CN112980882A - Application of Crbn gene in construction of GSPT1 sensitive model - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to the use of Crbn gene in constructing a GSPT1 sensitive model. The present invention provides the use of CRBN gene in constructing a GSPT1-sensitive cell model or a GSPT1-sensitive animal model, and further provides a GSPT1-sensitive cell model and a GSPT1-sensitive animal model and their construction methods, these cell models and animal models have the ability to degrade GSPT1 The obvious response can be used in the research of GSPT1 degrader, which can save material cost, labor cost and time cost, and has a good industrialization prospect.

Description

Application of Crbn gene in construction of GSPT1 sensitive model

Technical Field

The invention relates to the technical field of biology, in particular to application of a Crbn gene in constructing a GSPT1 sensitive model.

Background

Immunomodulatory Drugs (IMiDs), including Thalidomide (Thalidomide), Lenalidomide (Lenalidomide), and Pomalidomide (Pomalidomide), among others. Talidomide was first marketed in 1957, when called "weaning" for the treatment of vomiting in early pregnancy in women. However, because of its severe teratogenicity, which results in the birth of a large number of defective infants, since 1961, the response has been prohibited from being used as an antiemetic for pregnant women, but research work on Thalidomide has not been stopped, and particularly, its antitumor effect has been attracting much attention. From 2006, FDA in the united states successively approved IMiDs for clinical treatment of diseases such as multiple myeloma.

After 2014, the action mechanism of IMiDs is solved, and the compound can enhance the ubiquitination degradation of blood cell survival-dependent protein by ubiquitin ligase CRL4(CRBN), so the compound is vividly called as molecular glue. A series of molecular glues hijacking CRBN are developed successively, and typically 2016, a new IMiDs compound CC-885 reported in Nature by the development team of Celgene corporation can promote the ubiquitination degradation of new substrate translation termination factor GSPT1 by CRL4(CRBN) to kill various blood tumor and solid tumor cells. GSPT1, also known as eRF3, when translation encounters a stop codon, the eRF1-eRF3-GTP complex occupies the A site of the ribosome. After GTP hydrolysis, eRF3 and GDP are dissociated from the complex, and eRF1 remaining on the ribosome undergoes a huge change in conformation, and finally the two subunits 60s and 40s of the ribosome are dissociated, and protein translation is terminated. GSPT1 is a protein essential for cell survival, and when it is deleted, the cell in proliferation state will generate a large amount of wrong protein in the cell to trigger intracellular pressure to finally go to apoptosis. The GSPT1 degrading agent is already in clinical experiments. Whether the CC-885 or other GSPT1 degrading agents can inhibit the growth of tumor cells by degrading GSPT1 so as to become the antitumor drug or not is worthy of deep research. However, there is no mouse model for studying GSPT1 degradants such as CC-885.

During the process of promoting the degradation of the substrate by IMiDs, a CRBN-IMiDs-CC-885 ternary complex is formed firstly. Mouse and human CRBN have more than 97 percent of homology, but the IMiDs are different from each other by 3 bases in the combination area, so that the IMiDs can not promote the degradation of the substrate by the wild CRBN of the mouse. This is also why the initial response stops unresponsive in mice and is strongly teratogenic in humans. Appropriate humanized mouse models are required to study the toxic side effects of IMiDs.

CC-885 in promoting the ubiquitination degradation of GSPT1, a ternary complex CRBN-CC885-GSPT1 is formed, wherein the E (glutamic acid) 377 site of CRBN is a key site. Experiments showed that mutation of the E377 site of CRBN to V (valine) did not cause ubiquitination degradation of GSPT1 by CC-885. The amino acid of the Crbn of the mouse corresponding to the CRBN E377 site of the human is V380, so that the Crbn of the mouse can not react to CC-885, and the wild-type mouse can not be used for researching the toxic and side effect of the CC-885. A mouse model is needed to research the toxic effect of CC-885, and further to research whether GSPT1 can be used as a target of an anti-tumor drug.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide the use of the Crbn gene in the construction of a GSPT1 sensitive model, for solving the problems in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a use of a Crbn gene in constructing a GSPT1 sensitive cell model or a GSPT1 sensitive animal model.

The invention further provides a GSPT1 sensitive cell model, wherein the Crbn gene of the GSPT1 sensitive cell model comprises a V380E mutation, and the GSPT1 sensitive cell model is derived from a mouse.

The invention also provides a construction method of the GSPT1 sensitive cell model, which comprises the following steps:

introduction of Crbn into cells^V380EMutations to provide the model of GSPT1 sensitive cells described above, which were derived from mice.

The invention also provides a construction method of the GSPT1 sensitive animal model, which comprises the following steps:

introduction of Crbn in mice^V380EMutations to provide the aforementioned GSPT1 sensitive animal model.

In another aspect, the invention provides a method for screening a candidate drug for a GSPT1 degradation agent, comprising:

and (3) applying the candidate drug to the GSPT1 sensitive cell model or the GSPT1 sensitive animal model obtained by the construction method of the GSPT1 sensitive animal model.

Drawings

FIG. 1 shows Crbn in example 1 of the present invention^V380ESchematic representation of the sequencing results of mouse genome.

Fig. 2 is a graph showing the expression of GSPT1 and cell killing results in examples 2 and 3 of the present invention.

Fig. 3 is a schematic diagram showing cell viability of MiaPaCa2 cells and Panc1 cells in example 4 of the present invention.

Fig. 4 shows a schematic diagram of the degradation of GSPT1 in example 5 of the present invention.

Fig. 5 is a schematic diagram showing the blood pressure measurement result in embodiment 6 of the present invention.

FIG. 6 is a graph showing the survival curves of the cells after CC-885 injection in example 7 of the present invention.

FIG. 7 is a schematic diagram showing the results of splicing Caspase3 expression in example 8 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

Through a large number of practical researches, the inventor of the invention unexpectedly discovers that the mutation of the Crbn gene can be used for constructing a GSPT1 sensitive cell model or a GSPT1 sensitive animal model. The GSPT1 sensitive cell model or the GSPT1 sensitive animal model obtained by construction has sensitivity to a GSPT1 degradation agent, so that the GSPT1 sensitive cell model or the GSPT1 sensitive animal model can be used for screening candidate drugs of the GSPT1 degradation agent, and the invention is completed on the basis.

The invention provides application of a Crbn gene in constructing a GSPT1 sensitive cell model or a GSPT1 sensitive animal model. Sensitive to GSPT1 generally refers to a cell or animal that is sensitive to a GSPT1 degrading agent. For example, in the case of administration of a GSPT1 degrading agent, GSPT1 in a cell or in an animal is significantly degraded (e.g., the expression level of GSPT1 is significantly reduced, etc.). As another example, a cell or animal may exhibit significant toxic effects when administered with a GSPT1 degrading agent (e.g., decreased cell viability, decreased animal blood pressure, decreased survival, increased expression of splice caspase3, etc.).

In the above uses, a gstt 1 degrading agent generally refers to a substance that can reduce the expression and/or function of gstt 1. For example, a GSPT1 degrading agent may be one that reduces the expression and/or function of GSPT1, or one that completely eliminates the expression and/or function of GSPT 1. It will be apparent to those skilled in the art which substances are capable of acting as GSPT1 degradants, or potentially as GSPT1 degradants. For example, a GSPT1 degradant may generally bind to CRBN and GSPT1 to form a CRBN-degradant-GSPT 1 ternary complex, thereby achieving the effect of reducing the expression and/or function of GSPT 1. For another example, the GSPT1 degradant may be an immunomodulator (IMiDs) or the like. In one embodiment of the invention, the GSTT 1 degrader may be CC-885(CAS 1010100-07-8), and the like.

In the applications provided by the present invention, the polypeptide fragment encoded by the Crbn gene may be: a) the coded amino acid sequence comprises a protein shown as a sequence in SEQ ID NO. 1; or b) a protein which has an amino acid sequence with more than 80 percent of sequence identity with the sequence shown in SEQ ID NO.1 and has the function of the protein defined by a). Specifically, the protein in b) is specifically: the protein having the amino acid sequence shown in SEQ ID No.1, which is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids in the amino acid sequence shown in SEQ ID No.1, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids in the N-terminal and/or C-terminal, and has the function of the protein shown in SEQ ID No.1, for example, a substrate receptor protein of a Cullin 4 ubiquitin ligase complex, or the like. The amino acid sequence of the protein of b) above may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% identity with SEQ ID No. 1. "sequence identity" between two polypeptides generally indicates the percentage of amino acids that are identical between the sequences. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software, and in particular identity can be determined using programs such as BLAST from the NCBI database. Generally, the above-mentioned Crbn gene can be derived from mouse (Mus musculus).

MAGEGDQQDAAHNMGNHLPLLPDSEDEDDEIEMEVEDQDSKEARKPNIINFDTSLPTSHTYLGADMEEFHGRTLHDDDSCQVIPVLPEVLMILIPGQTLPLQLSHPQEVSMVRNLIQKDRTFAVLAYSNVQEREAQFGTTAEIYAYREEQEFGIEVVKVKAIGRQRFKVLELRTQSDGIQQAKVQILPECVLPSTMSAVQLESLNKCQVFPSKPISWEDQYSCKWWQKYQKRKFHCANLTSWPRWLYSLYDAETLMDRIKKQLREWDENLKDDSLPENPIDFSYRVAACLPIDDVLRIQLLKIGSAIQRLRCELDIMNKCTSLCCKQCQETEITTKNEIFSLSLCGPMAAYVNPHGYVHETLTVYKASNLNLIGRPSTVHSWFPGYAWTIAQCKICASHIGWKFTATKKDMSPQKFWGLTRSALLPTIPETEDEISPDKVILCL(SEQ ID No.1)

In the application, when a GSPT1 sensitive cell model or a GSPT1 sensitive animal model is constructed, a V380E mutation can be generally introduced into a Crbn gene. Due to the differences in the amino acid sequences of mouse Crbn and human Crbn, wild-type mice or cells thereof are generally insensitive to GSPT1 degradants. And Crbn is introduced into cells of mice^V380EAfter mutation, the obtained mouse cell model has sensitivity to a GSPT1 degradation agent, so that the mouse cell model can be used as a GSPT1 sensitive cell model. After V380E mutation is introduced into Crbn gene of mouse, the obtained mouse animal model has sensitivity to GSPT1 degradation agent, so that the mouse animal model can be used as GSPT1 sensitive animal model.

In the above applications, the GSPT 1-sensitive cell model and/or GSPT 1-sensitive animal model is usually derived from a mouse, and can be usually obtained by constructing a wild-type mouse, or a humanized mouse obtained by modification, or a cell thereof. For example, the cells may be mouse visceral cells, mouse spleen cells, mouse liver cells, mouse embryonic fibroblasts, and the like.

The second aspect of the invention provides a GSPT1 sensitive cell model, wherein a V380E mutation exists in a Crbn gene of the GSPT1 sensitive cell model, and the GSPT1 sensitive cell model is derived from a mouse. Crbn was introduced into cells of mice as described above^V380EFollowing mutation, mouse cell models are directed to GSPThe T1 degradant is sensitive, so that the T1 degradant can be used as a GSPT1 sensitive cell model.

Of the above GSPT 1-sensitive cell models, the GSPT 1-sensitive cell model can be usually constructed from mouse visceral cells. For example, applicable mouse cells may include a combination of one or more of mouse visceral cells, mouse spleen cells, mouse liver cells, mouse embryonic fibroblasts, and the like. Generally speaking, a model of GSPT 1-sensitive cells may be derived from a humanized mouse of its corresponding genotype.

Among the above GSPT1 sensitive cell models, Crbn in GSPT1 sensitive cell model^V380EThe mutation may be heterozygous (Crbn)^V380E/+) May also be homozygous (Crbn)^V380E/V380E) Preferably homozygous. Generally, Crbn^V380E/V380EHomozygous mutant GSPT1 sensitive cell model vs Crbn^V380E/+The hybrid mutant GSPT1 sensitive cell model has stronger sensitivity to GSPT1 degradation agent.

The third aspect of the invention provides a method for constructing a model of GSPT1 sensitive cells provided by the second aspect of the invention, comprising: introduction of Crbn into cells^V380EMutations to provide the model of GSPT1 sensitive cells described above, which were derived from mice. One skilled in the art can select an appropriate method to construct Crbn^V380EA mutant cell. For example, it can be determined by Crbn^V380EA mutant mouse animal model providing Crbn^V380EA mutant cell. For another example, the applicable mouse cells may be mouse visceral cells, and specifically may include one or a combination of more of mouse visceral cells, mouse spleen cells, mouse liver cells, mouse embryo fibroblasts, and the like.

The fourth aspect of the invention provides a GSPT1 sensitive animal model, wherein V380E mutation exists in Crbn gene of the GSPT1 sensitive animal model, and the GSPT1 sensitive animal model is from mouse. Crbn was introduced in vivo in mice as described above^V380EAfter mutation, the mouse animal model has sensitivity to a GSPT1 degradation agent, so that the mouse animal model can be used as a GSPT1 sensitive animal model.

Among the above-mentioned GSPT1 sensitive animal models, the GSPT1 sensitive animal model can be generally obtained by mouse construction. Generally speaking, a GSPT1 sensitive animal model may be derived from a wild type mouse.

Among the GSPT1 sensitive animal models, Crbn in GSPT1 sensitive animal model^V380EThe mutations may be heterozygous or homozygous, preferably homozygous. Generally, Crbn^V380E/V380EHomozygous mutant GSPT1 sensitive animal model relative to Crbn^V380E/+The heterozygous mutant GSPT1 sensitive animal model has stronger sensitivity to GSPT1 degradation agent.

The fifth aspect of the invention provides a method for constructing a GSPT1 sensitive animal model provided by the fourth aspect of the invention, which comprises the following steps: introduction of Crbn in mice^V380EMutations to provide the aforementioned GSPT1 sensitive animal model. One skilled in the art can select an appropriate method to construct Crbn^V380EA mutated mouse animal model (i.e., a GSPT1 sensitive animal model). For example, the subject to be administered may be mouse fertilized egg cells or the like by a method such as CRISPR Cas9 gene editing technology. In particular, Crbn can be edited by CRISPR Cas9 gene editing technology^V380EIntroducing mouse fertilized egg cell, breeding and/or screening to obtain Crbn^V380EThe animal model of mutation (for example, the fertilized egg cells obtained by the construction can be implanted into the uterus of female mouse, and then fed and subjected to gene identification, so that the animal model with Crbn can be obtained^V380EThe Crbn can be obtained by further breeding and passage of F0 mouse of chromosome^V380E/V380EAnimal model of homozygous mutation). As another example, the construction can be carried out by wild-type mouse cells.

The sixth aspect of the present invention provides a method for screening a drug candidate for a GSPT1 degradation agent, comprising: administering a candidate drug to a GSPT1 sensitive cell model provided by the second aspect of the invention, or a GSPT1 sensitive animal model provided by the fourth aspect of the invention. Generally, following administration of a candidate drug, a candidate drug for a GSPT1 degrader may be screened for by changes in the GSPT 1-sensitive cell model and/or the GSPT 1-sensitive animal model to which the candidate drug is administered. For example, a candidate drug can be screened by comparing a GSPT 1-sensitive cell model and/or a GSPT 1-sensitive animal model after administration of the candidate drug to a control model (e.g., a GSPT 1-sensitive cell model and/or a GSPT 1-sensitive animal model without administration of the candidate drug) and observing a change in the GSPT 1-sensitive cell model and/or a GSPT 1-sensitive animal model after administration of the candidate drug (e.g., whether a GSPT 1-sensitive cell model and/or a GSPT 1-sensitive animal model after administration of the candidate drug exhibits significant sensitivity to a GSPT1 degrading agent).

In the method for screening the candidate drugs of the GSPT1 degradation agent, the screened candidate drugs can further form a screening library, and further screening experiments (such as cell experiments, animal experiments, clinical experiments and the like) can be carried out on the substances so as to further confirm the potential of the substances as the GSPT1 degradation agent.

The invention provides the application of CRBN gene in constructing a GSPT1 sensitive cell model or a GSPT1 sensitive animal model, and further provides a GSPT1 sensitive cell model, a GSPT1 sensitive animal model and construction methods thereof, wherein the cell models and the animal models have obvious response to a GSPT1 degradation agent, so that the cell models and the animal models can be used for researching the GSPT1 degradation agent, can save material cost, labor cost and time cost, and have good industrialization prospect.

The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989 and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1

Crbn^V380EConstruction of mice

1. Construction of Crbn by CRISPR technique^V380EHumanized mouse model

For the mouse Crbn gene sequence: ACAGTGCACAGCTGGTTTCCCGG (SEQ ID No.12), designing and constructing sgRNA vector for mediating Crbn gene cutting, and constructing the following sequence on px459 vector:

sg#1：F：5’–CACCGACAGTGCACAGCTGGTTTCC–3’(SEQ ID No.2)

R:3’–CTGTCACGTGTCGACCAAAGGCAAA–5’(SEQ ID No.3)

system for annealing oligo:

oligo 1	(100μM)
		oligo 2	(100μM)
10X T4 Ligation Buffer(NEB)	1μl
		ddH2O	make up to 10. mu.l

The procedure of annealing was:

37℃30min

95℃5min and then ramp down to 25℃at 5℃/min

cleavage of PX459 vector:

Plasmid	1μg
		FastDigest BbsI(Fermentas)	1μl
FastAP(Fermentas)	1μl
		10X FastDigest Buffer	2μl
ddH2O	make up to 20. mu.l

Digesting for 3h at 37 ℃, and then cutting and recovering.

Linking the annealed product to PX459 vector:

16 ℃ overnight. Then, the vector is transformed into DH5a competence, and finally, a constructed sgRNA vector (PX459 with sgRNA) is obtained, and sequencing and verification are carried out.

2. Preparing a transcription template, transcribing the complete sgRNA and Cas9 mRNA:

2.1, preparation of sgRNA template:

T7 promoter tagged primer

F：taatacgactcactataggACAGTGCACAGCTGGTTTCC；(SEQ ID No.4)

R：AAAAGCACCGACTCGGTGCC；(SEQ ID No.5)

reaction system:

the PCR procedure was:

To step2 for 29cycles

72℃ 3min

the PCR product was recovered.

2.2 transcription of sgRNA and Cas9 mRNA

The template DNA of sgRNA was obtained from 2.1 above, and the template DNA of Cas9 was a plasmid purchased from addge inc. After 4 hours at 37 ℃ the reaction was carried out by adding 1. mu.L of TURBO DNase and reacting at 37 ℃ for 15 min. Then using MEGAclear^TMKit (thermo Fisher) recovered the transcript for use.

3. The transcribed sgRNA, Cas9 mRNA and synthetic repair template ssDNA were injected into fertilized eggs 3.5 days old (sgRNA:10 ng/. mu.L, Cas9 mRNA: 10 ng/. mu.L, ssDNA: 10 ng/. mu.L). Fertilized eggs were cultured in vitro to the two-cell stage using KSOM mouse embryo medium (Millipore). Then transplanted into an ICR female rat uterus of 0.5 days of pseudopregnancy. F0 mouse can be obtained after 19-21 days.

AAAGCGTCCAACCTGAATCTGATAGGCCGGCCTTCTACTGAGCACAGCTGGTTTCCCGGGTAATACAGCTGTTTACTTTTCTTGTTGACTCTTCATTTAGTTTTAGATGAACTTTCTAGGAAGATACAAAACAAACAGGACAGGAATAGTTTGATCACTTCATGAATGGGTTAAAAGCAGGGACATGAGA(SEQ ID No.6)

190nt，AIs a mutation of V380E which,Tis a synonymous mutation introduced, and is convenient to identify later.

4. Identification and screening of F0 mouse.

Primer F：GCAACAGAATATCTGCCAACCTC(SEQ ID No.7)；

Primer R：GCCACTTCCAAAGTAAAAGGGA(SEQ ID No.8)。

The genotype test results of F0 mouse are shown in FIG. 1 a.

5. After identifying the F0 generation, we selected a heterozygote and a wild type mouse for propagation, and their progenySelfing to obtain Crbn^V380E/V380EA mouse. During the breeding process, wild type, heterozygote and homozygote are obtained, and the genotypes are shown in figure 1 b.

6. Meanwhile, the influence of gene editing on the development of the mice is evaluated, and the gene editing is found not to influence the development of the mice. The birth rate of the mice followed Mendelian's Law of inheritance, and the results are shown in FIG. 1 c.

Example 2

Crbn^V380EHumanized mice are responsive to CC-885

From wild type mice and Crbn^V380E/+Spleen tissue was removed from the mice and spleen cells were ground on a 40 μm mesh screen and incubated with RPMI 1640 medium (containing 10% HIFBS, 20mM HEPES, 1mM sodium pyroltate, 0.05mM 2-mercaptoethanol, 1 XGluMax, 1 Xpenicillin streptomycin diabody), 5% CO2 at 37 ℃. Spleen divided in 6-well plates (2.5X 10)⁷Perwell) and cultured with human HL60 cells ((1X 10)⁷Well), 1640 culture medium, 10% fetal bovine serum, 1 XPS double antibody) as positive control, adding CC-885 with gradient concentration of 0, 10 and 100nM directly into each group of culture medium, treating at 37 ℃ for 24h, collecting cells, extracting total cell protein with RIPA, and detecting the expression of GSPT1 by immunoblotting. The cells were harvested and denatured with protein loading buffer (50mM Tris-HCl, 2% SDS, 0.025% BPB, 1% beta-mercaptoethanol, 10% glycerol) for 10min at 98 ℃. Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore, IPVH00010), blocked with 5% skimmed milk (in TBST buffer), incubated overnight at 4 ℃ with primary antibodies GSPT1(Abcam, ab49878) and Vincultin (Sigma, V9131), incubated with secondary antibodies at room temperature for 1 hour, and bands detected on the membrane by chemiluminescence (ECL chemiluminescence kit, Byunnan Bio, P001 0018 AM). The results are shown in FIG. 2 a.

Separating mouse embryo from mother mouse with pregnancy duration of 12.5 days, removing head and viscera, cutting limbs with surgical scissors, and digesting with pancreatin for 20min to obtain wild type Crbn^V380E/+Heterozygous mouse, Crbn^V380E/V380EHomozygous mouse, Crbn^I391V/I391VMEF cells of homozygous mice. MEF cellsAmplification was performed in DMEM medium (10% FBS, penicillin streptomycin double antibody) at 37 ℃ in an incubator. MEFs of each genotype were split into 6-well plates (4X 10)⁵Per well), gradient concentrations of 0, 10, 100nM CC-885 were added directly to each group of culture medium, cells were collected after treatment at 37 ℃ for 24h and total cell protein was extracted with RIPA, and expression of GSPT1 was detected using the above immunoblotting (Western blot), the results are shown in fig. 2 b.

As can be seen from FIGS. 2a and 2b, only in Crbn^V380EGSPT1 was degraded by CC-885 in mouse cells. The result shows that wild type mice do not respond to CC-885, the V380E mutation can make the mice sensitive to CC-885, and Crbn^V380EHomozygous mutations were more sensitive to CC-885 and more significantly degraded by GSPT1 than heterozygous mutant mice.

Example 3

CC-885 to Crbn^V380EThe mouse cell has killing effect

Crbn was extracted and cultured in the same manner as in FIG. 2b^V380E/+Hybrid mouse MEF cells and Crbn^I391V/I391VHomozygous mouse MEF cells were plated in 6-well plates at the same density, treated for 60h with 2-fold gradient CC-885, and examined for 450nm absorbance wavelength using CCK-8 kit and microplate reader, and the survival curve (plotted by GraphPad Prism 5 software) is shown in FIG. 2c, where<0.001. As can be seen from FIG. 2c, Huh7 is very sensitive to CC-885, Hep1-6 (negative control, Gibco DMEM medium, 10% fetal bovine serum, 1 XPS streptomycin double antibody, 4X 10⁵/well) no response to CC-885, Crbn^I391V/I391VHomozygous mouse MEF cells did not respond to CC-885, whereas Crbn^V380E/+MEF cells of heterozygous mice are sensitive to CC-885, and more than 50% of Crbn can be obtained by 1 mu M of CC-885 acting for 72h^V380ECell killing. Illustrating humanized Crbn^V380EMice can be made to respond to CC-885.

Extraction and cultivation of wild type, Crbn, Using the same method as in FIG. 2b^V380E/+Hybrid and Crbn^V380E/V380EMEF cells of homozygous mice are treated for 72h by CC-885 with gradient concentration of 10 times at intervals, and then a CCK-8 kit and an enzyme-linked immunosorbent assay (ELISA) instrument are used for detecting the absorption wavelength of 450nmTo determine cell viability and to plot the survival curves, the results are shown in FIG. 2d, where<0.001. Can be seen in CC-885 to Crbn^V380EMEF cells from homozygous mice are more sensitive than heterozygous MEF cells.

Example 4

The toxic effect of CC-885 is achieved by degrading GSPT1

The plasmid and cell strain construction method comprises the following steps:

construction of GSPT1 knockout plasmids and cell lines: the sgRNA sequence GATGCTGGCAAGTCAACCAT (SEQ ID No.9) corresponding to the GSPT1 gene was cloned into Celleca # SVCRU6T16-L vector (as per the instructions). The above vectors were co-infected with psPAX2 and VSVG plasmids into 293FT cells for packaging of the virus. The plasmid dosage during infection was: 3.75 μ g of SVCRU6T16 plasmid, 5.5 μ g of psPAX2 plasmid, 3.75 μ g of VSVG, and 1.2X 10 cell dose⁷Transfection reagents (EZ Trans reagents, Life iLAB Bio, Shanghai) were used in 55. mu.l, and after 60 hours virus was harvested to infect MiaPaCa2 and Panc1 cells (from ATCC, cultured in DMEM medium, 10% FBS, 1 Xpenicillin streptomycin double antibody). Fresh medium was changed 24 hours after virus infection. After one day of recovery, the cells were induced with 200ng/ml Dox for 3 days, and samples of the cells were harvested for expression detection of GSPT1 for subsequent experiments.

Connecting a codon-optimized GSPT1 sequence (synonymous mutation and GC content reduction) to a pHBLV vector, adding a FLAG label to the GSPT1 sequence during cloning, packaging viruses together with the pHBLV, psPAX2 and VSVG plasmid (the virus packaging method refers to the construction method of GSPT1 knockout viruses), infecting MiaPaCa2 and Panc1 cells, screening by using 1 mu g/ml puromycin, and collecting the expression condition of a cell detection gene. Construction of GSPT 1G 575N mutant: circular PCR was performed (TaKaRa,

max DNA Polymerase), primer sequence: G575N-F: 5"GACAAAAAATCAaatGAAAAAAGTAAGACCCGACCCC (SEQ ID No.10), G575N-R: TCattTGATTTTTTGTCTACCAAGCAGATTAA (SEQ ID No.11), PCR conditions were: 3min at 98 ℃, 35 cycles (30 s at 98 ℃, 30s at 55 ℃, 1min at 72 ℃), 1min at 72 ℃, dpn1(NEB) digestion for 2 hours after PCR, mutantsTransformed into STAB3 competent, single clones were picked and sequenced.

Various constructed cells were seeded into 96-well plates, 1000 cells/well. Adding CC-885 with different concentrations, detecting the 450nm absorption wavelength by a CCK-8 kit and a microplate reader after 72 hours to judge the cell activity, and drawing a curve, wherein the result is shown in figure 3. In the figure, KO _ GSPT1 represents a cell which overexpresses wild type GSPT1 in a GSPT1 gene knockout cell, KO _ GSPT 1G 575N represents a cell which overexpresses GSPT 1G 575N mutation in a GSPT1 gene knockout cell, NT _ GSPT1 represents a cell which overexpresses wild type GSPT1 in a CRISPR empty plasmid infected cell, NT _ GSPT 1G 575N represents an cell which overexpresses CRISPR empty plasmid, GSPT 1G 575N in a CRISPR infected cell, NT _ Vec represents an cell which infects CRISPR empty plasmid and overexpresses pHBLV empty vector, and KO _ Vec represents a cell which infects GSPT1 gene knockout cell and overexpresses pHBLV empty vector.

Previous studies have demonstrated that GSPT 1G 575N is not degradable by CC-885, but retains the biological function of wild-type GSPT 1. As shown in figure 3, when transferred to GSPT 1G 575N, the sensitivity of the cells to CC-885 was significantly reduced. This suggests that CC-885 is toxic by degrading GSPT 1. Mice that respond to CC-885 are therefore useful for studying the toxic side effects of GSPT1 degradation.

Example 5

CC-885 can degrade GSPT1 of mouse tissues

For wild type and Crbn^V380E/+Mice were injected intraperitoneally with 5mg/kg of CC-885, respectively, at Crbn^V380EMice were sacrificed at different time points by injection of CC-885 and spleen tissue harvested. Spleen tissues were lysed using RIPA and cryo-mill (shanghai Jingxin), and the protein samples obtained were immunoblotted to detect the expression of GSPT1, as shown in fig. 4.

As can be seen in FIG. 4, CC-885 resulted in a decrease in the abundance of GSPT1 expression in mice.

Example 6

Crbn-885 agonist^V380EGenotype mice blood pressure drop

Crbn^V380E(two Crbn)^V380E/+Heterozygous mice and two Crbn^V380E/V380EHomozygous mice) and Crbn^I391VMice (control group, 6 mice) were each intraperitoneally injected with 5mg/kg of CC-885, and blood pressure was measured at 10h, 20h, and 30h using a noninvasive blood pressure meter (Kent Scientific Corporation), and the results are shown in FIG. 5.

As can be seen from FIG. 5, Crbn^V380EMice developed a phenotype of significantly reduced blood pressure, whereas Crbn^I391VAfter the mice are injected with CC-885, the blood pressure is stable.

Example 7

CC-885 to Crbn^V380EToxicity in mice

To Crbn^V380E(two Crbn)^V380E/+Heterozygous mice and two Crbn^V380E/V380EHomozygous mice) and Crbn^I391V(control group, 6 mice) were injected with 5mg/kg of CC-885, and the death of the mice was observed and survival curves were plotted, and the results are shown in FIG. 6.

As can be seen from FIG. 6, Crbn^V380EMice died successively within CC-88548 h of injection, while Crbn^I391VNo abnormal phenotype was observed in mice intraperitoneally injected with CC-885.

Example 8

Crbn^V380EMouse intestinal tissue splicing Caspase3 expression is increased

For Crbn^V380EMice were dissected immediately at death (provided in example 6), while Crbn^I391VMice were maintained in all Crbn^V380EMice were dissected after death. Taking small intestine tissues for paraffin embedding and immunohistochemistry, wherein the kit used for immunohistochemistry is a hypersensitivity two-step method immunohistochemistry kit (Bioss, PV-0024), and the primary antibody is anti-cleared Caspase3(CST), and the result is shown in figure 7,

as can be seen from FIG. 7, Crbn^V380EIn tissue sections from small intestine, elevated expression of cleaved Caspase3 was observed, and in the figure, Scale bar was 50 μm.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

<110> Shanghai science and technology university

Application of <120> Crbn gene in construction of GSPT1 sensitive model

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 444

<212> PRT

<213> Mus musculus

<400> 1

Met Ala Gly Glu Gly Asp Gln Gln Asp Ala Ala His Asn Met Gly Asn

1 5 10 15

His Leu Pro Leu Leu Pro Asp Ser Glu Asp Glu Asp Asp Glu Ile Glu

20 25 30

Met Glu Val Glu Asp Gln Asp Ser Lys Glu Ala Arg Lys Pro Asn Ile

35 40 45

Ile Asn Phe Asp Thr Ser Leu Pro Thr Ser His Thr Tyr Leu Gly Ala

50 55 60

Asp Met Glu Glu Phe His Gly Arg Thr Leu His Asp Asp Asp Ser Cys

65 70 75 80

Gln Val Ile Pro Val Leu Pro Glu Val Leu Met Ile Leu Ile Pro Gly

85 90 95

Gln Thr Leu Pro Leu Gln Leu Ser His Pro Gln Glu Val Ser Met Val

100 105 110

Arg Asn Leu Ile Gln Lys Asp Arg Thr Phe Ala Val Leu Ala Tyr Ser

115 120 125

Asn Val Gln Glu Arg Glu Ala Gln Phe Gly Thr Thr Ala Glu Ile Tyr

130 135 140

Ala Tyr Arg Glu Glu Gln Glu Phe Gly Ile Glu Val Val Lys Val Lys

145 150 155 160

Ala Ile Gly Arg Gln Arg Phe Lys Val Leu Glu Leu Arg Thr Gln Ser

165 170 175

Asp Gly Ile Gln Gln Ala Lys Val Gln Ile Leu Pro Glu Cys Val Leu

180 185 190

Pro Ser Thr Met Ser Ala Val Gln Leu Glu Ser Leu Asn Lys Cys Gln

195 200 205

Val Phe Pro Ser Lys Pro Ile Ser Trp Glu Asp Gln Tyr Ser Cys Lys

210 215 220

Trp Trp Gln Lys Tyr Gln Lys Arg Lys Phe His Cys Ala Asn Leu Thr

225 230 235 240

Ser Trp Pro Arg Trp Leu Tyr Ser Leu Tyr Asp Ala Glu Thr Leu Met

245 250 255

Asp Arg Ile Lys Lys Gln Leu Arg Glu Trp Asp Glu Asn Leu Lys Asp

260 265 270

Asp Ser Leu Pro Glu Asn Pro Ile Asp Phe Ser Tyr Arg Val Ala Ala

275 280 285

Cys Leu Pro Ile Asp Asp Val Leu Arg Ile Gln Leu Leu Lys Ile Gly

290 295 300

Ser Ala Ile Gln Arg Leu Arg Cys Glu Leu Asp Ile Met Asn Lys Cys

305 310 315 320

Thr Ser Leu Cys Cys Lys Gln Cys Gln Glu Thr Glu Ile Thr Thr Lys

325 330 335

Asn Glu Ile Phe Ser Leu Ser Leu Cys Gly Pro Met Ala Ala Tyr Val

340 345 350

Asn Pro His Gly Tyr Val His Glu Thr Leu Thr Val Tyr Lys Ala Ser

355 360 365

Asn Leu Asn Leu Ile Gly Arg Pro Ser Thr Val His Ser Trp Phe Pro

370 375 380

Gly Tyr Ala Trp Thr Ile Ala Gln Cys Lys Ile Cys Ala Ser His Ile

385 390 395 400

Gly Trp Lys Phe Thr Ala Thr Lys Lys Asp Met Ser Pro Gln Lys Phe

405 410 415

Trp Gly Leu Thr Arg Ser Ala Leu Leu Pro Thr Ile Pro Glu Thr Glu

420 425 430

Asp Glu Ile Ser Pro Asp Lys Val Ile Leu Cys Leu

435 440

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

caccgacagt gcacagctgg tttcc 25

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctgtcacgtg tcgaccaaag gcaaa 25

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctgtcacgtg tcgaccaaag gcaaa 25

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aaaagcaccg actcggtgcc 20

<210> 6

<211> 190

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaagcgtcca acctgaatct gataggccgg ccttctactg agcacagctg gtttcccggg 60

taatacagct gtttactttt cttgttgact cttcatttag ttttagatga actttctagg 120

aagatacaaa acaaacagga caggaatagt ttgatcactt catgaatggg ttaaaagcag 180

ggacatgaga 190

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gcaacagaat atctgccaac ctc 23

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gccacttcca aagtaaaagg ga 22

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gatgctggca agtcaaccat 20

<210> 10

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gacaaaaaat caaatgaaaa aagtaagacc cgacccc 37

<210> 11

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcatttgatt ttttgtctac caagcagatt aa 32

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

acagtgcaca gctggtttcc cgg 23

Claims (10)

1. Use of Crbn gene in construction of GSPT1 sensitive cell model or GSPT1 sensitive animal model.
2. 2. The use of claim 1, wherein the Crbn gene V380E mutation is used in the construction of a GSPT1 sensitive cell model or a GSPT1 sensitive animal model.
3. 3. Use according to claim 1, wherein the cell model and/or animal model is derived from a mouse.
4. 4. A GSPT1 sensitive cell model, the Crbn gene of the GSPT1 sensitive cell model comprising a V380E mutation, the GSPT1 sensitive cell model derived from a mouse.
5. 5. The GSPT 1-sensitive cell model of claim 1, wherein the GSPT 1-sensitive cell model is derived from a combination of one or more of mouse visceral cells, mouse spleen cells, mouse liver cells, mouse embryonic fibroblasts;

   and/or, the GSPT1 sensitive cell model is derived from a humanized mouse.
6. 6. The method of constructing a GSPT1 sensitive cell model of any of claims 4-5, comprising:

   introduction of Crbn into cells^V380EMutations to provide the model of GSPT1 sensitive cells described above, which were derived from mice.
7. 7. The method of claim 6, wherein Crbn is introduced into the cells as a model of GSPT1 sensitive cells^V380EThe method of mutation is selected from the group consisting of by Crbn^V380EProviding Crbn in a mutated animal model^V380EA mutant cell;

   and/or, the cell is derived from one or more of mouse visceral cells, mouse spleen cells, mouse liver cells and mouse embryo fibroblasts.
8. 8. A method for constructing a GSPT1 sensitive animal model comprises the following steps:

   introduction of Crbn in mice^V380EMutations to provide the aforementioned GSPT1 sensitive animal model.
9. 9. The method of constructing a GSPT1 sensitive animal model of claim 8, wherein Crbn is introduced into mice^V380EThe mutation method is selected from CRISPR Cas9 geneEditing technology;

   preferably, the method comprises the following steps:

   mixing Crbn^V380EIntroducing mutation into mouse fertilized egg cells;

   breeding and/or screening to provide a GSPT1 sensitive animal model.
10. 10. A method of screening for a drug candidate for a GSPT1 degrader comprising:

    administering a candidate drug to the GSPT1 sensitive cell model of any one of claims 4-5 or the GSPT1 sensitive animal model obtained by the construction method of the GSPT1 sensitive animal model of any one of claims 8-9;

    preferably, the method further comprises the following steps:

    screening for candidate drugs for a GSPT1 degradant by changes in the GSPT1 sensitive cell model and/or the GSPT1 sensitive animal model to which the candidate drug was administered.

Images