Construction method of mouse model capable of rapidly degrading MSTN protein

Abstract

The invention belongs to the technical field of gene engineering, and relates to a construction method of a mouse model capable of rapidly degrading MSTN protein, which comprises the following steps: sequentially inoculating a 5'homologous arm, 3xEAAAK, an FKBP1A gene synonymous mutation sequence, 3xEAAAK, a red fluorescent protein mCherry sequence and a 3 'homologous arm on a Donor vector to obtain a Donor recombinant vector; designing gRNA based on a third exon of the Mstn gene, mixing the gRNA, Cas9 protein and a Donor recombinant vector, injecting the mixture into a mouse fertilized egg, and performing in-vitro culture and transplantation to obtain an F0-generation mouse; and hybridizing the F0-generation mouse with a wild type, or continuously breeding the filial generation to obtain the knock-in mouse model. According to the invention, a dTAG targeted protein degradation technology is utilized, the obtained homozygous knock-in mouse is subjected to dTAG administration, and rapid and reversible degradation and removal of MSTN protein can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and relates to establishing a mouse MSTN-FKPB-mCherry knock-in model based on CRISPR / Cas9-mediated gene editing technology, and specifically relates to a method for constructing a transgenic mouse with a human FKBP1A gene sequence knocked in. Background Art

[0002] Over the past few decades, molecular regulation technologies have continued to advance and have become an important means to study gene function in different biological contexts. In general, these technologies can be classified according to their targets: DNA, RNA or protein. However, since proteins play key roles in many biological processes, interventions targeting only DNA and RNA have some shortcomings. These factors may affect experimental design and hinder the accurate understanding of protein function in situations where rapid and reversible interventions are required. Therefore, technologies that can directly target and regulate protein levels have become particularly attractive, which may provide faster time resolution than DNA / RNA targeting technologies. The latest degradation tag (dTAG) protein degradation system provides the possibility of targeting any protein in the cell in a rapid, inducible and reversible manner.

[0003] The dTAG system is an innovative target confirmation method that uses special bifunctional small molecules to manipulate the protein degradation mechanism in cells to achieve degradation of specific proteins. This system was developed by Dr. Behnam Nabet and his team at the Dana Farber Cancer Institute. It not only has broad application prospects, but is also one of the effective strategies for achieving targeted protein degradation (TPD). The dTAG system utilizes the endogenous proteasome degradation pathway of the cell to induce the degradation of the target protein by fusing the target protein with the FKBP tag and then using a specific small molecule degrader dTAG. The prior art proposes two methods for constructing targeted protein degradation: the CRISPR-Cas9 system achieves genome-specific FKBP tag knock-in or the lentiviral system exogenously expresses the FKBP tag fused to the target target, and the target protein is expressed as a chimera with a FKBP tag mutant. After the addition of the dTAG ligand, a ternary complex can be formed between the target protein fused with the FKBP tag and the E3 ligase, triggering the polyubiquitination of the target protein, thereby causing the target protein to be degraded by the proteasome. Rapid and selective processing of labeled proteins is achieved. To verify the potential of the dTAG system to regulate protein levels in living mice, the existing technology injected the human leukemia cell line MV4-11 carrying a luciferase (luc-FKBP12F36V) reporter gene fused to the FKBP tag into the bone marrow of mice. After treatment with dTAG molecules, the activity of the reporter gene in the mice was observed to decrease significantly within 4 hours and rebound within 28 hours. This finding confirms that the dTAG system can achieve rapid and reversible evaluation of the function of target proteins in living mice.

[0004] Myostatin (MSTN) is a secreted signal transduction molecule that was originally discovered during the screening of new members of the TGF-β superfamily. MSTN is mainly expressed in skeletal muscle and is a negative regulator of muscle growth and development. Multiple species (including cattle, sheep, dogs, rabbits, rats, pigs, goats, humans, and mice) have been shown to cause muscle hypertrophy, increased number of muscle fibers, and increased muscle fiber size without causing serious adverse consequences. In addition, in animal models of cancer malignancy, inhibition of MSTN signal transduction or Mstn gene mutations has been shown to offset the loss of muscle mass and strength and prolong survival without affecting tumor growth. Therefore, extensive efforts have been made to develop effective strategies to block MSTN expression in order to increase muscle mass in animals. A lot of effort has been invested in developing drugs that can modulate MSTN signal transduction for clinical applications. However, MSTN inhibitors tested in clinical trials to date are limited to protein drugs, such as neutralizing antibodies, peptidomimetics, monomers, or decoy receptors. The Mstn gene in the mouse genome is located on chromosome 1, with a total length of 6.44 kb and 3 exons. The start codon ATG is located in the first exon, and the stop codon TGA is located in the third exon.

[0005] The dTAG system is fast, adjustable and reversible in the degradation of target proteins, and has been widely used in various cell lines and mouse models to study the function of target proteins. The successful construction of MSTN-FKBP-mCherry knock-in mice is crucial for using the dTAG system to study the function of MSTN proteins and discover and verify new targets. Compared with drug inhibitors, dTAG small molecule degraders have obvious advantages, including simpler production processes, more convenient clinical applications, and overcoming drug resistance to inhibitors; the degree of protein knockdown can be adjusted by changing the dosage; and the effect can be achieved more quickly.

[0006] In summary, constructing MSTN-FKPB-mCherry fusion protein-expressing mice is particularly critical for the rapid and reversible degradation and clearance of MSTN protein in mice. Summary of the invention

[0007] The purpose of the present invention is to provide a method for constructing a mouse model that rapidly degrades MSTN protein. The method utilizes CRISPR / Cas9 technology and microinjection technology to obtain a mouse model that can stably and efficiently express MSTN-FKPB-mCherry fusion protein.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a method for constructing a mouse model for rapidly degrading MSTN protein, comprising the following steps:

[0010] The Donor vector backbone is connected with a 5' homology arm, 3xEAAAK, a synonymous mutation sequence of the FKBP1A gene, 3xEAAAK, a red fluorescent protein mCherry sequence and a 3' homology arm arranged in sequence to obtain a Donor recombinant vector; the nucleotide sequence of the synonymous mutation sequence of the FKBP1A gene is shown in SEQ ID NO.4, the nucleotide sequence of 3xEAAAK is shown in SEQ ID NO.3, the nucleotide sequence of the red fluorescent protein mCherry is shown in SEQ ID NO.5, the nucleotide sequence of the 5' homology arm is shown in SEQ ID NO.2, and the nucleotide sequence of the 3' homology arm is shown in SEQ ID NO.6;

[0011] gRNA is designed based on the third exon of the Mstn gene on mouse chromosome 1, and the nucleotide sequence of the gRNA is shown in SEQ.ID.NO.1;

[0012] The gRNA, Cas9 protein and the Donor recombinant vector are mixed and co-injected into mouse fertilized eggs to knock the FKBP1A gene synonymous mutation sequence and the red fluorescent protein mCherry sequence into the mouse genome;

[0013] The injected fertilized eggs are cultured in vitro and then transplanted into surrogate mice to continue to develop until pups are born, thus obtaining F0 generation mice;

[0014] The F0 generation mice are hybridized with wild-type mice, or the hybrid offspring are further bred to obtain mice expressing MSTN-FKPB-mCherry fusion protein, that is, a mouse model that rapidly degrades MSTN protein.

[0015] The TGA stop codon of the mouse Mstn gene is located in exon 3. After the knock-in sequence is introduced, the TGA stop codon will be replaced by "3xEAAAK-mutated human FKBP1A CDS (without ATG start codon)-3xEAAAK-mCherry".

[0016] As a preferred embodiment of the present invention, the Donor recombinant vector is constructed according to the following steps:

[0017] Using the BAC plasmid as a template, amplifying the fragment containing the 5' homology arm and the fragment containing the 3' homology arm from the mouse genome, respectively, to obtain fragment-1 and fragment-3;

[0018] The 3xEAAAK, the FKBP1A gene synonymous mutation sequence, 3xEAAAK and the red fluorescent protein mCherry sequence are sequentially connected to synthesize fragment-2;

[0019] The fragment-1, the fragment-2, the fragment-3 and the Donor vector backbone are connected, the obtained connection products are incubated, and transformed into competent cells, cultured, and extracted to obtain the Donor recombinant vector. The nucleotide sequence of the Donor recombinant vector is shown in SEQ.ID.NO.21.

[0020] Furthermore, the primer sequences for amplifying the fragment containing the 5' homology arm are shown in SEQ.ID.NO.7 and SEQ.ID.NO.8, and the primer sequences for amplifying the fragment containing the 3' homology arm are shown in SEQ.ID.NO.9 and SEQ.ID.NO.10.

[0021] Furthermore, when the fragment-1, the fragment-2, the fragment-3 and the Donor vector backbone were connected, the 20 μL connection system contained: 80.0 ng Donor vector backbone, 7.19 ng fragment-1, 21.28 ng fragment-2, 27.54 ng fragment-3, and 10 μL NEBuilder HiFi DNAAssembly MasterMix.

[0022] Furthermore, the amplification system is: template DNA 1uL, 10uM upstream and downstream primers 1uL, 2×Phanta MaxMasterMix (Dye Plus) 12.5uL, and water is added to 25uL.

[0023] Furthermore, the amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 58°C for 35 s, extension at 72°C for 30-60 s, 35 cycles; and final extension at 72°C for 5 min.

[0024] As a preferred embodiment of the present invention, the competent cells are DH5-alpha competent cells.

[0025] As a preferred embodiment of the present invention, after the gRNA and the Cas9 protein are mixed and incubated in a metal bath, the Donor recombinant vector is added, mixed evenly, and co-injected into mouse fertilized eggs.

[0026] Furthermore, the incubation is carried out in a metal bath at 25 degrees Celsius for 10 minutes.

[0027] As a preferred embodiment of the present invention, the concentration of the gRNA is 90-110 pmol / uL, the concentration of the Cas9 protein is 18-22 μM, the concentration of the Donor recombinant vector is 12-18 ng / uL, 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 and the Donor recombinant vector is 20 uL.

[0028] In a second aspect, the present invention provides a mouse model for rapid degradation of MSTN protein constructed according to the above method.

[0029] As a preferred embodiment of the present invention, the obtained MSTN-FKPB-mCherry knock-in mice are administered with dTAG to achieve rapid and reversible degradation and clearance of MSTN protein in the mice.

[0030] The beneficial effects of the present invention are embodied in:

[0031] The present invention is based on CRISPR / Cas9-mediated gene editing technology, knocks in human FKBP1A protein coding sequence and red fluorescent protein mCherry sequence (marked as FKPB-mCherry tag sequence) in mouse Mstn gene, and introduces synonymous mutation of FKBP1A, avoids endogenous splicing site interference, avoids off-target effect, etc., thereby establishing a mouse model of rapid degradation of MSTN protein, i.e., mouse MSTN-FKPB-mCherry knock-in model. The fusion expression of mCherry fluorescent protein after FKBP tag plays an indicating role, and it can be judged whether the target protein is degraded by observing whether the fluorescence becomes weak. The mouse MSTN-FKPB-mCherry knock-in model can stably and efficiently express the MSTN protein (abbreviated as MSTN-FKPB-mCherry fusion protein) fused with FKPB-mCherry tag protein. In addition, the modeling process of the mouse MSTN-FKPB-mCherry knock-in model in the present invention has the advantage of short operation cycle.

[0032] The present invention promotes the expression of MSTN-FKPB-mCherry fusion protein by adding a fusion protein linker 3xEAAAK sequence between Mstn, FKBP1A synonymous mutation sequence and mCherry insertion sequence.

[0033] The present invention achieves rapid and reversible degradation and clearance of MSTN protein in the mouse by administering dTAG to the knock-in mouse obtained in the later stage.

[0034] The present invention is directed to F0 generation positive mice constructed using CRISPR / Cas9 technology (i.e., positive F0 generation heterozygous mice with knock-in completed). Due to the different genetic lineages that will appear due to the cutting efficiency and possible non-homologous repair, the F0 generation positive mice are mated with wild-type mice to obtain F1 generation positive mice with stable genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1This is a schematic diagram of a transgenic mouse model expressing the FKPB-mCherry tag constructed using CRISPR / Cas9 technology in an embodiment of the present invention.

[0036] Figure 2 This is the plasmid map of the Donor vector used for homologous recombination repair.

[0037] Figure 3 This is the F0 generation mouse breeding scheme in the embodiment of the present invention.

[0038] Figure 4 This is a diagram of the PCR identification strategy for the F0 and F1 generation mouse genotypes in the examples of the present invention.

[0039] Figure 5 The electrophoresis diagram of PCR identification of F1 generation mouse genotype in the embodiment of the present invention. A, 5'HA homologous recombination positive genome; B, 3'HA homologous recombination positive genome.

[0040] Figure 6 This is the sequencing diagram of the knock-in (Knockin, KI) sequence of the F1 generation mouse in the example of the present invention.

[0041] Figure 7 This is the electrophoresis diagram for PCR identification of the F2 generation mouse genotype in the embodiment of the present invention.

[0042] Figure 8 This is a Western blot analysis of the MSTN protein expressed in the present invention. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention, but not to limit the scope of protection of the present invention.

[0044] Example 1

[0045] Establishment of MSTN-FKPB-mCherry knock-in model in C57BL / 6J mice using CRISPR / Cas9-mediated gene editing technology

[0046] A mouse transgenic strategy was designed for the human gene FKBP1A. The CDS sequence of FKBP1A was knocked into the third exon of the Mstn gene (NCBI reference sequence: NM_010834.3) on mouse chromosome 1 using CRISPR / Cas9 technology. Figure 1 As shown. Specifically including the following steps:

[0047] 1. Design gRNA based on the CRISPR / Cas9 system. The sequence of the gRNA action site specific to the third exon of the Mstn gene is shown in Table 1.

[0048] Table 1. gRNA action sites

[0049]

[0050] Note: The italic bases in the sequence are PAM.

[0051] 2. According to the principle of homologous recombination repair and in combination with the knock-in target sequence (referred to as knock-in sequence), a Donor vector was designed. The Donor vector comprises a 5' homology arm, 3xEAAAK, a synonymous mutation sequence of the FKBP1A gene (labeled as Mutant HumanFKBP1A CDS-p.F37V), 3xEAAAK, a red fluorescent protein mCherry sequence and a 3' homology arm. Its main components are shown in Table 2, and the nucleotide sequences of the corresponding components are shown in Table 3.

[0052] Table 2. Main components of Donor vector

[0053]

[0054] Table 3. Partial nucleotide sequence of Donor vector

[0055]

[0056]

[0057]

[0058]

[0059] Note: The underlined bases are the introduced synonymous mutations. The sequence before mutation is TTT, and the sequence after mutation is GTT.

[0060] 3. Using In-Fusion technology, the above 3xEAAAK, Mutant Human FKBP1ACDS-p.F37V (TTTto GTT), and mCherry were assembled into a Donor vector, that is, the 5' homology arm, fusion protein linker 3xEAAAK, FKBP1A gene synonymous mutation sequence, 3xEAAAK, red fluorescent protein mCherry sequence, and 3' homology arm were connected to the Donor vector backbone in sequence. The specific process is as follows:

[0061] (1) Using high-fidelity Taq DNA polymerase and BAC (RPCI-23.C) plasmid (thermofisher) as a template, fragment-1 (i.e., the fragment containing the 5' homology arm) and fragment-3 (i.e., the fragment containing the 3' homology arm) containing homologous sequences from the mouse genome were amplified respectively. The sequences of the amplification primers are specifically shown in Table 4, the amplification system is shown in Table 5, and the amplification program is shown in Table 6.

[0062] The 3xEAAAK-Mutant Human FKBP1A CDS (without the ATG start codon)-3xEAAAK-mCherry sequence (denoted as fragment-2) was synthesized (Shanghai Bioengineering Company).

[0063] Table 4 Amplification primer nucleotide sequences

[0064]

[0065] Table 5. PCR system

[0066]

[0067]

[0068] Table 6. PCR program

[0069]

[0070] (2) Acc65I (NEB, R0599) and AscI (NEB, R0558) endonucleases were used to digest the existing plasmid (customized from Saiye Bio, internal catalog number Cat: VB139; specifically, it was modified from the commercial plasmid addgene #50005 to provide a vector backbone). The digestion system is shown in Table 7. The digestion product was subjected to gel electrophoresis and the target band was cut out. The DNA gel recovery kit (OMEGA, D2000) was used to recover and purify it to obtain a linearized plasmid (4201 bp, i.e., the vector backbone).

[0071] Table 7. Enzyme digestion system

[0072] Components Dosage Plasmid DNA 1.0 μg <![CDATA[10×rCutSmart TM Buffer]]> 5.0μL AscI 1μL Acc65I 0.5μL <![CDATA[ddH 2 The]]> To 50.0μL

[0073] (3) Using a seamless cloning kit (NEB, E2621), the PCR amplified fragment was connected to the linearized plasmid according to the reaction system shown in Table 8. The reaction was performed using a PCR instrument and incubated at 50° C. for 60 min; then placed on ice for transformation.

[0074] Table 8. In-Fusion System

[0075]

[0076] (4) The In-Fusion ligation product was transformed into DH5-alpha competent cells, and then the competent cells were added to LB liquid culture medium and cultured in a shaking incubator at 37°C for 30 min. The resulting culture was spread on LB solid culture medium containing ampicillin and cultured overnight. After ampicillin resistance (AmpR) screening, the monoclonal clones grown on the LB solid culture medium were selected and cultured in LB liquid culture medium. After that, PCR identification of the bacterial solution was performed, and after confirming that the ligation was successful, the bacterial solution was sent for sequencing. According to the sequencing results, the monoclonal clones with successful ligation and correct sequence were selected for shaking, and the bacterial solution was collected for plasmid extraction to obtain the Donor recombinant vector. Its plasmid map is shown as follows: Figure 2 shown.

[0077] The nucleotide sequence of the Donor recombinant vector is shown in SEQ ID NO.21:

[0078]

[0079] 4. Preparation of injection compound

[0080] Tube 1 solution: Add 0.8uL 100pmol / uL gRNA to RNase-free water, then add 0.2uL 20μM Cas9 protein (NEB, catalog number: M0646M) and mix well. Incubate in a metal bath at 25 degrees Celsius for 10 minutes to obtain tube 1 solution;

[0081] Tube 2 solution: Donor recombinant vector with a final concentration of 15 ng / uL;

[0082] The solution in tube 1 and the solution in tube 2 were mixed to obtain an injection complex, and the total volume of the injection complex was 20 μL.

[0083] 5. Microinjection and embryo transfer

[0084] After superovulation of female C57BL / 6J mice, fertilized eggs of C57BL / 6J mice were obtained by in vitro fertilization. Then, the obtained injection complex was injected into the fertilized eggs of C57BL / 6J mice using a glass microinjection needle with an extremely fine tip (0.1-0.5 μm), and the fertilized eggs after microinjection were then sent to the oviduct of surrogate C57BL / 6J mice through embryo transfer. After the pups were born, F0 generation mice were obtained, and the genotype of the F0 generation mice was identified by PCR method, such as Figure 3 shown.

[0085] The specific operation of the above embryo transplantation is as follows: the fertilized eggs after microinjection are cultured in vitro to morulae and quality tested; a small incision is made in the oviduct capsule of the surrogate mother mouse, and the direction of the oviduct fimbria is clearly seen, and the transplant tube is inserted from the opening into the oviduct fimbria 2 to 3 mm, and the embryos with good development quality are blown into the magnum, and the transplant tube is pulled out after a short pause.

[0086] 6. The positive F0 mice were mated with C57BL / 6J mice (Wild type, Wt) to obtain F1 mice, and their genotypes were identified by PCR. If positive mice (F1 heterozygotes) were born, it means that the knock-in sequence has been integrated into the germ cells, such as Figure 4 shown.

[0087] 7. The F1 generation heterozygous mice were self-pollinated, and the resulting F2 generation mice were genotyped to obtain MSTN-FKPB-mCherry homozygous knock-in mice, that is, mice expressing MSTN-FKBP-mCherry fusion protein were obtained.

[0088] 8. By administering dTAG to the knock-in mice, rapid and reversible degradation and clearance of MSTN protein in mice can be achieved.

[0089] Example 2

[0090] Mouse genotype PCR identification results

[0091] 1. DNA extraction

[0092] Select newborn mice aged 3 to 4 weeks, cut off the tail of the mouse about 0.3 cm long, place it in a 1.5 ml EP tube (the corresponding number of the mouse is marked on the tube cap) containing 50ul lysis buffer and 1ul proteinase K, centrifuge until the mouse tail is at the bottom of the EP tube, and place it in a 55°C metal bath overnight. The next day, set the metal bath temperature to 95°C for 5 minutes to inactivate proteinase K, vortex oscillator for 15 seconds to completely lyse the mouse tail, centrifuge at 12000rpm for 5 minutes, and the supernatant can be directly used as a template for genotype identification, or stored at -20°C for later use.

[0093] 2. PCR identification results of F1 generation mouse genotype

[0094] DNA was extracted from the mouse tail tip and its genotype was identified by PCR amplification. Figure 4 The PCR identification strategy in the amplification is that the 5'HA homologous recombination positive genome should be amplified with a 2.8kb fragment, and the negative genome should have no bands when amplified with the F1 / R1 primers; the 3'HA homologous recombination positive genome should be amplified with a 3.5kb fragment, and the negative genome should have no bands when amplified with the F2 / R2 primers. The amplification system is shown in Table 9, and the amplification program is shown in Table 10. The specific primers used for PCR identification are shown in Table 11.

[0095] Table 9. PCR system

[0096]

[0097] Table 10. PCR program

[0098]

[0099]

[0100] Table 11. PCR amplification primers

[0101]

[0102] The identification results are as follows Figure 5 As shown, mice No. 15, 18, 19, 22, 23, 24, and 27 are F1 mice that are positive for homologous recombination in both arms.

[0103] Note: If the DNA sample is not very pure or if there is not enough PCR extension time, it may not be possible to amplify long PCR products. Figure 4 In other PCR identification strategies, the target allele should be amplified as a 460 bp fragment using the F4 / R5 primers. The F4 / R5 primer sequences are shown in Table 13.

[0104] 3. Sequencing results of knock-in sequences of F1 mice

[0105] Primers F3 / R3 were used for PCR amplification, and the product was sent for sequencing.

[0106] The sequencing results are as follows Figure 6 The results showed that the knock-in site of the positive F1 generation No. 15 mouse was correct and the knock-in sequence was correct without mutation. Among them, the specific primers used for PCR amplification are shown in Table 12:

[0107] Table 12. PCR amplification primers

[0108]

[0109]

[0110] 4. PCR identification results of F2 generation mouse genotype

[0111] DNA was extracted from the mouse tail tip and its genotype was identified by PCR amplification. Figure 4 The PCR identification strategy in the PCR was used to perform PCR using primers F5 / R3 and F4 / R5. The genotype of the F2 generation of mice is divided into three categories based on the length of the PCR fragment: knock-in homozygous (Homozygous, Ho), knock-in heterozygous (Heterozygous, He), and wild type (Wildtype, Wt). The PCR fragment lengths corresponding to different genotypes are: two bands of 460bp and 1619bp (knock-in homozygous); three bands of 460bp, 503bp and 1619bp (knock-in heterozygous), and a 503bp band (wild type). Among them, the specific primers used for PCR identification are shown in Table 13:

[0112] Table 13. PCR amplification primers

[0113]

[0114] Example 3

[0115] Evaluation of targeting strategies for transient knockout of MSTN

[0116] The expression of MSTN protein in the muscle tissue of F2 mice was detected by Western blot.

[0117] The identification results are as follows Figure 8As shown, after dTAG administration to F2 heterozygous mice, the MSTN-FKPB-mCherry fusion protein was degraded, while the MSTN protein in the muscle tissue of mice not administered dTAG was normally expressed.

[0118] The above results show that the present invention uses In-Fusion technology to construct a Donor vector, and co-injects the Donor vector, gRNA and Cas9 mRNA into mouse fertilized eggs, thereby knocking the human FKBP1A gene sequence into the mouse genome using CRISPR / Cas9-mediated gene editing technology, and using it as the FKPB-mCherrytag sequence fused with the mouse Mstn gene for expression, and then through embryo transplantation and breeding of the resulting positive F0 generation mice, finally a parent capable of stably and efficiently expressing the MSTN-FKPB-mCherry fusion protein is obtained, so that the dTAG protein rapid degradation system model mouse can be used to achieve rapid, complete and reversible MSTN protein degradation induced by small molecule degraders, which provides a new idea and approach to solve the problem that traditional gene knockout animal models cannot explore the direct regulatory mechanism of the protein of interest.

Claims

1. A method for constructing a mouse model for rapid degradation of MSTN protein, characterized in that: The following steps are involved: The Donor vector backbone is connected with a 5' homology arm, 3xEAAAK, a synonymous mutation sequence of the FKBP1A gene, 3xEAAAK, a red fluorescent protein mCherry sequence and a 3' homology arm arranged in sequence to obtain a Donor recombinant vector; the nucleotide sequence of the synonymous mutation sequence of the FKBP1A gene is shown in SEQ ID NO.4, the nucleotide sequence of 3xEAAAK is shown in SEQ ID NO.3, the nucleotide sequence of the red fluorescent protein mCherry is shown in SEQ ID NO.5, the nucleotide sequence of the 5' homology arm is shown in SEQ ID NO.2, and the nucleotide sequence of the 3' homology arm is shown in SEQ ID NO.6; gRNA is designed based on the third exon of the Mstn gene on mouse chromosome 1, and the nucleotide sequence of the gRNA is shown in SEQ.ID.NO.1; The gRNA, Cas9 protein and the Donor recombinant vector are mixed and co-injected into mouse fertilized eggs to knock the FKBP1A gene synonymous mutation sequence and the red fluorescent protein mCherry sequence into the mouse genome; The injected fertilized eggs are cultured in vitro and then transplanted into surrogate mice to continue to develop until pups are born, thus obtaining F0 generation mice; The F0 generation mice are hybridized with wild-type mice, or the hybrid offspring are further bred to obtain mice expressing MSTN-FKPB-mCherry fusion protein, that is, a mouse model that rapidly degrades MSTN protein.

2. The construction method according to claim 1, characterized in that: The Donor recombinant vector is constructed according to the following steps: Using the BAC plasmid as a template, amplifying the fragment containing the 5' homology arm and the fragment containing the 3' homology arm from the mouse genome, respectively, to obtain fragment-1 and fragment-3; The 3xEAAAK, the FKBP1A gene synonymous mutation sequence, 3xEAAAK and the red fluorescent protein mCherry sequence are sequentially connected to obtain fragment-2; The fragment-1, the fragment-2, the fragment-3 and the Donor vector backbone are connected, the obtained connection products are incubated, and transformed into competent cells, cultured, and extracted to obtain the Donor recombinant vector. The nucleotide sequence of the Donor recombinant vector is shown in SEQ.ID.NO.

21.

3. The construction method according to claim 2, characterized in that: The primer sequences for amplifying the fragment containing the 5' homology arm are shown in SEQ.ID.NO.7 and SEQ.ID.NO.8, and the primer sequences for amplifying the fragment containing the 3' homology arm are shown in SEQ.ID.NO.9 and SEQ.ID.NO.

10.

4. The construction method according to claim 2, characterized in that: When the fragment-1, the fragment-2, the fragment-3 and the Donor vector backbone were connected, the 20 μL connection system contained: 80.0 ng Donor vector backbone, 7.19 ng fragment-1, 21.28 ng fragment-2, 27.54 ng fragment-3, and 10 μL NEBuilder HiFi DNAAssembly MasterMix.

5. The construction method according to claim 2, characterized in that: The competent cells are DH5-alpha competent cells.

6. The construction method according to claim 1, characterized in that: After the gRNA and the Cas9 protein are mixed and incubated in a metal bath, the Donor recombinant vector is added, mixed evenly, and co-injected into mouse fertilized eggs.

7. The construction method according to claim 6, characterized in that: The concentration of the gRNA is 90-110 pmol / uL, the concentration of the Cas9 protein is 18-22 μM, the concentration of the Donor recombinant vector is 12-18 ng / uL, 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 and the Donor recombinant vector is 20 uL.

8. A mouse model of rapid degradation of MSTN protein constructed according to the method of claim 1.

9. The mouse model for rapid degradation of MSTN protein according to claim 8, characterized in that: The obtained MSTN-FKPB-mCherry knock-in mice were administered dTAG to achieve rapid and reversible degradation and clearance of MSTN protein in mice.

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