CN119351470A

CN119351470A - Construction method and application of humanized COL3A1 gene mouse model

Info

Publication number: CN119351470A
Application number: CN202411965387.4A
Authority: CN
Inventors: 史培良; 吴正中; 陈曦; 吴俊�; 王雪
Original assignee: Chengdu Yaokang Biotechnology Co ltd
Current assignee: Chengdu Yaokang Biotechnology Co ltd
Priority date: 2024-12-30
Filing date: 2024-12-30
Publication date: 2025-01-24
Anticipated expiration: 2044-12-30
Also published as: CN119351470B

Abstract

The application belongs to the field of animal models, and particularly relates to a construction method and application of a humanized COL3A1 gene mouse model. The application is based on a gene editing means to insert the humanized COL3A1 gene at fixed points, fertilized eggs with the mutation can develop into a humanized COL3A1 gene mouse model, and provides a favorable model for researching the structure and biological effects of type III collagen, related diseases such as skin, heart and tissue fibrosis, related medicine, cosmetics and pharmaceutical evaluation of the diseases.

Description

Construction method and application of humanized COL3A1 gene mouse model

Technical Field

The application belongs to the field of animal models, and particularly relates to a construction method and application of a humanized COL3A1 gene mouse model.

Background

Collagen (Collagen) is the most abundant protein in humans, accounting for about one third of the protein composition, and has at least 16 distinct subtypes. Type III collagen was first discovered and described in 1971 in a prior study by Miller EJ et al. Type III collagen is encoded in humans by the COL3A1 gene. Collagen interacts with cells or cell surface receptors and regulates their adhesion, migration, proliferation and differentiation mainly through multiple receptor families. Collagen has a broad tissue distribution and thus plays a major role in physiological and pathological processes.

Type III collagen is one of the members of fibrillar collagen, can be widely expressed in various elastic human tissues, including dermis, ligament, blood vessel, gastrointestinal tract, uterus and various internal organs such as lung and liver, and is about 5% -20% of the total collagen content of human body, and is widely considered as an important structural protein by researchers. Among the numerous collagen family members, type III collagen has been found to possess critical homeostatic properties, such as maintaining the structural integrity of various organs and tissues. In addition to its steady state properties, abnormal expression of type III collagen has been demonstrated in a variety of pathologies. Type III collagen supports tissue and wound healing, participates in normal brain development during embryonic development and embryogenesis, acts as a modifier of fibril network, participates in platelet aggregation, initiates blood clotting cascade, mediates immune response, participates in inflammatory processes, and the like.

In the past studies, the effect of type III collagen on cell biology and the association of type III collagen with human diseases have been the focus of substantial research. Type III collagen was originally described as the major structural component of hollow organs. After a while, type III collagen has also been reported to be expressed in other tissues such as bone, gall bladder, placenta, bladder, gastrointestinal tract, fat, heart and endometrium. In addition, differential expression of type III collagen in various diseases is also revealed, and although a series of researches are developed for biological effects of type III collagen at present, the research of type III collagen in various diseases has the problem of poor accuracy due to the difference between COL3A1 amino acid sequences in animals and corresponding proteins in human beings, and the research cannot be attached to human diseases.

Therefore, the development of a humanized COL3A1 gene mouse model is of great importance, and the application is particularly proposed in view of the fact.

Disclosure of Invention

Based on the above, an embodiment of the application provides a construction method and application of a humanized COL3A1 gene mouse model.

The application also provides a construction method of a humanized COL3A1 gene mouse model, which comprises the following steps:

Knocking in a human COL3A1 gene at an H11 site of a chromosome 11 of a mouse fertilized egg by utilizing a gene editing technology to construct a recombinant fertilized egg, wherein the sequence of the human COL3A1 gene is shown as SEQ ID NO. 1.

And (3) introducing the recombinant fertilized eggs into a surrogate mouse for inoculation, and selecting positive mice from the young mice to obtain F0 mice.

The F0 generation mice are mated with wild mice, and positive heterozygote F1 generation mice are screened from the young mice to be used as humanized COL3A1 gene mouse models.

In some of these embodiments, the gene editing technique comprises one or more of BE3, CRISPR/Cas9, TALEN, and ZFN.

In some of these embodiments, constructing a recombinant fertilized egg comprises:

cloning the DNA sequence of gRNA shown as SEQ ID NO.2 into a vector to construct a recombinant plasmid.

And carrying out PCR amplification by taking the recombinant plasmid as a template to obtain the template, and carrying out transcription on the template to obtain the gRNA.

And co-injecting the gRNA, the Cas9 protein and/or mRNA of the Cas9 protein and a homologous recombinant vector containing a human COL3A1 gene into a mouse fertilized egg to construct a recombinant fertilized egg.

In some of these embodiments, the human COL3A1 gene homologous recombination vector is Col3A1-hCOL A1-WPRE-PolyA.

In some embodiments, the step of screening comprises PCR amplification of genomic DNA of the birth pups, detection of the amplified products and screening of a humanized COL3A1 gene mouse model based on the detection results.

The sequence of the PCR amplified primer is shown as SEQ ID NO. 3-SEQ ID NO. 8.

The application also provides a tissue or organ of the humanized COL3A1 gene mouse model constructed by the method for constructing the humanized COL3A1 gene mouse model.

The application provides an application of a humanized COL3A1 gene mouse model in screening and safety evaluation of medicines, medical instruments or cosmetics.

In some embodiments, the medicine comprises one or more of collagen medicines and anti-fibrosis medicines, and the medical instrument or the cosmetic comprises one or more of collagen fillers, injection and smearing preparations.

In another aspect, the application provides a gRNA of the H11 site of a target mouse, wherein the gRNA of the H11 site of the target mouse is shown as SEQ ID NO. 2.

In another aspect, the application provides a humanized COL3A1 gene editing system comprising a gRNA targeting the H11 site of a mouse, a homologous recombination vector, and a Cas9 protein and/or mRNA of the Cas9 protein.

The homologous recombination vector is Col3A1-hCOL A1-WPRE-PolyA.

The application is based on a gene editing means to insert the humanized COL3A1 gene at fixed points, and fertilized eggs with the mutation can develop into hCOL A1 gene mouse models, thereby providing a favorable model for researching the structure and biological effects of III type collagen, and screening and evaluating the safety of tissue fibrosis, skin and heart diseases and related pharmaceutical instruments.

Additional features, objects, and advantages of the application will be apparent from the description and claims, and from one or more embodiments of the application will be set forth in the description which follows.

Drawings

In order to more clearly illustrate the technical solution in the embodiments of the present application and to more fully understand the present application and its advantageous effects, the following brief description will be given with reference to the accompanying drawings, which are required to be used in the description of the embodiments. It is evident that the figures in the following description are only some embodiments of the application, from which other figures can be obtained without inventive effort for a person skilled in the art.

FIG. 1 is a schematic view of a model construction strategy according to the present application;

FIG. 2 is a diagram of a plasmid vector according to an embodiment of the present application;

FIG. 3 is a model building genotyping strategy of the application;

FIG. 4 shows the genotyping results of F0 mice of hCOL A1 mice model of the application;

FIG. 5 shows the genotyping results of F1-generation mice of hCOL A1 mouse model of the application;

FIG. 6 shows the detection result of human COL3A1 gene in hCOL A1 mice of the present application;

FIG. 7 is a graph showing neck ulcers in WT mice and hCOL A1 mice after two weeks of subcutaneous injection of recombinant human collagen in accordance with the present application;

FIG. 8 shows the test results of hCOL A1 mice test sample of the present application.

Detailed Description

The present application will be described in further detail with reference to embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order that the present disclosure may be more thorough and complete. It will also be appreciated that the present application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but may be modified or altered by persons skilled in the art without departing from the spirit of the application, and equivalents thereof are also intended to fall within the scope of the application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the application, it being understood that the application may be practiced without one or more of these details.

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 application belongs.

Terminology

Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:

The term "and/or," "and/or," as used herein, includes any one of two or more of the listed items in relation to each other, as well as any and all combinations of the listed items in relation to each other, including any two of the listed items in relation to each other, any more of the listed items in relation to each other, or all combinations of the listed items in relation to each other. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical schemes of all "logical or" connections), also include any and all combinations of A, B, C, D, i.e., the combinations of any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical schemes of all "logical and" connections).

The terms "plurality", "plural", "multiple", and the like in the present application refer to, unless otherwise specified, an index of 2 or more in number. For example, "one or more" means one kind or two or more kinds.

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

The "suitable" in the "suitable combination manner", "suitable manner", "any suitable manner" and the like herein refers to the fact that the technical scheme of the present application can be implemented, the technical problem of the present application is solved, and the technical effect expected by the present application is achieved.

In the present application, "further", "still further", "particularly" and the like are used for descriptive purposes to indicate differences in content but should not be construed as limiting the scope of the application.

In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.

In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.

In the present application, a numerical range (i.e., a numerical range) is referred to, and optional numerical distributions are considered to be continuous within the numerical range and include two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range and each numerical value between the two numerical endpoints unless otherwise specified. Unless otherwise indicated, when a numerical range merely refers to integers within the numerical range, both end integers of the numerical range are included, as well as each integer between the two ends, herein, each integer is recited directly, such as t is an integer selected from 1-10, and t is any integer selected from the group of integers consisting of 1,2,3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or may vary within a predetermined temperature range. It should be appreciated that the constant temperature process described allows the temperature to fluctuate within the accuracy of the instrument control. Allows for fluctuations in a range such as + -5 deg.C, + -4 deg.C, + -3 deg.C, + -2 deg.C, + -1 deg.C.

In the present application,% (w/w) and wt% each represent weight percent,% (v/v) represents volume percent, and% (w/v) represents mass volume percent.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Unless otherwise indicated to the contrary by the intent and/or technical aspects of the present application, all references to which this application pertains are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in the present application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated. In the case of the cited documents, examples and preferred modes of the cited relevant technical features are also incorporated into the present application by reference, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description of the application in conflict with the description, the application is modified in light of or adaptive to the description of the application.

The term "hCOL A1/Human COL3A1", i.e., the humanized COL3A1 gene, hCOL A1 is a gene encoding a type III collagen alpha 1 chain in humans, which is all called collagen TYPE III ALPHA 1 chain. This gene is located on the long arm of chromosome 2 (2q32.2), and the genome coordinates are chr2:188974373-189012746. The hCOL A1 gene was about 38kb in length and contained 51 exons. The protein encoded by this gene is an important structural protein in the extracellular matrix, mainly in the more elastic tissues such as skin, lung, uterus, intestine and vascular system, and is usually co-present with type I collagen.

The term "gRNA", which is fully referred to as guide RNA, is a key component in the CRISPR-Cas9 system. In the CRISPR-Cas9 system, the gRNA consists of two parts, crRNA (CRISPR RNA) and tracrRNA (trans-ACTIVATING CRRNA). The crRNA and tracrRNA together constitute a gRNA that binds to human EMX1 gene DNA via a 20nt long guide RNA, guiding Cas9 endonuclease to create DNA double strand breaks at the fragment of interest. To simplify the experimental design and improve the stability of the gRNA, crRNA and tracrRNA can be joined together by genetic engineering means to form a one-way guide RNA (sgRNA). The design of the gRNA is critical to the success of the CRISPR-Cas9 system. In designing the gRNA, it is necessary to determine the sequence of the target gene and design it by an online tool or by itself according to the CDS sequence of the target gene. The gRNA designed needs to be complementary to the target DNA sequence in order to be able to specifically guide the Cas9 enzyme to a specific location in the genome. The 5' end of the gRNA has a section of anchoring region which is complementary with the pre-mRNA editing sequence in a special G-U pairing mode, and the anchoring sequence promotes the binding of the gRNA and target DNA.

The previous literature reports indicate that most homozygous mice with type III collagen knockouts die after several days of birth, and that very small parts of homozygous mice manifest severe brain abnormalities and developmental retardation as well as vascular Ehlers-Danlos syndrome (vEDS), with about six months of age, die from aortic or intestinal rupture, while heterozygous mice survive normally. The application firstly performs humanized replacement on the mouse COL3A1 gene, constructs an in-situ replacement COL3A1 mouse model, and aims to obtain a mouse model capable of expressing the human COL3A1 gene but not expressing the mouse COL3A1 gene, but after the mouse COL3A1 gene is replaced in situ, the mouse offspring cannot be obtained stably.

Based on this, the application provides a gene editing cell, the genome of which comprises a human COL3A1 gene inserted at fixed point, and the human COL3A1 gene has a fragment shown as SEQ ID NO. 1.

In a specific example, the gene editing cell comprises a vector of the human COL3A1 gene.

Alternatively, the gene editing cell comprises a gRNA as shown in SEQ ID No.2 that targets the H11 site of the mouse. Wherein, H11 is located on mouse chromosome 11, which is a safe site for inserting exogenous gene, and exogenous gene integrated into the site can be expressed stably and efficiently without destroying the function of endogenous gene.

gRNA:CTGAGCCAACAGTGGTAGTA(SEQ ID NO.2)

In a specific example, the gene editing cell comprises a vector of a human COL3A1 gene and a gene editing system of gRNA targeting the H11 site of the mouse.

Wherein the gene editing cells comprise mouse fertilized eggs and embryos.

In another aspect, the application provides a gRNA of the H11 site of the target mouse, wherein the gRNA of the H11 site of the target mouse is shown as SEQ ID NO. 2.

In another aspect, the application provides a vector comprising a murine COL3A1 promoter, a kozak sequence, a human COL3A1 gene, a WPRE sequence, and a PolyA sequence.

The murine COL3A1 promoter refers to the gene promoter region encoding the type III collagen alpha 1 chain (Col 3 A1) in mice (Mus musculus). A promoter is a portion of a gene that is located upstream of the transcription initiation point and contains sequences that recognize and bind RNA polymerase, as well as sites for transcription factors that activate or repress transcription of the gene.

The Kozak sequence is a specific sequence flanking the initiation codon AUG on eukaryotic mRNA and has an important role in translation initiation. It is generally considered ACCACCAUGG, where the first base, G, after AUG and the third base, A or G, before AUG are critical for translation initiation. In addition, T is generally absent from the 15 bases upstream of AUG. The Kozak sequence is not a Ribosome Binding Site (RBS), but rather, in combination with a translation initiation factor, mediates translation initiation of mRNA containing a 5' cap structure. The Kozak sequence is used to ensure accurate and efficient translation of the gene.

The mechanism of action of the WPRE sequences includes improving polyadenylation of RNA, RNA nuclear transport and/or RNA translation. It is a powerful tool that can increase transgene expression. The WPRE sequence also increased mRNA, siRNA, shRNA and miRNA expression.

In a specific example, the vector is Col3A1-hCOL A1-WPRE-PolyA.

In another aspect, the application provides a humanized COL3A1 gene editing system comprising a gRNA targeting the H11 site of a mouse, a vector, a Cas9 protein, and mRNA of the Cas9 protein.

The CRISPR/Cas9 system consists of a recognition component CRISPR and a cutting component Cas, and can complete RNA-guided DNA recognition and editing. CRISPR/Cas9 technology systems use a sequence-specific guide RNA (sgRNA) molecule to guide endonucleases to target sites, thereby completing genome editing. For knockout/knock-in of different genes, sgrnas for the gene or corresponding sites need to be designed, thereby achieving an accurate gene editing effect.

In another aspect, the present application provides a method for constructing the gene editing cell, wherein the method comprises performing gene editing on an H11 site in a target cell, introducing the humanized COL3A1 gene editing system into the target cell, and preparing the gene editing cell by using the genome of the target cell to contain the inserted humanized COL3A1 gene, wherein the target cell comprises a mouse fertilized egg.

The application also provides a construction method of hCOL A1 gene mouse model, which comprises the following steps:

Knocking in a human COL3A1 gene at an H11 site of a chromosome 11 of a fertilized egg of a C57BL/6 mouse by utilizing a gene editing technology to construct a recombinant fertilized egg, wherein the sequence of the human COL3A1 gene is shown as SEQ ID NO. 1.

The F0 generation mice are mated with C57BL/6 mice, and positive heterozygote F1 generation mice are screened from the young mice to be used as humanized COL3A1 gene mouse models. It is understood that transplantation includes, but is not limited to, microinjection.

In one specific example, the gene editing technique includes one or more of BE3, CRISPR/Cas9, TALEN, and ZFN.

In a specific example, constructing a recombinant fertilized egg comprises cloning the DNA sequence of the gRNA shown in SEQ ID NO.2 into a vector to construct a recombinant plasmid;

Performing PCR amplification by taking the recombinant plasmid as a template to obtain a template, and transcribing the template to obtain gRNA;

And co-injecting the gRNA, the Cas9 protein and/or mRNA of the Cas9 protein and a homologous recombination vector containing a humanized COL3A1 gene into a mouse fertilized egg to construct a recombinant fertilized egg.

In a specific example, the step of screening comprises PCR amplification of genomic DNA of the birth pups, detection of the amplified products and screening of a humanized COL3A1 gene mouse model based on the detection.

In a specific example, the sequence of the PCR primer is shown as SEQ ID NO. 3-SEQ ID NO. 6.

In another aspect, the application provides a tissue or organ comprising a gene editing cell or a tissue or organ of a humanized COL3A1 gene mouse model constructed by the method of constructing a humanized COL3A1 gene mouse model.

The application also provides the gene editing cell, the vector, the humanized COL3A1 gene editing system, the hCOL A1 gene mouse model constructed by the method for constructing the hCOL A1 gene mouse model and the application of the tissue or organ in screening medicines, medical instruments or cosmetics and evaluating safety.

The clinical comprehensive evaluation of the medicine comprises evaluation of the medicine in multiple dimensions of safety, effectiveness, economy, innovation, suitability and the like. The safety evaluation mainly focuses on safety information before and after the medicine is marketed, including medicine instruction content, adverse event and adverse reaction information, medicine quality and curative effect stability.

In a specific example, the medicine comprises one or more of collagen medicines and anti-fibrosis medicines, and the medical instrument or the cosmetic comprises one or more of collagen fillers, injection and smearing preparations.

Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to the guidelines given in the present application, and may be according to the experimental manual or conventional conditions in the art, the conditions suggested by the manufacturer, or the experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

It should be understood that, in various embodiments of the present application, the sequence numbers of the foregoing processes do not mean the order of execution, and the order of execution of the processes should be determined by the functions and internal logic thereof, and should not constitute any limitation on the implementation process of the embodiments of the present application.

Example 1

The embodiment provides a method for constructing a humanized mouse model modified by COL3A1 genes, which specifically comprises the following steps:

1. a specific construction design strategy diagram is shown in fig. 1.

Referring to FIG. 1, FIG. 1 shows site-directed insertion of a fragment of interest at the H11 site in a wild-type mouse.

2. Determination of sequence of human fragment insertion

In this example, mCol A1-hCOL A1-WPRE-PolyA gene fragment was inserted at the site of H11 in mice, and the inserted fragment was about 8.2kb in length. According to the structure and function of human COL3A1, the sequence information after recombination is shown in figure 2, and the inserted hCOL A1 sequence is shown in SEQ ID NO. 1.

3. The mCol A1-hCOL A1-WPRE-poly a gene fragment was site-directed inserted into the H11 site of mice using CRISPR CAS a, CDS length 4401nt, encoding 1466aa. H11 is located on mouse chromosome 11, is a safe site for inserting exogenous gene, and exogenous gene integrated into the site can be expressed stably and efficiently without destroying the function of endogenous gene.

4. Vector construction

According to the design scheme, gRNA is designed, constructed and transcribed in vitro, and a homologous recombination vector (Donor vector) is constructed at the same time, and the correctness of the vector sequence is verified by sequencing, wherein the gRNA sequence information is shown in table 1:

TABLE 1 gRNA information

5. Microinjection

Microinjection of CRISPR/Cas9 system and Donor vector samples into fertilized eggs of mice with C57BL/6 background, transplanting the fertilized eggs surviving after injection into pseudopregnant female mice, and waiting for pregnancy.

6. Identification of F0 mice

The F0 generation mice generated by the recipient mice are cut at the age of 5-7 days and numbered by cutting the tail and the toe, genome DNA is extracted, two pairs of primers are respectively used for carrying out two-end PCR identification after middle targeting on the obtained rat tail genome DNA of the F0 mice, the primers GJS 022022076642-01-mCol 3a1-5tR1/H11-tF2 are respectively positioned outside a 5 '-end homology arm and inside a humanized fragment of the donor, if the pair of primers are amplified to generate PCR products, the target donor is effectively inserted at the 5' -end of the genome of the mice, and the BGH-pA-tF1/H11-tR2 is respectively positioned inside a humanized fragment of the donor outside a3 '-end homology arm, if the pair of primers are amplified to generate PCR primers, the target donor is effectively inserted at the 3' -end of the genome of the mice. The results were verified as:

Wild type ①② PCR reactions did not yield a Target band, ③ PCR reactions allowed for a single WT band.

Heterozygotes ①② PCR reactions gave a Target band and ③ PCR reactions gave a single WT band.

The Target band was obtained by ①② PCR reaction on homozygote, and the single WT band was not obtained by ③ PCR reaction.

Specific identification strategies are shown in FIG. 3, and identification methods are shown in tables 2 to 5. The 5 'and 3' identification electrophoreses of COL3A1-KI-target F0 mice are shown in FIG. 4.

The COL3A1-KI-target F0 mice were identified as positive in 5 'and 3' of the human COL3A1 gene, and the mice were identified as positive in sequence, as indicated by no mutation, no. 19, no. 21, no. 25, no. 43, no. 49, no. 51, no. 53, no. 55, no. 65, no. 77, and No. 80.

TABLE 2

The specific PCR reaction system is shown in Table 3:

TABLE 3 Table 3

The sequencing primer sequences are shown in table 4:

TABLE 4 Table 4

The PCR reaction conditions are shown in Table 5:

TABLE 5

7. Breeding of positive F0 mice

Mating the positive F0 generation mice with wild background mice after sexual maturity, cutting tails and cutting toe numbers of the born F1 generation mice at 5-7 days old, extracting genome DNA, carrying out PCR and sequencing identification, and confirming genotype.

8. F1 generation mouse identification results

The 5 'and 3' identification electrophoreses of COL3A1-KI-target F1 mice are shown in FIG. 5. The identification results show that the identification of the 5 'end and the 3' end of the mouse humanized COL3A1 gene with the number of 51 and 53 is positive, and the sequencing has no mutation, so that the obtained mice are positive mice for correctly carrying out gene recombination. Wherein, WT is C57BL/6, negative control, N is positive blank control, no template control, P is positive control, the number is the rat tail number, M is DNA MARKER, TRANS 2K PLUS II band is 5000bp, 2000bp,1000bp,750bp,500bp,250bp,100bp.

Example 2 test of hCOL A1 Gene mouse model constructed according to the present application verifies that:

1. Detection of humanized COL3A1 Gene in humanized COL3A1 mice

Heart, liver, spleen, lung, kidney, bladder, stomach, fat and skin tissues of WT mice and hCOL A1 mice were taken and expression of the human COL3A1 gene was detected by real-time fluorescent quantitative PCR. Real-time fluorescent quantitative PCR detection was performed on WT mice and hCOL A1 mice using the Human COL3A1 primer, and the results are shown in FIG. 6.

The results showed that no expression of hCOL A1 gene was detected in the WT mice and hCOL A1 gene was detected in the hCOL A1 mice, indicating successful establishment of the humanized COL3A1 mice.

2. Test of COL3A1 humanized mouse test

The application carries out drug effect test on human hCOL A1 mice and WT mice, subcutaneous injection of recombinant human collagen, human placenta matrix protein and solvent, collection of mouse peripheral blood after 30 days for detecting blood routine, flow cytometry for detecting spleen immune cell proportion of the mice, and analysis of immune response of the mice to the human collagen.

As shown in FIG. 7, after two weeks of subcutaneous injection of recombinant human collagen, WT mice developed ulcerations and crusts on the skin at the injection site of the cervical and dorsal parts, while hCOL A1 mice did not develop significant adverse reactions.

Blood routine data show that, as shown in figure 8, compared with the corresponding vehicle-injected mice, after 30 days of subcutaneous injection of recombinant human collagen, the NEU% of WT mice is significantly reduced, LYM% is significantly increased, WBC and MON% are not significantly changed, and the WBC, NEU, LYM and MON% of peripheral blood of hCOL A1 mice are not significantly changed. After 30 days of subcutaneous injection of human placental matrix protein, WT mice showed no significant changes in WBC, NEU, LYM, MON% from the peripheral blood of hCOL A1 mice.

Further, compared with the corresponding solvent-injected mice, the spleen cells are detected by flow cytometry, and after 30 days of subcutaneous injection of recombinant human collagen, the spleen M1 type macrophages, M2 type macrophages, CD4+ T cells and CD8+ T cells of the WT mice are not obviously changed, the M1 type macrophages of the hCOL A1 mice are obviously increased, and the proportions of the M2 type macrophages, the CD4+ T cells and the CD8+ T cells of the WT mice are not obviously changed. After 30 days of subcutaneous injection of human placenta matrix protein, the proportion of M1 type macrophages in spleen of WT mice is obviously reduced, the proportion of M2 type macrophages, CD4+ T cells and CD8+ T cells is not obviously changed, the proportion of M2 type macrophages in spleen of hCOL A1 mice is obviously reduced, and the proportion of M1 type macrophages, CD4+ T cells and CD8+ T cells is not obviously changed.

The data indicate that hCOL A1 mice and WT mice do not respond consistently to human collagen and differ from human collagen from different sources.

In conclusion, the hCOL A1 mouse provided by the application can express the humanized COL3A1 gene, and the safety of the product can be evaluated more scientifically.

The above examples merely illustrate a few embodiments of the present application, which are convenient for a specific and detailed understanding of the technical solutions of the present application, but should not be construed as limiting the scope of the claims. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Further, it is understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above teachings, and equivalents thereof are intended to fall within the scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art obtain technical solutions through logical analysis, reasoning or limited experiments, all of which are within the scope of protection of the appended claims. The scope of the patent of the application should therefore be determined with reference to the appended claims, which are to be construed as in accordance with the doctrines of claim interpretation.

Claims

1. A method for constructing a humanized COL3A1 gene mouse model, comprising:

The human COL3A1 gene was knocked into the H11 site of chromosome 11 of the mouse fertilized egg using gene editing technology to construct a recombinant fertilized egg; the sequence of the human COL3A1 gene is shown in SEQ ID NO.1;

The recombinant fertilized eggs are introduced into surrogate mice for gestation, and positive mice are selected from the born mice to obtain F0 generation mice;

The F0 generation mice were mated with wild-type mice, and positive heterozygous F1 generation mice were screened out from the newborn mice as humanized COL3A1 gene mouse models.

2. The method for constructing a humanized COL3A1 gene mouse model according to claim 1, characterized in that the gene editing technology includes one or more of BE3, CRISPR/Cas9, TALEN and ZFN.

3. The method for constructing a humanized COL3A1 gene mouse model according to claim 1, wherein constructing a recombinant fertilized egg comprises:

The DNA sequence of the gRNA shown in SEQ ID NO.2 was cloned into the vector to construct a recombinant plasmid;

Performing PCR amplification using the recombinant plasmid as a template to obtain a template, and transcribing the template to obtain gRNA;

The gRNA, Cas9 protein and/or mRNA of Cas9 protein and a homologous recombination vector containing the human COL3A1 gene are co-injected into mouse fertilized eggs to construct recombinant fertilized eggs.

4. The method for constructing a humanized COL3A1 gene mouse model according to claim 3, characterized in that the homologous recombination vector of the human COL3A1 gene is Col3a1-hCOL3A1-WPRE-PolyA.

5. The method for constructing a humanized COL3A1 gene mouse model according to any one of claims 1 to 4, characterized in that the screening step comprises: performing PCR amplification on the genomic DNA of the newborn mice, detecting the obtained amplification product and screening out the humanized COL3A1 gene mouse model according to the obtained detection result;

The sequences of the primers for PCR amplification are shown in SEQ ID NO.3 to SEQ ID NO.8.

6 . The tissue or organ of the humanized COL3A1 gene mouse model constructed by the method for constructing a humanized COL3A1 gene mouse model according to any one of claims 1 to 5 .

7. The method for constructing a humanized COL3A1 gene mouse model according to any one of claims 1 to 5, and use of the constructed humanized COL3A1 gene mouse model in the screening and safety evaluation of drugs, medical devices or cosmetics.

8. The use according to claim 7 is characterized in that the medicine includes one or more of collagen drugs and anti-fibrosis drugs; the medical device or cosmetic includes one or more of collagen fillers, injections and smear preparations.

9. A gRNA targeting the H11 site of mice, characterized in that the gRNA targeting the H11 site of mice is as shown in SEQ ID NO.2.

10. A humanized COL3A1 gene editing system, characterized in that the humanized COL3A1 gene editing system comprises: the gRNA targeting the H11 site of mice according to claim 9, a homologous recombination vector, and a Cas9 protein and/or mRNA of the Cas9 protein;

The homologous recombination vector is Col3a1-hCOL3A1-WPRE-PolyA.