2, and the nucleotide sequence of Dcaf17-Sg1R is shown in SEQ ID NO:3.

[0010] SEQ ID NO: 2: TAGGGCTTCTTCGCACGCGACGC.

[0011] SEQ ID NO: 3: AAACGCGTCGCGTGCGAAGAAGC.

[0012] Furthermore, the Cas9 mentioned is Cas9 mRNA or Cas9 protein.

[0013] Furthermore, the specific steps of the above construction method are as follows:

[0014] (1) Under red light, active sgRNA and Cas9 mRNA or Cas9 protein 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 Dcaf17 gene-modified golden hamsters.

[0015] (2) The Dcaf17 gene-modified golden hamsters were purified by hybridization and genotyping of F0, F1 and F2 generations: F0 generation Dcaf17 gene-modified golden hamsters were hybridized with normal wild-type golden hamsters to obtain F1 generation Dcaf17 gene-modified golden hamsters. The obtained F1 generation golden hamsters were identified by PCR and sequenced. The F1 generation heterozygous golden hamsters were hybridized to obtain F2 generation golden hamsters. The obtained F2 generation golden hamsters were identified by PCR and sequenced. Homozygous males in the F2 generation golden hamsters were selected as Dcaf17 gene-modified golden hamster animal models.

[0016] Furthermore, in step (1), the genomic DNA of the F0 generation Dcaf17 gene-modified golden hamster was identified by PCR.

[0017] Furthermore, the specific primer pairs for PCR identification of the golden hamster and its offspring include: Decaf17-TOF, Decaf17-TOR, Decaf17-TIF, and Decaf17-TIR;

[0018] The nucleotide sequence of the Decaf17-TOF is shown in SEQ ID NO:4, the nucleotide sequence of the Decaf17-TOR is shown in SEQ ID NO:5, the nucleotide sequence of the Decaf17-TIF is shown in SEQ ID NO:6, and the nucleotide sequence of the Decaf17-TIR is shown in SEQ ID NO:7.

[0019] In the technical solution of this invention, the golden hamster is kept in an SPF-grade or equivalent environment.

[0020] In the technical solution of this invention, the experimental process is carried out at room temperature of 28.5℃ under red light.

[0021] 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.

[0022] 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%.

[0023] In the technical solution of this invention, the target site of sgRNA is located in exon 1 of Dcaf17. 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.

[0024] As a specific embodiment of the present invention, the method for constructing the Dcaf17 gene-modified golden hamster model of the present invention includes the following steps:

[0025] (1) Structure of the Dcaf17 gene and design of the target site of sgRNA: The target site of the gene to be knocked out in golden hamster was determined, and an sgRNA targeting exon 1 of the golden hamster Dcaf17 gene was designed. The nucleotide sequence of the sgRNA is shown in SEQ ID NO:1.

[0026] The designed sgRNA was amplified in vitro to construct a plasmid expressing the sgRNA, and the sgRNA was transcribed in vitro. Preferably, the primers for constructing the plasmid expressing the sgRNA include: gDcaf17-Sg1F with nucleotide sequences as shown in SEQ ID NO:2 and gDcaf17-Sg1R with nucleotide sequences as shown in SEQ ID NO:3.

[0027] (2) Donor preparation: Select 6-8 week old golden female mice. On the first day of estrus, at 9:00 AM, inject pregnant mare serum gonadotropin (PMSG) (15 IU / 100g) intraperitoneally to induce superovulation. On the fourth day at 6:00 PM, mate with male mice in a 1:1 ratio. On the second day at 9:00 AM, examine vaginal secretions under a microscope for sperm. The presence of sperm indicates mating.

[0028] (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, sperm were examined under a microscope in vaginal secretions. The presence of sperm indicated that the recipient was a 0.5-day true pregnancy recipient.

[0029] (4) Embryo Acquisition: Golden hamster fertilized eggs are sensitive to pH, temperature and light. All experiments were performed at room temperature (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 oviduct was excised from the abdomen and placed in culture droplets. Under a stereomicroscope, the ampulla of the oviduct 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.

[0030] (5) Embryo injection: 20 ng / μl sgRNA and 50 ng / μl cas9 mRNA were injected into the cytoplasm of two-cell embryos through a micromanipulation system. The injected embryos were cultured in a three-gas incubator at 37.5℃, 10% carbon dioxide, 5% oxygen and 85% nitrogen.

[0031] (6) Embryo transfer: 1.25% of recipient female mice were anesthetized with afodin. After making an incision in the middle of the back skin, the abdominal wall muscle layer was opened between the abdominal ribs and iliac bone. The fat pad was removed by forceps and one ovary and fallopian tube were pulled out. 14-20 embryos were aspirated for use. Under a stereomicroscope, ophthalmic forceps were used to longitudinally tear the ampulla and fimbriae of the fallopian tube. The embryo was blown into the ampulla of the fallopian tube through the transfer tube.

[0032] (7) A method for constructing a Dcaf17 gene knockout mutant golden hamster model, comprising: constructing a Dcaf17 gene knockout mutant golden hamster model by using gDcaf17-Sg1 (sgRNA) alone. The Dcaf17 gene mutation includes: ① deletion of the nucleotide sequence after amino acid 23 in the entire exon 1 sequence of the Dcaf17 gene.

[0033] (8) Breeding and screening of the Dcaf17 gene-modified golden hamster model, which includes: ① injecting a mixture of Cas9 mRNA (or Cas9 protein) and sgRNA into golden hamster fertilized eggs and then transferring them to surrogates to obtain F0 generation golden hamsters, and screening the positive golden hamsters among them as the F0 generation Dcaf17 gene-modified golden hamsters; ② screening the offspring obtained by crossing the F0 generation Dcaf17 gene-modified 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 homozygous males among the F2 generation golden hamsters are selected as the Dcaf17 gene-modified golden hamster animal model.

[0034] In one embodiment, the specific primer pairs used for PCR identification and gene sequencing include:

[0035] Dcaf17-TOF:CGTCAGCCTTCCCTTGAGAGTA (SEQ ID NO:4)

[0036] Dcaf17-TOR:CCAGCGTAATGCCTGAACCTT (SEQ ID NO:5)

[0037] Dcaf17-TIF:CCTTGAGAGTAGCTGGTGGC (SEQ ID NO:6)

[0038] Dcaf17-TIR: AACTACGGTCAGGTGAATTACAGAA (SEQ ID NO:7).

[0039] (9) Application of the Dcaf17 gene-modified golden hamster model

[0040] This invention protects the method for producing Dcaf17 gene-modified golden hamsters by genetically modifying fertilized eggs or two-cell embryos and then transplanting them into surrogate mother mice for development and production, as well as the method for creating pseudopregnant mice based on these genes.

[0041] As one specific implementation, the plasmid construction and in vitro transcription process includes the following steps:

[0042] (1) Constructing sgRNA expression plasmid

[0043] Vector backbone preparation: The PUC57-CRISP9 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.

[0044] Oligo annealing: The two synthesized specific primers, gDcaf17-Sg1F and gDcaf17-Sg1R, were mixed. First, they were treated with T4 polynucleotide kinase (T4 PNK) to ensure 5' end 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).

[0045] Ligation and Transformation: The annealed double-stranded Oligo fragment (after dilution) was mixed with the purified linearized vector backbone, and ligation was performed 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.

[0046] Positive clone identification: Single colonies were picked and cultured. After plasmid extraction, PCR amplification and sequencing were performed using universal primers M13F / M13R to verify whether the inserted sgRNA sequence was correct.

[0047] (2) In vitro transcription to synthesize sgRNA

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] Fertility tests were conducted on Dcaf17- / - animals obtained using the method of this invention and wild-type golden hamsters over a 4-month period. The results showed that Dcaf17- / - males were infertile. Compared with wild-type golden hamsters, there was no significant difference in body weight and testicular weight in Dcaf17-deficient golden hamsters, but there was a significant difference in epididymal size, with the epididymis being smaller. PAS staining showed a low sperm concentration in the epididymal tail, containing some giant cells. By preparing sperm smears from the epididymal tail and examining sperm morphology with hematoxylin and eosin (H&E) staining, sperm from Dcaf17- / - golden hamsters exhibited severe morphological defects, including amorphous heads and excess cytoplasm, abnormal head shape, and multiple flagellar malformations (shortened, curled, bent, or absent sperm flagella). Pseudopregnant females obtained by mating with Dcaf17- / - golden hamsters could be accurately determined by microscopic examination of sperm, and offspring were successfully obtained through embryo transfer. Therefore, it is confirmed that the Dcaf17 gene-modified golden hamster model was successfully constructed and can be applied to the preparation of pseudopregnant mice for assisted reproduction.

[0053] Secondly, this invention seeks to protect the application of the Dcaf17 gene-modified golden hamster model constructed by the above method in studying the related functions and mechanisms of action of the Dcaf17 gene.

[0054] Thirdly, this invention seeks to protect the use of male rats in the Dcaf17 gene-modified golden hamster model constructed by the above method in the preparation of assisted reproductive-related pseudopregnancy recipient rats.

[0055] The advantages of the Dcaf17 gene-modified golden hamster model constructed in this invention are as follows:

[0056] (1) The method for constructing the golden hamster model of the present invention is highly efficient and has a stable reproductive transmission rate. Golden hamster offspring modified with the Dcaf17 gene are obtained through gene modification of two-cell embryos via 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 exploring the regulatory mechanism of the Dcaf17 gene and the preparation of pseudopregnancy recipients, among other assisted reproductive technologies.

[0057] (2) Compared with wild-type golden hamsters, the sperm of the Dcaf17- / - golden hamsters constructed in this invention exhibits severe morphological defects, including amorphous head and excess cytoplasm, abnormal head shape and multiple flagellar malformations (sperm flagella are shortened, curled, bent or absent), which is helpful for male reproductive research.

[0058] (3) Based on the present invention, the sperm in the vaginal secretions of the Dcaf17- / - golden hamster can be observed under a microscope after mating with the female to determine whether the mating was successful. It can stably and efficiently prepare pseudopregnancy receptors required for various assisted reproduction, and significantly improve the golden hamster assisted reproduction technology system. Attached Figure Description

[0059] Figure 1 This is a schematic diagram of the construction of a golden hamster modified with the Dcaf17 gene, as described in an embodiment of the present invention.

[0060] Figure 2 This is a comparison of the morphology of the testes, body weight, and weight of the testes and epididymis in golden hamsters modified with the Dcaf17 gene, as shown in this embodiment of the invention.

[0061] Figure 3 This is a comparison image of PAS staining in the epididymal tail of a golden hamster modified with the Dcaf17 gene, as described in this embodiment of the invention.

[0062] Figure 4 This is a comparison of H&E staining of epididymal sperm from golden hamsters modified with the Dcaf17 gene, as described in an embodiment of the present invention.

[0063] Figure 5 This is a comparative image showing the implantation of pseudopregnant recipient embryos prepared from golden hamsters with the Dcaf17 gene modified according to an embodiment of the present invention.

[0064] Figure 6 This is a structural diagram of the U6-T7-GFP-trcRNA sequence. Detailed Implementation

[0065] 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.

[0066] Example 1

[0067] This embodiment provides a method for constructing a Dcaf17 gene-modified golden hamster animal model via microinjection. The specific steps of the construction method are as follows:

[0068] (1) Structure of the Dcaf17 gene (Gene ID: 101841040; protein ID: XP_040601479.1, as shown in SEQ ID NO:8) and design of the target site for sgRNA: The target site of the gene to be knocked out in golden hamster was determined, and an sgRNA targeting the golden hamster Dcaf17 gene was designed. The sgRNA was gDcaf17-Sg1 (SEQ ID NO:1: GGCTTCTTCGCACGCGACGCGGG). The designed sgRNA was amplified in vitro to construct a plasmid expressing the sgRNA, and the sgRNA was transcribed in vitro. The primers for expressing the sgRNA plasmid included: gDcaf17-Sg1F (SEQ ID NO:2: TAGGGCTTCTTCGCACGCGACGC) and gDcaf17-Sg1R (SEQ ID NO:3: AAACGCGTCGCGTGCGAAGAAGC).

[0069] The specific process for constructing a plasmid expressing sgRNA is as follows:

[0070] (a) Construction of PUC57-CRISP9-sgRNA-GFP plasmid

[0071] 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. The desired fragment of 2608 bp was used as the backbone of the PUC57-CRISP9 vector.

[0072] 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 6 As shown, the sgRNA was inserted into the PUC57-CRISP9 backbone (2608 bp) to construct PUC57-CRISP9-sgRNA-GFP, and then sequenced to verify it.

[0073] (b) PUC57-CRISP9 vector backbone: The PUC57-CRISP9-sgRNA-GFP plasmid was digested with BsaI restriction endonuclease at 37°C for 2 hours, and purified by gel excision. The required fragment of 2965 bp was used as the PUC57-CRISP9 vector backbone.

[0074] (c) Oligo fragment acquisition: 1 μl of gDcaf17-Sg1F (100 μM), 1 μl of gDcaf17-Sg1R (100 μM), 1 μl of 10X T4 Ligation Buffer (NEB), 1 μl of T4 PNK (NEB), and 6 μl of RNase-free water were added. The mixture was annealed at 37°C for 30 minutes, 95°C for 5 minutes, and at a rate of -6°C / min, then cooled to 25°C. This sample was diluted 100-fold. 1 μl of this solution was added to 1 μl of 50 ng PUC57-CRISP9 vector backbone (2965 bp), 1 μl of 10X T4 Ligation Buffer (NEB), 1 μl of T4 Ligase (NEB), and 6 μl of RNase-free water. The mixture was incubated at room temperature (25±5°C) for 30 minutes. The mixture was then transformed into DH5α E. coli. + Resistance screening was performed, and the plasmid was identified using universal primers M13F / M13R (M13R: CAG GAA ACA GCT ATG ACC; M13F: TGT AAA ACG ACGGCC AGT), followed by sequencing using M13F primers.

[0075] The successfully sequenced plasmids were amplified by M13F / M13R PCR. The amplification product was gel-purified and transcribed into sgRNA using the HiScribe T7 in vitro transcription kit (NEB, E2040S) Protocol. 200 ng of the PCR product was added to a 10 μl reaction mixture along with 1 μl of T7 10X Reaction Buffer, 0.5 μl of T7 Enzyme Mix, and 3 μl of 75 mM dNTPs. The mixture was incubated at 37°C for 2 hours. Then, 0.5 μl of TURBO Dnase was added, the reaction mixture was mixed, and the mixture was incubated at 37°C for 15 minutes.

[0076] Ethanol-based homozygous RNA: Add 57.5 μL of nucleic acid-free water and 7.5 μL of ammonium acetate to the above reaction mixture and mix well. Add 160 μL of 95% ethanol, mix well, and incubate at -20°C on ice for 15 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C and collect the RNA. Redissolve the RNA in 20 μL of RNase-free water, take 1 μL and dilute 10-fold, and determine the RNA concentration using a spectrophotometer. Dilute to 1000 ng / μL with RNase-free water and store the RNA at -80°C.

[0077] (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.

[0078] (3) Recipient preparation: Eight-week-old female golden hamsters were selected and mated with male hamsters in a 1:1 ratio at 6 pm on the fourth day of estrus. At 9 am on the second day, sperm were examined under a microscope in vaginal secretions. The presence of sperm indicated that the recipient was a true pregnancy recipient at 0.5 days. The method for preparing pseudopregnancy recipients in male golden hamsters with Dcaf17 gene modification was the same.

[0079] (4) Embryo Acquisition: Golden hamster fertilized eggs are sensitive to pH, temperature and light. All experiments were performed at room temperature of 28.5℃ under red light. After mating, the female mice were anesthetized by intraperitoneal injection of 1.25% aphthylamine (1.8 ml / 100 g), and the donor female mice were euthanized by cervical dislocation. The oviduct was removed from the abdomen and placed in a culture droplet. Under a stereomicroscope, the ampulla of the oviduct 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 operation was completed within 30 minutes.

[0080] (5) Embryo injection: 20 ng / μl sgRNA and 50 ng / μl cas9 mRNA were injected into the cytoplasm of two-cell embryos through a micromanipulation system. 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%.

[0081] (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.

[0082] (7) The identification primers are any one or a combination of two of the outer identification primer pairs and the inner identification primers;

[0083] For the F0 generation, nested primers were used to ensure accurate identification. First, PCR amplification of F0 generation tail DNA was performed using DCaf17-TOF and DCaf17-TOR primers. One μl of the amplification product was then directly used for a second PCR amplification using DCaf17-TIF and DCaf17-TIR primers. The second amplification product was then bidirectionally sequenced using DCaf17-TIF and DCaf17-TIR primers. For F1 and later generations of hamsters, amplification was performed directly using DCaf17-TIF and DCaf17-TIR primers, followed by sequencing using DCaf17-TIF.

[0084] Dcaf17-TOF:CGTCAGCCTTCCCTTGAGAGTA (SEQ ID NO:4)

[0085] Dcaf17-TOR:CCAGCGTAATGCCTGAACCTT (SEQ ID NO:5)

[0086] Dcaf17-TIF:CCTTGAGAGTAGCTGGTGGC (SEQ ID NO:6)

[0087] Dcaf17-TIR: AACTACGGTCAGGTGAATTACAGAA (SEQ ID NO:7).

[0088] F0 generation positive heterozygous golden hamsters were selected by sequencing and then mated with wild-type golden hamsters to obtain F1 generation golden hamsters. F2 generation golden hamsters were obtained by hybridization among F1 generation heterozygous golden hamsters. The F2 generation golden hamsters were then identified by PCR and sequenced to obtain homozygotes. Sequencing results showed that the Decaf17 deficiency (Dcaf17- / -) involved a 5-nucleotide deletion (del5), leading to translation errors in the Decaf17 protein (e.g., ...). Figure 1 (As shown). The short-chain screening and identification primer pairs include: Decaf17-TOF, Decaf17-TOR, Decaf17-TIF, and Decaf17-TIR.

[0089] (8) Comparative Experiment: The experiment consisted of model construction and unilateral oviduct embryo transfer in pseudopregnant mice. ① Screening results using F0 generation sequencing showed that a 5-nucleotide deletion (mut5) was present in the Dcaf17 gene-modified golden hamster strain, leading to translation errors in the Dcaf17 protein. ② The testicular morphology of 8-week-old WT and Dcaf17- / - golden hamsters was similar. There was no significant difference in body weight and testes between WT and Dcaf17- / - golden hamsters, but the epididymal weight was significantly reduced (n=3 for each genotype, ***P<0.0001) (e.g. Figure 2 As shown). ③ PAS staining showed low sperm concentration in the epididymal tail of Dcaf17- / - golden hamsters, and some giant cells were observed (such as...). Figure 3 (As shown). ④ Unlike normal sperm with sickle-shaped heads, sperm from Dcaf17- / - golden hamsters exhibit severe morphological defects, including amorphous heads and excess cytoplasm, abnormal head shape, and multiple flagellar malformations (sperm flagella are shortened, curled, bent, or absent). Numerous headless sperm were scattered throughout the smear, indicating that the neck region of these sperm is very fragile (e.g., Figure 4 (As shown). ⑤ Pseudopregnant female mice prepared from male golden hamsters modified with the Dcaf17 gene underwent unilateral oviduct embryo transfer. Post-operative laparotomy at 15 days showed unilateral pregnancy on the transfer side, demonstrating the effectiveness of using Dcaf17 gene-modified golden hamsters to prepare pseudopregnant mice (as shown). Figure 5(As shown).

[0090] Example 2:

[0091] Three pseudopregnant female hamsters were prepared by unilateral oviduct embryo transfer, with two pseudopregnant hamsters each receiving 25 fertilized eggs and one receiving 15 fertilized eggs.

[0092] Offspring were observed in recipient female mice on day 17 after embryo transfer. A total of 43 offspring were born, with a birth rate of 66%. This demonstrates the effectiveness of using the Dcaf17 gene to modify golden hamsters to prepare pseudopregnant mice.

[0093] sequence list

[0094] The amino acid sequence of the protein encoded by the Dcaf17 gene (SEQ ID NO:8):

[0095] MGPTPKANVCRRLSRRTLGFFARDAGVVQRTNLGILRELVCQESTKFKNVWTTHSKSPIAYERGRIYFDNYRCCVSSVASEPRKLYEMPKCSKSEKIEDALLWECPVGEILPNPSDYKSSLIALTAHNWL LRISATTGEILEKIYLASYCKFRYLSWDTPQEVIAVKSAQNKGSAMARQAGTQQPVLLYLAVFRVLPFSLVGILEINKKVFANVTDATLSHGILIVMYSSGLVRLYSFQAIIEQFMQQKLDLGCACSQGG TTGTVGEAPFGIPCNVKITDSPSPLFEVSSLENAFQIGGHPWHYIITPNKKKQKGVFHICALKDNSLAKNGIQEMECCSLESDWIYFHPDASGRIIHVGPNQVKVLKLSEVENDSSQHQISEDFVIWANR EDRNENLVTVTASGRVVKRNVNLLDDDPEQETFKIVDYEDELDLLSVVAVTQIDAEGKAHLDFHCNEYGTLLKSIPLVESWDVTYSHEVYFDRDLVLHIEQKPNRVFSCYVYQMVCDPGEEEDIVNRKD*

[0096] Nucleotide sequence of U6-T7-GFP-trcRNA (SEQ ID NO:9):

[0097]

Claims

1. A method for constructing a Dcaf17 gene-modified golden hamster model, characterized in that, The method involves injecting Cas9 functional elements and sgRNA targeting exon 1 of the Dcaf17 gene into two-cell embryos under a red light source, followed by embryo transfer into a recipient to obtain Dcaf17 gene-modified golden hamsters.

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 DCaf17 gene in golden hamster was designed. The nucleotide sequence of the sgRNA is shown in SEQ ID NO:

1.

3. The construction method according to claim 2, characterized in that, The designed sgRNA was amplified in vitro to construct a plasmid expressing the sgRNA, and the sgRNA was transcribed in vitro. The primers for constructing the plasmid expressing the sgRNA included Dcaf17-Sg1F and gDcaf17-Sg1R. The nucleotide sequence of Dcaf17-Sg1F is shown in SEQ ID NO:2, and the nucleotide sequence of Dcaf17-Sg1R is shown in SEQ ID NO:

3.

4. The construction method according to claim 1, characterized in that, The Cas9 mentioned is either Cas9 mRNA or Cas9 protein.

5. The construction method according to any one of claims 1-4, characterized in that, The specific steps of this method are as follows: (1) Under red light, active sgRNA and Cas9 mRNA or Cas9 protein 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 Dcaf17 gene-modified golden hamsters. (2) F0 generation Dcaf17 gene-modified golden hamsters were crossed with normal wild-type golden hamsters to obtain F1 generation Dcaf17 gene-modified golden hamsters. The F1 generation golden hamsters were identified by PCR and sequenced. The F1 generation heterozygous golden hamsters were crossed to obtain F2 generation golden hamsters. The F2 generation golden hamsters were identified by PCR and sequenced. Homozygous males in the F2 generation golden hamsters were selected as Dcaf17 gene-modified golden hamster animal models.

6. The construction method according to claim 5, characterized in that, In step (1), the genomic DNA of F0 generation Dcaf17 gene-modified golden hamsters was identified by PCR.

7. The construction method according to claim 5 or 6, characterized in that, The specific primer pairs for PCR identification of the golden hamster and its offspring include: Decaf17-TOF, Decaf17-TOR, Decaf17-TIF, and Decaf17-TIR; the nucleotide sequence of Decaf17-TOF is shown in SEQ ID NO:4, the nucleotide sequence of Decaf17-TOR is shown in SEQ ID NO:5, the nucleotide sequence of Decaf17-TIF is shown in SEQ ID NO:6, and the nucleotide sequence of Decaf17-TIR is shown in SEQ ID NO:

7.

8. The application of the Dcaf17 gene-modified golden hamster model constructed by any of the methods described in claims 1-7 in studying the related functions and mechanisms of action of the Dcaf17 gene.

9. The use of male rats in the Dcaf17 gene-modified golden hamster model constructed by any of the methods described in claims 1-7 in the preparation of assisted reproductive-related pseudopregnancy recipient rats.

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