A mouse model specifically degrading p300 protein and a construction method thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a mouse model for specifically degrading p300 protein and a construction method thereof, steps of which are as follows: 5' homologous arms, 3xGGGGS, FKBP1A a gene synonymous mutation sequence, P2A, a green fluorescent protein EGFP sequence and 3' homologous arms are sequentially arranged on a Donor vector skeleton to obtain a Donor recombinant vector; gRNA is designed based on Ep300 the 31st exon of a gene; the gRNA, Cas9 protein and the Donor recombinant vector are mixed and co-injected into a mouse fertilized egg to make the FKBP1A gene synonymous mutation sequence and the green fluorescent protein EGFP sequence be knocked into a mouse genome; after a transplantable embryo is obtained by in-vitro culture, the embryo is transplanted into a surrogate mouse uterus to continue development until a young mouse is born; F0 generation mice are crossed with wild-type mice to obtain mice expressing a p300-FKPB-P2A-EGFP fusion protein, that is, a mouse model for specifically degrading p300 protein is obtained. The mouse model is administered with dTAG in a later stage, so that rapid and reversible specific degradation and removal of p300 protein in the mouse in vivo are achieved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a mouse model that specifically degrades p300 protein and its construction method. Background Technology

[0002] Proteins are the direct executors of various biological processes within cells, and the dynamic regulation of their function is crucial for understanding cellular physiological and pathological mechanisms. However, traditional molecular regulatory techniques mainly focus on the gene level, indirectly affecting protein expression through gene knockout or RNA interference. This approach has limitations, such as the irreversibility of gene knockout, which prevents dynamic regulation of protein function; and while RNA interference can reduce protein expression levels to some extent, its effect is relatively slow and it has off-target effects, potentially affecting the expression of other non-target genes. Therefore, research into techniques that can directly target and regulate protein levels is particularly important.

[0003] p300 is a multifunctional transcriptional coactivator belonging to the KAT3 histone acetyltransferase family, initially identified in eukaryotic gene expression regulation studies. Through histone acetyltransferase activity and a protein-protein interaction network mediated by its domains, p300 participates extensively in core biological processes such as chromatin remodeling, transcriptional activation, DNA damage repair, and cell fate determination. In the field of cancer, aberrant activation of p300 is closely associated with various malignant tumors, including prostate cancer, breast cancer, and leukemia. For example, it drives the androgen receptor AR signaling pathway, promoting prostate cancer progression; or it maintains tumor cell survival by activating oncogenic transcriptional programs such as c-Myc. Furthermore, p300 can mediate the overactivation of inflammatory factors such as NF-κB in inflammatory diseases and exacerbate neurofibrillary tangles caused by Tau protein acetylation in neurodegenerative diseases, closely related to the development of Alzheimer's disease. In the mouse genome... Ep300 Located on chromosome 15, it is approximately 87kb in length. The start codon ATG is located in exon 1, and the stop codon TGA is located in exon 31.

[0004] Despite the growing importance of p300 as a therapeutic target, current intervention methods still face significant challenges. Existing small molecule inhibitors, such as C646 and CCS1477, primarily exert their effects by targeting the bromodomain (BRD) or histone acetyltransferase (HAT) domain of p300. However, these domains are highly conserved in the cyclic adenosine monophosphate (cAMP) response element-binding protein (CBP), making it difficult for inhibitors to distinguish between the two and preventing them from degrading the protein itself. PROTAC technology recruits both the target protein and E3 ligases simultaneously using bifunctional molecules, but its design depends on the selectivity of the target protein ligand. Existing PROTAC degraders targeting p300, such as XYD129, XYD190, XYD198, and QC-182, all exhibit cross-degradation of CBP. Gene editing technologies, such as CRISPR-Cas9 knockout of p300, are prone to causing embryonic lethality or severe developmental defects, while RNA interference technology is limited by its transient nature and delivery efficiency.

[0005] To address the aforementioned limitations, there is an urgent need to develop new technologies that can dynamically and reversibly regulate the level of p300 protein specifically. Summary of the Invention

[0006] To achieve rapid, reversible, and specific degradation and clearance of p300 protein in vivo, this invention provides a method for constructing a mouse model that specifically degrades p300 protein.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a method for constructing a mouse model that specifically degrades p300 protein, comprising the following steps:

[0009] A 5' homologous arm and 3×GGGGS were sequentially attached to the Donor vector framework. FKBP1A The gene synonymous mutant sequence, P2A, green fluorescent protein EGFP sequence, and 3' homologous arm were used to obtain the Donor recombinant vector;

[0010] The nucleotide sequence of the 5' homologous arm is shown in SEQ.ID.NO.2; the nucleotide sequence of the 3×GGGGS is shown in SEQ.ID.NO.3; FKBP1A The gene synonymous mutation sequence is shown in SEQ.ID.NO.4; the nucleotide sequence of P2A is shown in SEQ.ID.NO.5; the nucleotide sequence of the green fluorescent protein EGFP sequence is shown in SEQ.ID.NO.6; and the nucleotide sequence of the 3' homologous arm is shown in SEQ.ID.NO.7.

[0011] Based on mouse chromosome 15 Ep300 A gRNA was designed from exon 31 of the gene, and the nucleotide sequence of the gRNA is shown in SEQ.ID.NO.1;

[0012] The gRNA, Cas9 protein, and Donor recombinant vector were mixed and co-injected into mouse zygotes to induce the... FKBP1A Synonymous mutant sequences and green fluorescent protein (EGFP) sequences were knocked into the mouse genome;

[0013] The fertilized eggs after injection were cultured in vitro and then transplanted into surrogate mice to continue developing until the pups were born, thus obtaining F0 generation mice.

[0014] The F0 generation mice are crossed with wild-type mice, or the offspring obtained from the cross are bred to obtain mice that specifically degrade p300 protein.

[0015] Preferably, the Donor recombinant vector is constructed according to the following steps:

[0016] Using BAC plasmid as a template, a fragment containing the 5' homologous arm from the mouse genome was amplified to obtain fragment-1, the sequence of which is shown in SEQ.ID.NO.2;

[0017] Using BAC plasmid as a template, a fragment containing the 3' homologous arm from the mouse genome was amplified to obtain fragment-3, the sequence of which is shown in SEQ.ID.NO.7;

[0018] The 3×GGGGS, FKBP1A The gene synonymous mutation sequence, P2A, and green fluorescent protein (EGFP) sequence were sequentially linked to obtain fragment-2;

[0019] By linking fragment-1, fragment-2, fragment-3 and the Donor vector backbone, the Donor recombinant vector is obtained, and the nucleotide sequence of the Donor recombinant vector is shown in SEQ.ID.NO.12.

[0020] Preferably, the primer sequences for amplifying fragment-1 are shown in SEQ.ID.NO.8 and SEQ.ID.NO.9;

[0021] The primer sequences for amplifying fragment-2 are shown in SEQ.ID.NO.10 and SEQ.ID.NO.11.

[0022] Preferably, when linking fragment-1, fragment-2, fragment-3 and the Donor carrier backbone, the 20 μL linking system contains:

[0023] 88.6 ng Donor vector backbone, 39.2 ng fragment-1, 23.1 ng fragment-2, 23.9 ng fragment-3, 10 μL NEBuilder HiFi DNA Assembly Master Mix.

[0024] Preferably, after the gRNA and the Cas9 protein are mixed and incubated at 25°C, the Donor recombinant vector is added, mixed well, and then co-injected into mouse zygotes.

[0025] Preferably, the concentration of the gRNA is 90 pmol / μL to 110 pmol / μL, the concentration of the Cas9 protein is 18 µM to 22 µM, and the concentration of the Donor recombinant vector is 12 ng / μL to 18 ng / μL.

[0026] The volume ratio of the gRNA to the Cas9 protein is 3-5:1, and the total volume of the mixture of gRNA and Cas9 protein with the Donor recombinant vector is 20 μL.

[0027] Preferably, the concentration of the gRNA is 100 pmol / μL, the concentration of the Cas9 protein is 20 µM, and the concentration of the Donor recombinant vector is 15 ng / μL.

[0028] The volume ratio of the gRNA to the Cas9 protein is 4:1.

[0029] The present invention also provides a mouse model for rapid degradation of p300 protein, which is obtained by the construction method described above.

[0030] Preferably, dTAG is administered to mice that specifically degrade p300 protein to achieve rapid and reversible degradation and clearance of p300 protein in mice.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] This invention provides a method for constructing a mouse model that specifically degrades p300 protein. This invention is based on CRISPR / Cas9-mediated gene editing technology in mice. Ep300 The human FKBP1A protein coding sequence, P2A, and green fluorescent protein (EGFP) sequence were knocked into the gene, labeled as the FKBP-P2A-EGFP tag sequence, and introduced... FKBP1ASynonymous mutations were used to avoid interference from endogenous cleavage sites and off-target effects, thereby establishing a mouse model for rapid and specific degradation of the p300 protein, namely the mouse p300-FKPB-P2A-EGFP knock-in model. EGFP fluorescent protein acts as an indicator, allowing for the determination of successful target sequence knock-in by observing fluorescence. Furthermore, the addition of a self-cleaving peptide P2A between the FKBP and EGFP sequences has minimal impact on protein function, ensuring that the target protein retains its native function after cleavage. Therefore, EGFP does not interfere with the structure of the p300-FKBP fusion protein, ensuring smooth binding of the dTAG ligand to FKBP. This mouse p300-FKPB-P2A-EGFP knock-in model can stably and efficiently express the p300 protein fused with the FKPB tag protein, referred to as the p300-FKPB fusion protein. In addition, the mouse p300-FKPB-P2A-EGFP knock-in model of this invention has the advantage of a short operation cycle.

[0033] This invention, through Ep300, FKBP1A Adding a 3×GGGGS sequence as a fusion protein linker between synonymous mutant sequences promotes the expression of the p300-FKPB fusion protein.

[0034] This invention achieves rapid, reversible, and specific degradation and clearance of p300 protein in mice by administering dTAG to the resulting knock-in mice in the later stage.

[0035] This invention addresses the F0 generation positive mice constructed using CRISPR / Cas9 technology. Due to cleavage efficiency and possible non-homologous repair, different genetic lineages may emerge. By mating F0 generation positive mice with wild-type mice, stable genotype F1 generation positive mice can be obtained. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of a transgenic mouse model expressing the FKPB tag constructed using CRISPR / Cas9 technology in an embodiment of the present invention.

[0037] Figure 2 This is a plasmid map of the Donor vector used for homologous recombination repair.

[0038] Figure 3 This is the breeding program for F0 generation mice.

[0039] Figure 4 A diagram illustrating the PCR identification strategy for F0 and F1 generation mice.

[0040] Figure 5 Electrophoresis images for PCR identification of F1 generation mice: A: 5'HA homologous recombination positive genome; B: 3'HA homologous recombination positive genome.

[0041] Figure 6 This is a sequencing image of the knock-in sequence in F1 generation mice.

[0042] Figure 7 Electrophoresis images for PCR identification of F2 generation mice: A: Identification results of the 506bp band; B: Identification results of the 1611bp and 459bp bands.

[0043] Figure 8 Western blot diagram to identify the expressed p300-FKBPP fusion protein. Detailed Implementation

[0044] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0045] The inventive concept of this invention is as follows:

[0046] The dTAG protein degradation system offers the possibility of targeting any protein within the cell in a rapid, inducible, and reversible manner.

[0047] The dTAG system is a targeted protein degradation technology based on genetically engineered tags and bifunctional small molecules. It utilizes CRISPR-Cas9 or lentiviral technology to fuse the target protein with an artificially modified FKBP tag, constructing a chimeric protein. The heterobifunctional small molecule degrader dTAG binds to the FKBP tag at one end and recruits E3 ubiquitin ligases such as CRBN or VHL at the other end, forming a ternary complex that induces ubiquitination and proteasome degradation of the target protein. dTAG regulation of target protein abundance exhibits rapid, reversible, and adjustable characteristics, providing a rapid dose-dependent effect on total cellular protein abundance with precise time control. Moreover, this effect is reversible after elution of the degrader. This avoids the problems of limited dynamic control, indeterminate effect sizes, and often complete irreversibility associated with methods such as RNAi or CRISPR-Cas9.

[0048] A specific sgRNA was designed targeting the C-terminus of the target protein gene, and a donor plasmid containing the FKBP tag sequence was constructed. This plasmid includes homologous arms to promote homologous recombination. The sgRNA and Cas9 protein were co-transfected with the donor plasmid into mouse embryonic stem cells (mESCs), allowing the FKBP tag to be inserted into the C-terminus of the target gene via homologous recombination. The edited embryonic stem cells were then injected into mouse embryos to generate chimeric mice, which were subsequently bred to obtain transgenic mice carrying the FKBP tag. The advantage of this method is that it enables genome-level specific modification, ensuring that the fusion of the FKBP tag with the target protein occurs within the cell's native gene expression framework, thus more closely resembling protein expression and function under physiological conditions.

[0049] The dTAG system can rapidly, tunably, and reversibly degrade target proteins and has been widely used in various cell lines and mouse models to study target protein function. This invention successfully constructed p300-FKBP-P2A-EGFP knock-in mice, which is crucial for using the dTAG system to study the specific function of p300 protein, discover and validate new targets, and achieve high spatiotemporal resolution specific target protein degradation in in vivo models. This strategy not only avoids dependence on the inherent ligands of the target but also allows for dose-modulation of protein function through the degrading agent, providing a potential breakthrough for elucidating the specific transient effects of p300 in diseases and developing precision therapies. Compared with drug inhibitors and degrading agents, dTAG small molecule degrading agents have significant advantages, including simpler manufacturing processes, more convenient clinical applications, overcoming resistance to inhibitors, the ability to adjust the degree of protein knockdown by changing the dosage, faster onset of action, and specific degradation of p300 protein alone.

[0050] In summary, constructing p300-FKPB fusion protein expression mice is crucial for the rapid, reversible, and specific degradation and clearance of p300 protein in mice.

[0051] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0052] Example 1

[0053] A mouse model that specifically degrades p300 protein and its construction method are as follows:

[0054] This invention utilizes CRISPR / Cas9-mediated gene editing technology to establish a C57BL / 6J mouse p300-FKPB-P2A-EGFP knock-in model.

[0055] 1. Targeting human genes FKBP1ADesign a mouse transgenic strategy, using CRISPR / Cas9 technology to... FKBP1A The CDS sequence was knocked into mouse chromosome 15. Ep300 In exon 31 of the gene, Ep300 The gene is the gene encoding the p300 protein. For example... Figure 1 As shown, Ep300 The reference sequence of the gene in NCBI is: NM_177821.6. The specific steps include:

[0056] 1.1 Design of gRNA based on the CRISPR / Cas9 system Ep300 The sequence of the specific gRNA action site of exon 31 of the gene is shown in Table 1.

[0057] Table 1 gRNA action sites

[0058]

[0059] Note: In Table 1, the italicized bases in the sequence are PAM.

[0060] 1.2. Based on the principle of homologous recombination repair and combined with the knock-in target sequence, a Donor vector was designed. This Donor vector contains a 5' homologous arm, 3×GGGGS, FKBP1A The gene synonymous mutation sequence, P2A, green fluorescent protein (EGFP) sequence, and 3' homologous arm are shown in Table 2, and their main components are shown in Table 3. The target sequence can also be referred to as the knock-in sequence. FKBP1A Gene synonymous mutation sequence marker: Mutant Human FKBP1A CDS-p.F37V.

[0061] 5' homologous arm, 3×GGGGS, FKBP1A The sequence information of the gene synonymous mutation sequence, P2A, green fluorescent protein EGFP sequence and 3' homologous arm is shown in SEQ.ID.NO.2~SEQ.ID.NO.7 respectively.

[0062] Table 2 Main Components of the Donor Carrier

[0063]

[0064] Table 3. Partial nucleotide sequences of Donor vectors

[0065]

[0066] Note: In Table 3, the underlined bases are introduced synonymous mutations. The sequence before the mutation is TTT, and the sequence after the mutation is GTT.

[0067] 1.3. Using In-Fusion technology, the above 5' homologous arms, 3×GGGGS, and Mutant Human were integrated. FKBP1A CDS-p.F37V, P2A, EGFP, and the 3' homologous arm were assembled into the Donor vector, which consists of a 5' homologous arm and a fusion protein linker 3×GGGGS sequentially inserted into the Donor vector backbone. FKBP1A Gene synonymous mutation sequence, P2A, green fluorescent protein (EGFP) sequence, and 3' homologous arm. The specific process is as follows:

[0068] (1) Using high-fidelity Taq DNA polymerase and BAC RP23-16K17 plasmid as template, fragment-1 and fragment-3 containing homologous sequences from the mouse genome were amplified, respectively. The specific sequences of the amplification primers are shown in Table 4, the amplification system is shown in Table 5, and the amplification program is shown in Table 6.

[0069] Fragment-1: Fragment containing a 5' homologous arm, i.e., SEQ.ID.NO.2; Fragment-3: Fragment containing a 3' homologous arm, i.e., SEQ.ID.NO.7.

[0070] A 3×GGGGS-Mutant Human FKBP1A CDS-P2A-EGFP sequence was synthesized and denoted as fragment-2, wherein FKBP1A CDS does not contain the ATG start codon.

[0071] Table 4 Nucleotide sequences of amplification primers

[0072]

[0073] Table 5 shows the PCR systems used for amplifying fragments 1 through 3.

[0074]

[0075] Table 6 shows the PCR procedures for amplifying fragments 1 through 3.

[0076]

[0077] (2) Using a seamless cloning kit, the PCR amplified fragment and the linearized plasmid were ligated according to the reaction system shown in Table 7. The reaction was performed using a PCR instrument and incubated at 50°C for 60 min; then transformed on ice.

[0078] The linearized plasmid is 4430bp, custom-made by Cyagen Biosciences, internal catalog number Cat: VB139; specifically, it is a linearized plasmid obtained by modifying the commercial plasmid addgene#50005, which is used to provide the vector backbone.

[0079] Table 7 In-Fusion System

[0080]

[0081] (3) The In-Fusion ligation product was transformed into DH5-alpha competent cells. The competent cells were then added to LB liquid medium and cultured in a shaker at 37°C for 30 min. The resulting culture was spread onto LB solid medium containing ampicillin and cultured overnight. After ampicillin resistance screening, single clones growing on LB solid medium were picked and cultured in LB liquid medium. Then, bacterial PCR identification was performed. After confirming successful ligation, the bacterial culture was sent for sequencing. Based on the sequencing results, single clones with successful ligation and correct sequences were selected for shaking culture. The bacterial culture was then collected for plasmid extraction to obtain the Donor recombinant vector. Its plasmid map is shown below. Figure 2 As shown.

[0082] The nucleotide sequences of the Donor recombinant vector are shown in SEQ.ID.NO.12 and SEQ.ID.NO.13, with 1bp~4749bp in sequence 12 of the nucleotide or amino acid sequence listing computer-readable vector and 4750bp~8731bp in sequence 13.

[0083] 1.4 Preparation of the injection complex.

[0084] Solution in tube 1: Add 0.8 μL of 100 pmol / μL gRNA to RNase-free water, then add 0.2 μL of 20 µM Cas9 protein and mix well. Incubate in a metal bath at 25 °C for 10 min to obtain solution in tube 1. The gRNA is the sequence shown in SEQ ID NO. 1.

[0085] Tube 2 solution: Donor recombinant vector with a final concentration of 15 ng / μL.

[0086] The solutions in tube 1 and tube 2 were mixed to obtain the injection complex, and the total volume of the injection complex was 20 μL.

[0087] 1.5 Microinjection and embryo transfer.

[0088] Following superovulation in female C57BL / 6J mice, zygotes were obtained via in vitro fertilization. The resulting injection complex was then injected into the zygotes using a 0.1 μm glass microinjection needle. The microinjected zygotes were then transferred to the oviducts of surrogate C57BL / 6J mice via embryo transfer. F0 generation mice were obtained after birth, and their genotypes were identified using PCR.

[0089] The specific procedures for embryo transfer are as follows: After microinjection, the fertilized eggs are cultured in vitro to the morula stage and quality is tested; a small opening is made in the fallopian tube capsule of the surrogate mother mouse, and after the direction of the fallopian tube fimbriae is clearly seen, the transfer tube is inserted into the fallopian tube fimbriae 3mm from the opening, and a well-developed embryo is blown into the enlarged part. After a short pause, the transfer tube is pulled out.

[0090] 1.6. Positive F0 generation mice were mated with Wildtype C57BL / 6J mice to obtain F1 generation mice. Genotyping was performed using PCR. If any positive mice were born (i.e., F1 generation heterozygotes), it indicates that the knock-in sequence had been integrated into the germ cells. Figure 3 and Figure 4 As shown.

[0091] 1.7. Self-cross the F1 generation heterozygous mice and perform genotyping on the resulting F2 generation mice to obtain p300-FKPB-P2A-EGFP homozygous knock-in mice, which are mice expressing the p300-FKBP fusion protein.

[0092] 1.8 By administering dTAG to the resulting knock-in mice in the later stage, rapid and reversible specific degradation and clearance of p300 protein in the mice were achieved.

[0093] 2. PCR identification results of mouse genotypes.

[0094] 2.1 DNA extraction.

[0095] Newborn mice aged 3 weeks were selected, and their tails, approximately 0.3 cm in length, were cut off and placed in a 1.5 mL EP tube containing 50 μL of lysis buffer and 1 μL of proteinase K. The tube was centrifuged until the tail was at the bottom, and incubated overnight at 55°C. The next day, the metal bath temperature was set to 95°C for 5 min to inactivate proteinase K. The tail was then vortexed for 15 s to ensure complete lysis, followed by centrifugation at 12000 rpm for 5 min. The supernatant can be used directly as a template for genotyping or stored at -20°C for later use.

[0096] 2.2 PCR identification results of F1 generation mice genotypes.

[0097] DNA was extracted from the tail tip of mice and its genotype was identified by PCR amplification.

[0098] according to Figure 4The PCR identification strategy used in this study employed primers F2 and R2 for amplification. A 3.4 kb fragment should be amplified from 5'HA homologous recombination-positive genomes, while no band should be observed in negative genomes. For amplification using primers F1 and R1, a 2.5 kb fragment should be amplified from 3'HA homologous recombination-positive genomes, while no band should be observed in negative genomes. The amplification system is shown in Table 8, and the amplification procedure is shown in Table 9. Specific primers used for PCR identification are shown in Table 10.

[0099] Table 8. PCR system for identifying positive genomes of homologous recombination with 5'HA and 3'HA.

[0100]

[0101] Table 9. PCR procedure for identifying positive genomes of homologous recombination with 5'HA and 3'HA.

[0102]

[0103] Table 10 Primers for PCR amplification of positive genomes for homologous recombination of 5'HA and 3'HA

[0104]

[0105] The results of the identification are as follows Figure 5 As shown, mice 17, 20, 21, 22, and 23 are F1 generation mice that are positive for homologous recombination in both arms.

[0106] Note: If the DNA sample is not very pure or does not have sufficient PCR extension time, long-fragment PCR products may not be amplified. You can use... Figure 4 Other PCR identification strategies used in this study employed F3 and R4 primers for amplification, aiming to amplify a 506 bp fragment of the target allele. The F3 and R4 primer sequences are shown in Table 12.

[0107] 2.3 Sequencing results of the knock-in sequence in F1 generation mice.

[0108] PCR amplification was performed using primers F3 and R3, and the product was then sequenced.

[0109] Sequencing results as follows Figure 6 As shown in Table 11, the knock-in sites in positive F1 generation 17 mice were correct, and the knock-in sequence was correct and without mutation. The specific primers used for PCR amplification are shown in Table 11.

[0110] Table 11 Primers for PCR amplification in F1 generation mice

[0111]

[0112] 2.4. PCR identification results of F2 generation mice genotypes.

[0113] DNA was extracted from the tail tip of mice and its genotype was identified by PCR amplification. Figure 4 The PCR identification strategy used primers F4 and R5, and F3 and R4 for PCR. The F2 generation mice were classified into three genotypes based on PCR fragment length: homozygous (Homozygous), heterozygous (Heterozygous), and wild-type (Wildtype). Different genotypes corresponded to different PCR fragment lengths: homozygotes had two bands (1611 bp and 506 bp); heterozygotes had three bands (1611 bp, 459 bp, and 506 bp); and wild-type mice had one band (459 bp). Specific primers used for PCR identification are shown in Table 12. Homozygotes are abbreviated as Ho, heterozygotes as He, and wild-type as Wt.

[0114] See results Figure 7 P34~P36, P38 and P39 are homozygous; P30~P33 and P37 are heterozygous; P40 is Wt.

[0115] Table 12 Primers for PCR amplification of F2 generation mouse genotype identification

[0116]

[0117] 3. Evaluation of the target strategy for instantaneous p300 knockout.

[0118] The expression of p300 protein in F2 generation mouse tissues was detected by Western blot. This invention uses muscle tissue as an example.

[0119] The results of the identification are as follows Figure 8 As shown, after F2 generation heterozygous mice were administered dTAG, the p300-FKPB fusion protein was degraded, and the target band was larger than that of p300 protein in WT mice, while p300 protein was normally expressed in the muscle tissue of mice that were not administered dTAG.

[0120] The above results demonstrate that this invention utilizes In-Fusion technology to construct the Donor vector. By co-injecting the Donor vector, gRNA, and Cas9 mRNA into mouse zygotes, it can transmit human-derived DNA to mice. FKBP1A The gene sequence was knocked into the mouse genome using CRISPR / Cas9-mediated gene editing technology and used as a marker for interaction with the mouse genome. Ep300By expressing the FKPB tag sequence through gene fusion, and then breeding the resulting positive F0 generation mice through embryo transfer, a parental model capable of stably and efficiently expressing the p300-FKPB fusion protein was finally obtained. This enabled the use of the dTAG protein rapid degradation system model mouse to achieve rapid, complete, and reversible p300 protein-specific degradation induced by small molecule degrading agents, providing a new approach and method to solve the problem that traditional gene knockout animal models cannot explore the direct regulatory mechanism of proteins of interest.

[0121] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0122] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model that specifically degrades p300 protein, characterized in that, Includes the following steps: A 5' homologous arm and 3×GGGGS were sequentially attached to the Donor vector framework. FKBP1A The gene synonymous mutant sequence, P2A, green fluorescent protein EGFP sequence, and 3' homologous arm were used to obtain the Donor recombinant vector; The nucleotide sequence of the 5' homologous arm is shown in SEQ.ID.NO.2; the nucleotide sequence of the 3×GGGGS is shown in SEQ.ID.NO.3; FKBP1A The gene synonymous mutation sequence is shown in SEQ.ID.NO.4; the nucleotide sequence of P2A is shown in SEQ.ID.NO.5; the nucleotide sequence of the green fluorescent protein EGFP sequence is shown in SEQ.ID.NO.6; and the nucleotide sequence of the 3' homologous arm is shown in SEQ.ID.NO.

7. Based on mouse chromosome 15 Ep300 A gRNA was designed from exon 31 of the gene, and the nucleotide sequence of the gRNA is shown in SEQ.ID.NO.1; The gRNA, Cas9 protein, and Donor recombinant vector were mixed and co-injected into mouse zygotes to induce the... FKBP1A Synonymous mutant sequences and green fluorescent protein (EGFP) sequences were knocked into the mouse genome; The fertilized eggs after injection were cultured in vitro and then transplanted into surrogate mice to continue developing until the pups were born, thus obtaining F0 generation mice. The F0 generation mice are crossed with wild-type mice, or the offspring obtained from the cross are bred to obtain mice that specifically degrade p300 protein.

2. The construction method according to claim 1, characterized in that, The Donor recombinant vector was constructed according to the following steps: Using BAC plasmid as a template, a fragment containing the 5' homologous arm from the mouse genome was amplified to obtain fragment-1, the sequence of which is shown in SEQ.ID.NO.2; Using BAC plasmid as a template, a fragment containing the 3' homologous arm from the mouse genome was amplified to obtain fragment-3, the sequence of which is shown in SEQ.ID.NO.7; The 3×GGGGS, FKBP1A The gene synonymous mutation sequence, P2A, and green fluorescent protein (EGFP) sequence were sequentially linked to obtain fragment-2; By linking fragment-1, fragment-2, fragment-3 and the Donor vector backbone, the Donor recombinant vector is obtained. The full-length nucleotide sequence of the Donor recombinant vector is 8731 bp, of which 1 bp to 4749 bp corresponds to SEQ.ID.NO.12 and 4750 bp to 8731 bp corresponds to SEQ.ID.NO.

13.

3. The construction method according to claim 2, characterized in that, The primer sequences for amplifying fragment-1 are shown in SEQ.ID.NO.8 and SEQ.ID.NO.9; The primer sequences for amplifying fragment-2 are shown in SEQ.ID.NO.10 and SEQ.ID.NO.

11.

4. The construction method according to claim 2, characterized in that, When linking fragment-1, fragment-2, fragment-3, and the Donor vector backbone, the 20 μL linking system contains: 88.6 ng Donor vector backbone, 39.2 ng fragment-1, 23.1 ng fragment-2, 23.9 ng fragment-3, 10 μL NEBuilder HiFi DNA Assembly Master Mix.

5. The construction method according to claim 1, characterized in that, After incubating the gRNA and Cas9 protein at 25°C, the Donor recombinant vector was added, mixed well, and then injected into mouse zygotes.

6. The construction method according to claim 5, characterized in that, The concentration of the gRNA is 90 pmol / μL to 110 pmol / μL, the concentration of the Cas9 protein is 18 µM to 22 µM, and the concentration of the Donor recombinant vector is 12 ng / μL to 18 ng / μL. The volume ratio of the gRNA to the Cas9 protein is 3-5:1, and the total volume of the mixture of gRNA and Cas9 protein with the Donor recombinant vector is 20 μL.

7. The construction method according to claim 5, characterized in that, The concentration of the gRNA is 100 pmol / μL, the concentration of the Cas9 protein is 20 µM, and the concentration of the Donor recombinant vector is 15 ng / μL. The volume ratio of the gRNA to the Cas9 protein is 4:1.

Images