CN114747541B

CN114747541B - Construction method and application of PSGL-1 humanized non-human animal model

Info

Publication number: CN114747541B
Application number: CN202210409835.7A
Authority: CN
Inventors: 杨志伟; 吴献贤; 刘星; 王智慧
Original assignee: Institute of Laboratory Animal Science of CAMS
Current assignee: Institute of Laboratory Animal Science of CAMS
Priority date: 2022-04-19
Filing date: 2022-04-19
Publication date: 2022-12-13
Anticipated expiration: 2042-04-19
Also published as: CN114747541A

Abstract

The invention discloses a construction method and application of a PSGL-1 humanized non-human animal model. The invention utilizes CRISPR/Cas9 technology to knock human PSGL-1 gene into the Rosa26loci site of a non-human animal at a fixed point so as to construct a non-human animal model capable of expressing human PSGL-1. Through related experiments, the invention successfully constructs a humanized PSGL-1 non-human animal model on the nucleic acid level and the protein level respectively. The non-human animal model can be used for researching the human PSGL-1 protein in the pathogenesis process of inflammation-related diseases, and the safety and effectiveness evaluation of humanized PSGL-1 antibody drugs are realized.

Description

Construction method and application of PSGL-1 humanized non-human animal model

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a construction method and application of a PSGL-1 humanized non-human animal model.

Background

Leukocyte binding and rolling are the initial steps in the development of an inflammatory response, and binding and rolling of inflamed vessels in vivo is mediated primarily by selectins (selectins). P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric, mucin-type glycoprotein ligand that is expressed persistently on the surface of all leukocytes (including monocytes, granulocytes, lymphocytes and some CD34+ stem cells) and is capable of binding to selectin. There are three subtypes of selectins, including P-selectin, E-selectin and L-selectin, where P-selectin is expressed on the surface of activated platelets and vascular endothelial cells, E-selectin is expressed on the surface of activated endothelial cells, and L-selectin is expressed on the surface of most leukocytes. PSGL-1 binds to three selectins, but the strongest P-selectin. After PSGL-1 is combined with activated vascular endothelial surface P-selectin/E-selectin, adhesion and rolling of leukocytes and vascular endothelium are started, infiltration of leukocytes to subintium is promoted, and secretion of inflammatory factors is promoted; PSGL-1 binds to P-selectin on the surface of activated platelets, leading to the formation of leukocyte-platelet complexes, further promoting the adhesion and infiltration of inflammatory cells; the combination of PSGL-1 and L-selectin on the surface of T cells can regulate the homing of T cells and promote the secretion of subsequent inflammatory factors.

The structure of human PSGL-1 protein includes a binding domain, a transmembrane domain and a cytoplasmic domain (PSGL-1 as a novel therapeutic target. Constantin G. Drug News Perspec 2004). Mucins form disulfide-bonded homodimers thereon. Human PSGL-1 is rich in serine, threonine and proline and contains 15 decamer repeats. Three NH of residues 46, 48 and 51 ₂ The terminal tyrosine is located in an anionic consensus sequence that favors tyrosine sulfation. Potential O-linked glycosylation sites are present in threonine 44, 57, 69 and 70. P-selectin binds to the N-terminus of PSGL-1 by stereospecific interaction with clustered tyrosine sulfate and nearby core 2O-glycans, with sialylated Lewis x (sLex) epitopes. Similarly, L-selectin binds with high affinity to the N-terminal region of PSGL-1 through a synergistic interaction with three sulfated tyrosine residues and an appropriately positioned C2-O-sLex O-glycan. Binding of E-selectin-PSGL-1 appears to be independent of sulfation, requiring fucosyltransferases to glycosylate sLex and PSGL-1. More and more researches show that PSGL-1 is closely related to the occurrence and development of inflammatory diseases, such as pancreatitis, colitis, ischemia-reperfusion injury, thrombosis, atherosclerosis and the like. PSGL-1 plays an important role in inflammation-related diseases.

Several antibodies against human PSGL-1 gene targets, such as Neihulizumab and SelK-2, have been developed. However, there was a large difference in the homology between the human PSGL-1 gene and the animal PSGL-1 gene. These antibodies or drugs directed against the target of the human PSGL-1 gene cannot recognize the target of the animal PSGL-1 gene, resulting in the inability to evaluate the effectiveness and safety of such antibodies using normal wild-type animals. Therefore, the PSGL-1 gene of a wild animal needs to be subjected to humanized transformation, so that the wild animal contains the human PSGL-1 gene and expresses the human PSGL-1 protein, and the wild animal PSGL-1 protein can be used for evaluating the in vivo effectiveness and safety of an antibody or a medicament.

Disclosure of Invention

Most of the studies on PSGL-1 are based on non-human animal models of PSGL-1 knockout, and the reports on humanized PSGL-1 non-human animal models are not found. The invention aims to provide an hPSGL-1 humanized non-human animal model and a construction method thereof, and the humanized non-human animal model can be used for researching human PSGL-1 protein in the pathogenesis process of inflammation-related diseases and realizing the safety and effectiveness evaluation of humanized PSGL-1 antibody drugs. The invention provides a mode for knocking in human PSGL-1 gene, constructs a non-human animal model capable of expressing human PSGL-1, and has reference value for realizing fixed-point knocking in other human genes.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a non-human animal model constructed based on CRISPR/Cas9 technology, which is a human PSGL-1 knock-in non-human animal model.

Further, the knock-in site of the non-human animal model is Rosa26 site.

Furthermore, the sequence of PSGL-1 is shown as SEQ ID NO. 1.

Further, the animal is a rodent.

Further, the rodent is a mouse.

Further, the mouse is a C57BL/6J mouse.

In a second aspect, the invention provides sgrnas for editing PSGL-1, wherein the sequences of the sgrnas are shown in SEQ ID nos. 3 to 13.

Furthermore, the sequence of sgRNA is shown in SEQ ID NO.3-4, SEQ ID NO. 10-13.

Furthermore, the sequence of sgRNA is shown in SEQ ID NO.3 and SEQ ID NO. 10.

In a third aspect of the invention, a targeting vector for expressing a human PSGL-1 gene is provided, the targeting vector comprising a 5 'homology arm, a human PSGL-1 gene, a stop codon, a WPRE element, poly A and a 3' homology arm.

Furthermore, the sequence of the PSGL-1 is shown as SEQ ID NO. 1.

In a fourth aspect, the invention provides a CRISPR/Cas9 system for site-directed knock-in of a human PSGL-1 gene at a Rosa26 site, the CRISPR/Cas9 system comprising the targeting vector of the third aspect of the invention.

In a fifth aspect, the present invention provides a method for constructing a human PSGL-1 non-human animal model by introducing PSGL-1 into the genome of a single-cell embryo or embryonic stem cell of a non-human animal to modify the genome of the non-human animal.

In some embodiments, the nucleic acid sequence is operably linked to an expression vector. The expression vectors are completely commercially available at present, such as some viral vectors, plasmids, and phages.

As an alternative embodiment, the expression vector can be introduced into cells by a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, or binding to a cell membrane-permeable peptide.

Further, the method comprises the steps of:

(1) Constructing a targeting vector;

(2) Preparing sgRNA and Cas9 mRNA;

(3) Mixing the targeting vector, cas9 and sgRNA, injecting the mixture into fertilized egg cells, and transplanting the fertilized egg cells into an oviduct of a pseudopregnant animal to obtain an F0 generation positive animal with human PSGL-1 knocked in;

(4) Hybridizing the positive animals of the F0 generation with wild animals to obtain positive heterozygous animals of the F1 generation;

(5) And selfing the F1 generation positive heterozygous animals to obtain a stably inherited human PSGL-1 non-human animal model.

Further, the knock-in site is the Rosa26 site.

Further, the genome sequence of Rosa26loci is shown in SEQ ID NO. 2.

Further, the targeting vector of step (1) comprises a 5 'homology arm, a human PSGL-1 gene, a stop codon, a WPRE element, poly A and a 3' homology arm.

Further, the sequence of the PSGL-1 is shown as SEQ ID NO. 1.

Further, the sgRNA sequence in step (2) is shown in SEQ ID NO. 3-13.

Furthermore, the sequence of sgRNA is shown in SEQ ID NO.3-4, SEQ ID NO. 10-13.

Furthermore, the sequence of sgRNA is shown in SEQ ID NO.3 and SEQ ID NO. 10.

Further, the method for identifying the knock-in human PSGL-1 animal is PCR identification.

Further, the sequences of primers used for identifying F0 positive animals by PCR are shown in SEQ ID NO. 14-19.

The sequence of the primer used for identifying the F1 generation positive animals by PCR is shown in SEQ ID NO. 16-19.

The sequences of primers used for identifying the human PSGL-1 non-human animal model with stable inheritance by PCR are shown in SEQ ID NO. 20-23.

According to a sixth aspect of the present invention, there is provided a method for obtaining a non-human animal into which a human PSGL-1 is knocked, wherein the human PSGL-1 knocked model animal constructed by the method according to the fifth aspect of the present invention is bred.

Further, offspring of the human PSGL-1 knock-in model animal were obtained.

Further, the animal is a rodent.

Further, the rodent is a mouse.

Further, the mouse is a C57BL/6J mouse.

The seventh aspect of the present invention provides a use of the method of the fifth aspect of the present invention or the model animal obtained by the method of the sixth aspect of the present invention in screening a medicament for preventing or treating inflammation-related diseases.

Further, the inflammation-related diseases include colitis, pancreatitis, abdominal aortic aneurysm, atherosclerosis.

An eighth aspect of the invention provides the use of a model animal obtained by the method of the fifth aspect of the invention or the method of the sixth aspect of the invention for the safety and efficacy assessment of a medicament.

Further, the drug is selected from antibody drugs.

The invention has the advantages and beneficial effects that: the invention provides an hPSGL-1 humanized non-human animal model and a construction method thereof, and the humanized non-human animal model can be used for researching human PSGL-1 protein in the pathogenesis process of inflammation-related diseases and realizing the safety and effectiveness evaluation of humanized PSGL-1 antibody drugs.

Drawings

FIG. 1 is a diagram showing the strategy of site-directed typing of a human PSGL-1 gene fragment;

FIG. 2 is a Cas9 in vitro cleavage result analysis diagram;

FIG. 3 is a gel electrophoresis analysis chart of F0 generation hPSGL-1 mice; wherein 3A is a gel electrophoresis chart for detecting a RD-KI-W32-2 (1-8) mouse transgenic strip, 3B is a gel electrophoresis chart for detecting a transgenic strip positive mouse RD-KI-W32-2 (2, 3,7,8) 5 'end and 3' knock-in strip, 3C is a gel electrophoresis chart for detecting a RD-KI-W32-3 (1-11, WT) mouse transgenic strip, 3D is a gel electrophoresis chart for detecting a transgenic strip positive mouse RD-KI-W32-3 (4,5,6) 5 'end and 3' knock-in strip, and 3E is a DNA strip indicator chart;

FIG. 4 is the F1 generation hPSGL-1 mouse gel electrophoresis analysis diagram; wherein, 4A is a figure for detecting 5 'end insertion by agarose gel electrophoresis, and 4B is a figure for detecting 3' end insertion by agarose gel electrophoresis;

FIG. 5 is a graph showing the relative expression levels of hSGL-1 mRNA in heart, spleen, lung and kidney tissues of different groups of mice;

FIG. 6 is a graph showing the expression levels of PSGL-1 protein in heart, liver, spleen and lung tissues of mice of different groups; wherein, 6A is a graph of the PSGL-1 protein expression level in heart tissues of different groups of mice; 6B is a graph of PSGL-1 protein expression levels in liver tissues of different groups of mice; 6C is a graph of the expression level of PSGL-1 protein in spleen tissues of different groups of mice; 6D is a graph of PSGL-1 protein expression levels in lung tissue of different groups of mice.

Detailed Description

The invention provides a mode of knocking in human PSGL-1 gene, and constructs a non-human animal model capable of expressing human PSGL-1.

PSGL-1 includes wild type, mutant or fragments thereof. The term encompasses full-length, unprocessed PSGL-1, as well as any form of PSGL-1 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of PSGL-1. The term encompasses, for example, the PSGL-1 gene, human PSGL-1 as well as PSGL-1 from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats). In a preferred embodiment, PSGL-1 is a human gene on chromosome 12q24.11 with gene ID 6404 in the present invention.

The term "knock-in" as used herein refers to a genetic modification produced by replacing genetic information encoded in a chromosomal locus with a different DNA sequence, that is, a technique of introducing a foreign functional gene (a gene whose genome is not present or has been inactivated) into a cell by homologous recombination with a homologous sequence in the genome, inserting the gene into the genome, and expressing the gene in the cell by homologous recombination.

The term "humanized" as used herein refers to a nucleic acid or protein whose structure (i.e., nucleotide or amino acid sequence) comprises portions that correspond substantially or identically to the structure of a particular gene or protein as found naturally in a non-human animal, and that also comprises portions that correspond more closely to equivalent structures found in the corresponding human gene or protein than are present in the particular non-human gene or protein of interest. In some embodiments, a "humanized" gene is a gene that encodes a polypeptide having substantially the amino acid sequence of a human polypeptide (e.g., a human protein or characteristic portion thereof).

The invention provides a method for constructing a human PSGL-1 non-human animal model, which modifies the genome of a non-human animal by introducing PSGL-1 into the genome of a single-cell embryo or embryonic stem cell of the non-human animal.

As used herein, the term "non-human animal model" refers to a non-human animal that has or displays characteristics of a disease or condition. By used as an animal model is meant any use of the animal for studying a disease or condition, such as for studying progression or development or response to a new or existing therapy.

As used herein, the term "non-human animal" includes non-human vertebrates, more preferably mammals, such as domesticated livestock (e.g., cattle, horses, pigs), pets (e.g., dogs, cats), or rodents.

As an alternative embodiment, the non-human animal is a rodent.

The term "rodent" refers to any and all members of a phylogenetic rodent (e.g., mouse, rat, squirrel, beaver, woodchuck, hamster, guinea pig, and guinea pig), including any offspring derived therefrom.

In some embodiments, rodents of the disclosure include, as non-limiting examples, mice, rats and hamsters. In some embodiments, rodents of the disclosure include, as non-limiting examples, mice and rats. In some embodiments, the rodent is selected from the superfamily murinus (Muroidea). In some embodiments, the rodents of the disclosure are from a family selected from the group consisting of: calomyidae (e.g. mouse-like hamsters), cricotidae (Cricetidae) (e.g. hamsters, new world rats and mice, voles), muridae (Muridae) (true mice and rats, gerbils, acanthos, coronaries), nesomyidae (Nesomyidae) (climbing mice, rock rats, tailed rats, madagassah rats and mice), cephalomyidae (placathoideae) (e.g. spiny squirrel) and Spalacidae (e.g. spalacina glaciens, bamboo rats and zokors). In some embodiments, the rodent of the present disclosure is selected from a true mouse or rat (muridae), a gerbil, an acantho, and a coronaria. In some embodiments, the mice of the present disclosure are from a member of the murine family (Muridae).

In a specific embodiment of the invention, the rodent is a mouse.

The term "embryonic stem cell" refers to a primitive (undifferentiated) cell derived from an embryo at a pre-implantation stage, which is capable of dividing in culture for a long period of time without differentiation, and is known to develop into cells and tissues of the three major germ layers.

The term "genome" generally refers to a complete set of genetic information in the form of one or more nucleic acid sequences, including textual or computer representations thereof. The genome may comprise DNA or RNA, depending on the organism from which it originates. Most organisms have a DNA genome, while some viruses have an RNA genome.

The term "genomic sequence" refers to a sequence present in a genome. Because RNA is transcribed from a genome, the term encompasses sequences that are present in the nuclear genome of an organism, as well as sequences that are present in cDNA copies of RNA (e.g., mRNA) transcribed from the genome.

In some embodiments of the invention, the method for constructing the non-human animal model of human PSGL-1 comprises the following steps:

(1) Constructing a targeting vector;

(2) Preparing sgRNA and Cas9 mRNA;

(4) Hybridizing the F0 generation positive animals with wild animals to obtain F1 generation positive heterozygous animals;

(5) And selfing the F1 generation positive heterozygous animals to obtain a human PSGL-1 non-human animal model with stable inheritance.

The term "zygote" describes a cell formed by the association of two gametes, which undergoes prokaryotic and prokaryotic fusion development and initiates first cell division, or more broadly, refers to a developing individual produced by a gamete.

The term "positive" means that the target marker is present in a large amount or at a high concentration compared to the amount or concentration of other wild-type markers as reference.

The present invention includes any art-available method for detecting the expression of the human PSGL-1 gene described herein. By "detecting expression" is meant determining the amount or presence of an RNA transcript of an intrinsic gene or an expression product thereof. Methods of detecting intrinsic gene expression, i.e., gene expression profiling, of the present disclosure include polynucleotide hybridization-based methods, polynucleotide sequencing-based methods, immunohistochemical methods, and proteomics-based methods. These methods generally detect the expression products (e.g., mRNA) of the intrinsic genes described herein. In a preferred embodiment, PCR-based methods, such as reverse transcription PCR (RT-PCR), and array-based methods, such as microarrays, are used. "microarray" refers to an ordered arrangement of hybridizable array elements, such as, for example, polynucleotide probes, on a substrate.

In a preferred embodiment of the present invention, the method for detecting the expression of the human PSGL-1 gene as described herein is PCR. The invention identifies whether the human PSGL-1 gene is knocked in by designing and synthesizing a primer aiming at the human PSGL-1.

The term "primer" refers to an oligonucleotide that is capable of hybridizing to (also referred to as "annealing") a nucleic acid and serving as a starting site for a nucleotide (RNA or DNA) polymerization reaction in a suitable buffer and at a suitable temperature under suitable conditions (i.e., in the presence of four different nucleoside triphosphates and a polymerization reaction reagent such as DNA or RNA polymerase or reverse transcriptase). The appropriate length of the primer depends on the intended use of the primer, but typically the primer is at least 7 nucleotides in length, more typically ranging from 10 nucleotides to 30 nucleotides, or even more typically from 15 nucleotides to 30 nucleotides in length. Other primers may be slightly longer, for example 30 to 50 nucleotides in length. In this context, "primer length" refers to the portion of an oligonucleotide or nucleic acid that hybridizes to a complementary "target" sequence and initiates nucleotide synthesis. Short primer molecules generally require cooler temperatures to form sufficiently stable hybridization complexes with the template. The primer does not necessarily reflect the exact sequence of the template but must be sufficiently complementary to hybridize with the template.

The invention provides application of the human PSGL-1 non-human animal model in screening medicaments for preventing or treating inflammation-related diseases.

In some embodiments, "inflammation-related disease" refers to any and all abnormalities associated with inflammation, including chronic and acute inflammatory diseases, including, but not limited to, immune-mediated inflammatory disease (IMID) and autoimmune diseases arthritis, glomerulonephritis, vasculitis, psoriatic arthritis, systemic Lupus Erythematosus (SLE), idiopathic Thrombocytopenic Purpura (ITP), psoriasis, still's disease (macrophage activation syndrome), uveitis, scleroderma, myositis, reiter's syndrome (Reiter's syndrome), and Wegener's syndrome. Other examples of conditions associated with inflammation include, but are not limited to, acne vulgaris, asthma, celiac disease, chronic prostatitis, hypersensitivity reactions, pelvic inflammatory disease, inflammatory bowel disease, reperfusion injury, sarcoidosis, transplant rejection, vasculitis, interstitial cystitis, crohn's disease, colitis, dermatitis, diverticulitis, hepatitis, parkinson's disease, atherosclerosis, alzheimer's disease, and cancer. In addition, chronic inflammatory diseases like rheumatoid arthritis, inflammatory bowel disease, psoriasis, and liver disease cause "pathological behavior" including fatigue, discomfort, and lack of social interest.

In the present invention, the term "prevention or treatment", wherein prevention refers to the complete or partial prevention or inhibition of symptoms of a disease or the frequency of such symptoms occurring, or to a reduction in the risk of acquiring a given symptom of a disease. In a specific embodiment of the invention, the disease is an inflammation-related disease. Prevention includes inhibiting or preventing symptoms associated with an inflammation-related disease, reducing the severity of symptoms associated with an inflammation-related disease, or ameliorating signs and symptoms associated with an inflammation-related disease, prevention includes inhibiting, preventing, or reducing the severity of symptoms associated with an inflammation-related disease, which term includes the effect that occurs before a patient begins to suffer from an inflammation-related disease or related disorder, i.e., delaying the onset of symptoms associated with an inflammation disease, or inhibiting or reducing the severity of symptoms associated with an inflammation disease; treatment refers to reducing or eliminating the severity of symptoms of an inflammatory disease, the frequency with which such symptoms occur, and the term includes the effect that occurs when a patient has an inflammatory disease or related condition, i.e., reducing the severity of one or more symptoms or effects of symptoms related to an inflammatory disease.

As an embodiment of the invention, the invention provides the use of the human PSGL-1 non-human model animal as described above in the evaluation of the safety and efficacy of a drug.

As a preferred embodiment, the drug is selected from the group consisting of antibody-based drugs.

The term "antibody" is meant to include whole antibody molecules, antigen-binding fragments, monovalent antibodies, and single chains thereof. Antibody molecules belong to the family of plasma proteins called immunoglobulins, the basic building blocks (immunoglobulin folds or domains) of which are used in various forms in many molecules of the immune system and other biological recognition systems. Natural antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH) consisting of three domains, CH1, CH2 and CH3, and a hinge region. The light chain constant region is aligned with the heavy chain first constant region (CH 1) and the light chain variable region is aligned with the heavy chain variable region, forming a so-called "Fab fragment". The heavy chains of CH1 and CH2 are separated from each other by a so-called hinge region which allows the Fab "arms" of the antibody molecule to rotate to some extent. The hinge region typically comprises one or more cysteines which are capable of forming a disulfide bridge with a cysteine of the hinge region of another heavy chain within the antibody molecule.

The term "antibody-based drug" as used in the present invention refers to a class of drugs consisting of antibody substances.

The invention is further illustrated below with reference to specific examples. It should be understood that the particular embodiments described herein are presented by way of example and not as limitations of the invention. The main features of the present invention can be applied to various embodiments without departing from the scope of the present invention.

Example 1 PSGL-1 humanized mouse model establishment

(1) Determining human source fragment typing-in position and human source sequence of typing-in

CDS of SELPLG-201 (ENST 00000228463.6) transcript corresponding to the mouse is selected to carry out knock-in of human-derived genes on the mouse, the knock-in position is Rosa26 site, the genome sequence of Rosa26loci is shown as SEQ ID NO.2, and the inserted human-derived PSGL-1 sequence is shown as SEQ ID NO. 1.

(2) Cas9/gRNA target design and activity detection

A sgRNA sequence (table 1) aiming at a target dna a oligonucleotide chain sequence is designed by using software, and a Cas9/gRNA target efficiency detection kit is used to detect the in vitro enzyme digestion activity of the gRNA target. Cas9 in vitro enzyme digestion results show that M-ROSA-L1 and M-ROSA-R3 have the highest activity (figure 2).

TABLE 1 sgRNA target sequences

(3) Construction of Donor plasmid

Constructing homology arms carrying target sites and an insertion sequence Donor vector, transforming the Donor vector into DH5a competent cells, carrying out amplification culture, extracting plasmids and purifying to obtain a Donor fragment product.

(4) Construction of F0-generation mice by microinjection of fertilized eggs

Cas9 mRNA synthesized by in vitro transcription, sgRNA and obtained donor segments are mixed and the concentration is adjusted, the mixture is injected into mouse fertilized eggs in a micro-injection way, then the fertilized eggs are transplanted into a female mouse oviduct of a pseudopregnant woman, and 19 mice of the F0 generation are born together after the F0 generation mice are born (table 2). After 2 weeks of birth of F0 mouse, the rat tail was cut, rat tail tissue DNA was extracted, target region primers (Table 3) were designed, genotype identification was performed by PCR, the PCR reaction system is shown in Table 4, and the PCR reaction program is shown in Table 5. Three positive mice of the F0 generation were obtained, namely RD-KI-W32-2-2#, RD-KI-W32-3-5#, RD-KI-W32-3-6# (FIG. 3).

TABLE 2 fertilized egg injection date, mouse birth date, and birth number

TABLE 3 F0 Generation mouse identification primers

TABLE 4 PCR reaction System

TABLE 5 PCR reaction procedure

(5) Establishment of stably inherited Rosa-SELPLG mouse strains

When the mice are 6-8 weeks old after the Rosa-SELPLG knock-in positive mice, the mice are mated with wild mice to obtain F1 generation mice. The F1 generation mice obtained by the primer pair in SEQ ID NO.16-19 are subjected to PCR identification. And (4) carrying out sequencing analysis on the mice identified as positive by PCR, wherein the clones which are correctly sequenced are positive F1 generation mice. The 2#,3# mice were positive F1 mice confirmed by PCR (fig. 4) and sequencing.

F1 generation positive heterozygous mice (PSGL-1 humanized gene knock-in mice) are selfed to obtain F2 homozygous mice.

Example 2 verification of the expression level of human PSGL-1mRNA in humanized PSGL-1 mice

1. Experimental methods

Tissue RNA extraction and RT-PCR: RNA was extracted from heart, liver, spleen, lung, and kidney tissues of F2 homozygous mice, total RNA was extracted using Trizol reagent (Invitrogen, CA), and PrimeScript RT Master Mix (RR 036A, taKaRa) was transcribed into cDNA. The cDNA was amplified with SYBR Green PCR Master Mix (Applied Biosystems, calif.) and detected using 7500 Fast Real-time PCR System Machine (Applied Biosystems, calif.) with amplification primers as shown in Table 6.

TABLE 6 F2 mouse generation amplification primers

2. Results of the experiment

The results are shown in FIG. 6, in which FIG. 6 shows the expression levels of human PSGL-1mRNA in heart, spleen, lung and kidney tissues of 4-month-old wild-type C57BL6J mice and humanized B6-hPSGL-1 mice, and the expression levels of PSGL-1mRNA are shown as RQ values, i.e., 2- Δ Ct. As can be seen from the figure, the wild-type mouse does not express the human PSGL-1mRNA, whereas the humanized PSGL-1 mouse highly expresses the PSGL-1 mRNA. Data are shown as Mean ± SD, N =3-5. The results show that the invention successfully constructs a humanized PSGL-1 mouse model from the nucleic acid level.

Example 3 verification of the expression level of human PSGL-1 protein in humanized PSGL-1 mice

1. Experimental methods

Tissue protein extraction and Western blot: total protein was extracted from heart, liver, spleen, lung, kidney tissues using RIPA buffer containing protease inhibitors (P0013B, byunnantian organisms), and the concentration was determined by BCA protein assay kit (P0010S, byunnantian organisms). Equal amounts of protein samples were loaded onto SDS-PAGE and then transferred to nitrocellulose membranes. After blocking with 5% skim milk, the membrane was incubated overnight at 4 ℃ with anti-human PSGL-1 protein (ab 78188, abcam) that only recognizes human, and then with horseradish peroxidase-labeled secondary antibody. Protein bands were imaged by the Tanon 5500 chemiluminescence imaging system.

2. Results of the experiment

The result of the Western blot experiment is shown in figure 6, and the B6-hPSGL-1 mouse highly expresses human PSGL-1 protein in heart, liver, spleen, lung and kidney tissues, while the C57BL/6J mouse does not express human PSGL-1 protein. Data are presented as Mean ± SD, N =3-5, p <0.01 (t-test), with gray scale values for PSGL-1 protein in C57BL/6J and B6-hPSGL-1 mouse heart tissue of 4.18, 57.42, respectively; the gray values of PSGL-1 protein in liver tissue are respectively 20.28 and 112.11; the gray scale values of spleen tissue PSGL-1 protein were 12.26 and 111.03, respectively, and the gray scale values of lung tissue PSGL-1 protein were 4.86 and 108.57, respectively. The results show that the invention successfully constructs a humanized PSGL-1 mouse model on the protein level.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, it is possible to make various improvements and modifications to the present invention, and those improvements and modifications will fall within the protection scope of the claims of the present invention.

Sequence listing

<110> institute of animal research for medical experiments of Chinese academy of medical sciences

<120> construction method and application of PSGL-1 humanized non-human animal model

<141> 2022-04-19

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1287

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggcagtgg gggccagtgg tctagaagga gataagatgg ctggtgccat gcctctgcaa 60

ctcctcctgt tgctgatcct actgggccct ggcaacagct tgcagctgtg ggacacctgg 120

gcagatgaag ccgagaaagc cttgggtccc ctgcttgccc gggaccggag acaggccacc 180

gaatatgagt acctagatta tgatttcctg ccagaaacgg agcctccaga aatgctgagg 240

aacagcactg acaccactcc tctgactggg cctggaaccc ctgagtctac cactgtggag 300

cctgctgcaa ggcgttctac tggcctggat gcaggagggg cagtcacaga gctgaccacg 360

gagctggcca acatggggaa cctgtccacg gattcagcag ctatggagat acagaccact 420

caaccagcag ccacggaggc acagaccact caaccagtgc ccacggaggc acagaccact 480

ccactggcag ccacagaggc acagacaact cgactgacgg ccacggaggc acagaccact 540

ccactggcag ccacagaggc acagaccact ccaccagcag ccacggaagc acagaccact 600

caacccacag gcctggaggc acagaccact gcaccagcag ccatggaggc acagaccact 660

gcaccagcag ccatggaagc acagaccact ccaccagcag ccatggaggc acagaccact 720

caaaccacag ccatggaggc acagaccact gcaccagaag ccacggaggc acagaccact 780

caacccacag ccacggaggc acagaccact ccactggcag ccatggaggc cctgtccaca 840

gaacccagtg ccacagaggc cctgtccatg gaacctacta ccaaaagagg tctgttcata 900

cccttttctg tgtcctctgt tactcacaag ggcattccca tggcagccag caatttgtcc 960

gtcaactacc cagtgggggc cccagaccac atctctgtga agcagtgcct gctggccatc 1020

ctaatcttgg cgctggtggc cactatcttc ttcgtgtgca ctgtggtgct ggcggtccgc 1080

ctctcccgca agggccacat gtaccccgtg cgtaattact cccccaccga gatggtctgc 1140

atctcatccc tgttgcctga tgggggtgag gggccctctg ccacagccaa tgggggcctg 1200

tccaaggcca agagcccggg cctgacgcca gagcccaggg aggaccgtga gggggatgac 1260

ctcaccctgc acagcttcct cccttag 1287

<210> 2

<211> 4529

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgcacgggga ttcccagtgc cggcgccagg ggcacgcggg acacgccccc tcccgccgcg 60

ccattggcct ctccgcccac cgccccacac ttattggccg gtgcgccgcc aatcagcgga 120

ggctgccggg gccgcctaaa gaagaggctg tgctttgggg ctccggctcc tcagagagcc 180

tcggctaggt aggggatcgg gactctggcg ggagggcggc ttggtgcgtt tgcggggatg 240

ggcggccgcg gcaggccctc cgagcgtggt ggagccgttc tgtgagacag ccgggtacga 300

gtcgtgacgc tggaaggggc aagcgggtgg tgggcaggaa tgcggtccgc cctgcagcaa 360

ccggaggggg agggagaagg gagcggaaaa gtctccaccg gacgcggcca tggctcgggg 420

gggggggggc agcggaggag cgcttccggc cgacgtctcg tcgctgattg gcttcttttc 480

ctcccgccgt gtgtgaaaac acaaatggcg tgttttggtt ggcgtaaggc gcctgtcagt 540

taacggcagc cggagtgcgc agccgccggc agcctcgctc tgcccactgg gtggggcggg 600

aggtaggtgg ggtgaggcga gctggacgtg cgggcgcggt cggcctctgg cggggcgggg 660

gaggggaggg agggtcagcg aaagtagctc gcgcgcgagc ggccgcccac cctccccttc 720

ctctggggga gtcgttttac ccgccgccgg ccgggcctcg tcgtctgatt ggctctcggg 780

gcccagaaaa ctggcccttg ccattggctc gtgttcgtgc aagttgagtc catccgccgg 840

ccagcggggg cggcgaggag gcgctcccag gttccggccc tcccctcggc cccgcgccgc 900

agagtctggc cgcgcgcccc tgcgcaacgt ggcaggaagc gcgcgctggg ggcggggacg 960

ggcagtaggg ctgagcggct gcggggcggg tgcaagcacg tttccgactt gagttgcctc 1020

aagaggggcg tgctgagcca gacctccatc gcgcactccg gggagtggag ggaaggagcg 1080

agggctcagt tgggctgttt tggaggcagg aagcacttgc tctcccaaag tcgctctgag 1140

ttgttatcag taagggagct gcagtggagt aggcggggag aaggccgcac ccttctccgg 1200

aggggggagg ggagtgttgc aatacctttc tgggagttct ctgctgcctc ctggcttctg 1260

aggaccgccc tgggcctggg agaatccctt ccccctcttc cctcgtgatc tgcaactcca 1320

gtctttctag aagatgggcg ggagtcttct gggcaggctt aaaggctaac ctggtgtgtg 1380

ggcgttgtcc tgcaggggaa ttgaacaggt gtaaaattgg agggacaaga cttcccacag 1440

attttcggtt ttgtcgggaa gttttttaat aggggcaaat aaggaaaatg ggaggatagg 1500

tagtcatctg gggttttatg cagcaaaact acaggttatt attgcttgtg atccgcctcg 1560

gagtattttc catcgaggta gattaaagac atgctcaccc gagttttata ctctcctgct 1620

tgagatcctt actacagtat gaaattacag tgtcgcgagt tagactatgt aagcagaatt 1680

ttaatcattt ttaaagagcc cagtacttca tatccatttc tcccgctcct tctgcagcct 1740

tatcaaaagg tattttagaa cactcatttt agccccattt tcatttatta tactggctta 1800

tccaacccct agacagagca ttggcatttt ccctttcctg atcttagaag tctgatgact 1860

catgaaacca gacagattag ttacatacac cacaaatcga ggctgtagct ggggcctcaa 1920

cactgcagtt cttttataac tccttagtac actttttgtt gatcctttgc cttgatcctt 1980

aattttcagt gtctatcacc tctcccgtca ggtggtgttc cacatttggg cctattctca 2040

gtccagggag ttttacaaca atagatgtat tgagaatcca acctaaagct taactttcca 2100

ctcccatgaa tgcctctctc ctttttctcc atttataaac tgagctatta accattaatg 2160

gtttccaggt ggatgtctcc tcccccaata ttacctgatg tatcttacat attgccaggc 2220

tgatatttta agacattaaa aggtatattt cattattgag ccacatggta ttgattactg 2280

cttactaaaa ttttgtcatt gtacacatct gtaaaaggtg gttccttttg gaatgcaaag 2340

ttcaggtgtt tgttgtcttt cctgacctaa ggtcttgtga gcttgtattt tttctattta 2400

agcagtgctt tctcttggac tggcttgact catggcattc tacacgttat tgctggtcta 2460

aatgtgattt tgccaagctt cttcaggacc tataattttg cttgacttgt agccaaacac 2520

aagtaaaatg attaagcaac aaatgtattt gtgaagcttg gtttttaggt tgttgtgttg 2580

tgtgtgcttg tgctctataa taatactatc caggggctgg agaggtggct cggagttcaa 2640

gagcacagac tgctcttcca gaagtcctga gttcaattcc cagcaaccac atggtggctc 2700

acaaccatct gtaatgggat ctgatgccct cttctggtgt gtctgaagac cacaagtgta 2760

ttcacattaa ataaataaat cctccttctt cttctttttt ttttttttaa agagaatact 2820

gtctccagta gaatttactg aagtaatgaa atactttgtg tttgttccaa tatggtagcc 2880

aataatcaaa ttactcttta agcactggaa atgttaccaa ggaactaatt tttatttgaa 2940

gtgtaactgt ggacagagga gccataactg cagacttgtg ggatacagaa gaccaatgca 3000

gactttaatg tcttttctct tacactaagc aataaagaaa taaaaattga acttctagta 3060

tcctatttgt ttaaactgct agctttactt aacttttgtg cttcatctat acaaagctga 3120

aagctaagtc tgcagccatt actaaacatg aaagcaagta atgataattt tggatttcaa 3180

aaatgtaggg ccagagttta gccagccagt ggtggtgctt gcctttatgc ctttaatccc 3240

agcactctgg aggcagagac aggcagatct ctgagtttga gcccagcctg gtctacacat 3300

caagttctat ctaggatagc caggaataca cacagaaacc ctgttgggga ggggggctct 3360

gagatttcat aaaattataa ttgaagcatt ccctaatgag ccactatgga tgtggctaaa 3420

tccgtctacc tttctgatga gatttgggta ttattttttc tgtctctgct gttggttggg 3480

tcttttgaca ctgtgggctt tctttaaagc ctccttcctg ccatgtggtc tcttgtttgc 3540

tactaacttc ccatggctta aatggcatgg ctttttgcct tctaagggca gctgctgaga 3600

tttgcagcct gatttccagg gtggggttgg gaaatctttc aaacactaaa attgtccttt 3660

aatttttttt ttaaaaaatg ggttatataa taaacctcat aaaatagtta tgaggagtga 3720

ggtggactaa tattaaatga gtccctcccc tataaaagag ctattaaggc tttttgtctt 3780

atacttaact ttttttttaa atgtggtatc tttagaacca agggtcttag agttttagta 3840

tacagaaact gttgcatcgc ttaatcagat tttctagttt caaatccaga gaatccaaat 3900

tcttcacagc caaagtcaaa ttaagaattt ctgactttta atgttaattt gcttactgtg 3960

aatataaaaa tgatagcttt tcctgaggca gggtctcact atgtatctct gcctgatctg 4020

caacaagata tgtagactaa agttctgcct gcttttgtct cctgaatact aaggttaaaa 4080

tgtagtaata cttttggaac ttgcaggtca gattctttta taggggacac actaagggag 4140

cttgggtgat agttggtaaa atgtgtttca agtgatgaaa acttgaatta ttatcaccgc 4200

aacctacttt ttaaaaaaaa aagccaggcc tgttagagca tgcttaaggg atccctagga 4260

cttgctgagc acacaagagt agttacttgg caggctcctg gtgagagcat atttcaaaaa 4320

acaaggcaga caaccaagaa actacagtta aggttacctg tctttaaacc atctgcatat 4380

acacagggat attaaaatat tccaaataat atttcattca agttttcccc catcaaattg 4440

ggacatggat ttctccggtg aataggcaga gttggaaact aaacaaatgt tggttttgtg 4500

atttgtgaaa ttgttttcaa gtgatagtt 4529

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cccatcttct agaaagactg g 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctggagttgc agatcacgag g 21

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgcagatcac gagggaagag g 21

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgcacccttc tccggagggg g 21

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agctgcagtg gagtaggcgg g 21

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcagtaaggg agctgcagtg g 21

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aacaggtgta aaattggagg g 21

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aggataggta gtcatctggg g 21

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

attgcttgtg atccgcctcg g 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tcgatggaaa atactccgag g 21

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gtctttaatc tacctcgatg g 21

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctgtccatgg aacctactac 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gatgagatgc agaccatctc 20

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cctcgtcgtc tgattggctc tc 22

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggacaggata agtatgacat catca 25

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gcatcgcatt gtctgagtag g 21

<210> 19

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cagatcaggc agagatacat agtg 24

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tgttgctgat cctactgggc 20

<210> 21

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cacagtggta gactcagggg t 21

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

aagaaggtgg tgaagcaggc 20

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

tccaccaccc agttgctgta 20

Claims

1. A non-human animal model constructed based on CRISPR/Cas9 technology is characterized in that the model is a human PSGL-1 knock-in non-human animal model;

the knock-in site of the non-human animal model is a Rosa26 site;

the sequence of PSGL-1 is shown as SEQ ID NO. 1;

the animal is a rodent;

the rodent is a mouse;

the mice are C57BL/6J mice.

2. An sgRNA used for editing PSGL-1, wherein the sequence of the sgRNA is shown in SEQ ID NO. 3-13.

3. The sgRNA of claim 2, wherein the sequence of the sgRNA is shown in SEQ ID nos. 3 to 4, 10 to 13.

4. The sgRNA of claim 3, wherein the sequence of the sgRNA is shown in SEQ ID No.3 and SEQ ID No. 10.

5. A targeting vector for expressing a human PSGL-1 gene, which is characterized by comprising a 5 'homology arm, a human PSGL-1 gene, a stop codon, a WPRE element, poly A and a 3' homology arm;

the sequence of the PSGL-1 is shown as SEQ ID NO. 1.

6. A CRISPR/Cas9 system for site-directed knock-in of a human PSGL-1 gene at Rosa26 site, wherein the CRISPR/Cas9 system comprises the targeting vector of claim 5.

7. A method for constructing a non-human animal model of human PSGL-1, wherein the method modifies the genome of a non-human animal by introducing PSGL-1 into the genome of a single-cell embryo or embryonic stem cell of the non-human animal;

the method comprises the following steps:

(1) Constructing a targeting vector;

(2) Preparing sgRNA and Cas9 mRNA;

(3) Mixing the targeting vector, cas9 mRNA and sgRNA, injecting the mixture into fertilized egg cells, and transplanting the fertilized egg cells into an oviduct of a pseudopregnant animal to obtain an F0 generation positive animal with human PSGL-1 knocked in;

(5) Selfing the F1 generation positive heterozygous animal to obtain a stably inherited human PSGL-1 non-human animal model;

the knock-in site is the Rosa26 site;

the genome sequence of Rosa26loci is shown as SEQ ID NO. 2;

the targeting vector in the step (1) comprises a 5 'homology arm, a human PSGL-1 gene, a stop codon, a WPRE element, poly A and a 3' homology arm;

the sequence of the PSGL-1 is shown as SEQ ID NO. 1;

the sgRNA sequence in the step (2) is shown in SEQ ID NO. 3-13.

8. The method according to claim 7, wherein the sgRNA sequences are shown in SEQ ID nos. 3-4, 10-13.

9. The method according to claim 8, wherein the sgRNA sequences are shown as SEQ ID No.3 and SEQ ID No. 10.

10. The method according to any one of claims 7 to 9, wherein the method for identifying a knock-in human PSGL-1 is PCR identification;

the sequence of the primer used for identifying the F0 generation positive animals by PCR is shown in SEQ ID NO. 14-19;

the sequence of the primer used for identifying the F1 generation positive animals by PCR is shown in SEQ ID NO. 16-19;

11. A method for obtaining a non-human animal into which PSGL-1 of human origin is knocked in, which comprises breeding a human PSGL-1 knocked-in model animal constructed by the method according to any one of claims 7 to 10.

12. The method of any one of claims 7-11, wherein the animal is a rodent;

the rodent is a mouse;

the mice are C57BL/6J mice.

13. Use of a model animal obtained by the method of any one of claims 7 to 12 for screening a medicament for preventing or treating an inflammation-related disease;

the inflammation related diseases include colitis, pancreatitis, abdominal aortic aneurysm, and atherosclerosis.