CN112280800A - Construct and application thereof in animal aging cell tracing and aging cell clearing - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a construct integrating functions of positioning, tracing, sorting, inducing apoptosis of senescent cells and/or clearing senescent cells, and a cell or animal model modified by the construct. The construct functions by targeting the P21 gene in the genome and inserting it in its proper location. The cells or animal models obtained by the method of the invention can be used for studying senescence and senescence-associated diseases.

Description

Construct and application thereof in animal aging cell tracing and aging cell clearing

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a construct and application thereof in animal senescence tracing and senescent cell elimination, and a cell or an animal model modified by the construct.

Background

Senescent cells refer to cells that exit the cell cycle, enter a non-dividing state, while maintaining cell function to some extent. The basic changes of senescent cells include swelling of the nucleus, increased nuclear to cytoplasmic ratio, changes in chromatin structure, decreased or lost ability of cells to replicate, decline of cell function, slowing of metabolism, etc. More typically characterized by positive staining with senescence-associated beta-galactosidase (SA-beta-Gal); the secretion Phenotype related to aging (SASP), namely secretion of various cytokines such as IL-1a, IL-1b, IL-6, TGF-beta 1, TNF-alpha, CCL5, CXCL10 and the like, appears. The detection indexes related to the cell senescence research comprise senescence-associated beta-galactosidase (SA-beta-gal), telomere length and telomerase activity, senescence-associated heterochromatin (SAHF), senescence-associated secretory phenotype, senescence pathway genes p53, p21 and p16INK4a and the like. SA- β -gal is the most commonly used marker of cellular senescence, but is not suitable as a tracer marker. By utilizing a lineage tracing technology, the aging cells are marked by the aging markers, and the activities such as proliferation, differentiation, migration and the like of the aging cells and the progeny cells thereof are tracked and observed, so that the method has important significance for developing the biological behavior research of the aging cells in development, aging and tumorigenesis.

Aging can occur throughout the life cycle, including the embryogenesis period. The number of senescent cells also increases with age, which also plays an important role in development and wound healing. Subjects in Zhongke department and park in Circulation, 2019, it was confirmed that CCN 1-induced senescent fibroblasts secrete a plurality of SASP cytokines. The senescent cells in the bodies of the aged mice are eliminated by utilizing the Senolytics drugs, so that the physiological state of the mice can be improved, and the life of the mice can be prolonged. Targeting aged bone marrow cells can increase the survival rate of leukemic mice. In recent years, a plurality of articles also prove that aged cells are closely related to bone aging, lumbar parashoulder disease and osteoarthritis regeneration. In the human body, part of organs suffering from chronic injury diseases, such as liver and kidney, have the existence of aged cells, and the existence of the aged cells has certain influence on the occurrence and development of tumors. The relationship between senescence and tumors is not clearly elucidated at present, and on the one hand it is believed that senescent cells recruit macrophages, activate the immune system and enhance their clearance of tumor cells. On the other hand, the existence of a large number of senescent cells can secrete a large number of senescent cell-associated secretory phenotype (SASP) factors, and a large number of inflammatory factors can promote the occurrence of tumors.

How to better explain tumorigenesis from the perspective of senescent cells, understanding the positive and negative roles that senescent cells play in different stages of tumorigenesis is a problem that needs to be solved in the art. In addition, many problems remain to be further researched on how to target and kill senescent cells without damaging adjacent healthy cells, how to block SASP negative factors and simultaneously retain the effects of positive factors, and how to popularize the research results of animal experiments to clinical diagnosis and treatment.

In order to better study the role and mechanism of aging in development, disease and tumor, researchers are trying to establish and study various aging animal models. However, in view of the complexity of the development of aging, the correlation and restriction of signal pathways in vivo is very complicated, and the causes, types and expression markers of aging cells have great differences, so that the research work is very difficult. Even if it is known that some genes have a correlation with senescence, it is not easy to successfully obtain a useful disease model or a useful anti-senescence method by modifying senescence-associated genes.

Disclosure of Invention

The invention aims to provide a genetically modified cell or animal model and application thereof in aging tracing and removing aging cells.

In a first aspect of the invention, there is provided an isolated construct, which is targeted to the P21 gene and comprises a reporter gene and a diphtheria toxin receptor gene; the constructs are capable of engineering the genome of a cell or animal, performing senescent cell localization, tracking, sorting, inducing apoptosis of senescent cells, and/or eliminating senescent cells in a cell or animal body.

In a preferred embodiment, the construct targets exon 3 of the P21 gene; preferably after the 47 th base of exon 3.

In another preferred embodiment, the reporter gene comprises a reporter gene selected from the group consisting of: luciferase gene (luciferase), tdTomato, green fluorescent protein Gene (GFP), enhanced green fluorescent protein coding gene, red fluorescent protein gene (RFP), beta-galactosidase coding gene, and the like; preferably, the reporter gene comprises: luciferase gene and tdTomato; more preferably, the luciferase is AkaLuc; more preferably, the construct comprises the following operably linked elements in order from 5 'to 3': AkaLuc, tdTomato, diphtheria toxin receptor gene.

In another preferred embodiment, the 5' end of AkaLuc, between AkaLuc and tdTomato, and between tdTomato and diphtheria toxin receptor gene, further comprises 2A sequence (ribosome skip sequence), P2A and/or T2A.

In another preferred embodiment, the diphtheria toxin receptor gene further comprises a terminator at the 3' end; preferably the terminator is poly a.

In another preferred embodiment, the construct further comprises an FLP recognition site (FRT); preferably, the FLP recognition sites are 2 and further include a positive selection marker gene (more preferably a Neo gene) therebetween: PGK-Neo and/or DDSDC-Neo.

In another preferred embodiment, the construct further comprises a herpes simplex virus thymidine kinase negative selection gene.

In another preferred embodiment, both ends of the construct, preferably upstream of the AkaLuc and downstream of the terminator (preferably 5 'of 2A and 3' of the positive selection marker gene), further comprise a 5 'homology arm sequence and a 3' homology arm sequence, respectively.

In another aspect of the invention, the use of the construct is provided for targeting the P21 gene, engineering the genome of a cell or animal, performing senescent cell localization, tracking, sorting, inducing apoptosis and/or eliminating senescent cells in a cell or animal body.

In another preferred embodiment, the construct is targeted in exon 3 of the P21 gene.

In another aspect of the invention, there is provided a cell having integrated into its genome said construct which is exogenous; preferably, the construct is integrated into the P21 gene of the cell, more preferably into exon 3 of the P21 gene; preferably, the cell comprises: embryonic stem cells, somatic cells, germ cells, or tumor cells.

In another preferred embodiment, the germ cells include fertilized eggs, oocytes, and sperm.

In another preferred embodiment, the somatic cell comprises: fibroblasts, epithelial cells, blood cells, cells of tissue or organ origin.

In another aspect of the invention, there is provided a method of engineering the genome of a cell, the method comprising: transfecting a cell with any of the constructs described above to obtain a cell having the exogenous construct integrated into its genome; preferably, the test cells comprise: embryonic stem cells, somatic cells, germ cells, or tumor cells; preferably, the germ cells include fertilized eggs, oocytes, sperm; preferably, the somatic cells comprise: fibroblasts, epithelial cells, blood cells, cells of tissue or organ origin.

In another aspect of the invention, there is provided a method of localizing (including fluorescently localizing), tracking, sorting, inducing apoptosis, and/or eliminating senescent cells, the method comprising:

(1) transfecting the construct into a test cell to obtain a cell with a foreign construct integrated in a genome;

(2) observing the expression of the reporter gene in the cell, thereby locating, tracking, sorting senescent cells, inducing apoptosis of the senescent cells and/or eliminating senescent cells;

preferably, the test cells comprise: embryonic stem cells, somatic cells, germ cells, or tumor cells; preferably, the germ cells include fertilized eggs, oocytes, sperm; preferably, the somatic cells comprise: fibroblasts, epithelial cells, blood cells, cells of tissue or organ origin.

In another preferred embodiment, the cell is an ex vivo cell.

In another preferred embodiment, the cells comprise primary or subcultured cell lines or cell strains.

In another preferred embodiment, the method is an ex vivo method.

In another preferred embodiment, the method of engineering the genome of a cell or animal is not for the direct purpose of diagnosis or therapy.

In another preferred embodiment, the method of localizing, tracking, sorting, inducing apoptosis and/or eliminating senescent cells is not for the direct purpose of diagnosis or treatment.

In another aspect of the invention, there is provided a method of preparing an animal in which senescent cells are able to be localized, traced, induced to undergo apoptosis and/or eliminated, the method comprising:

(1) introducing said construct into an embryonic stem cell or a germ cell to obtain an embryonic stem cell or germ cell having a genome into which said construct is integrated;

(2) using said embryonic stem or germ cells to produce an animal body that is capable of undergoing senescent cell tracking, or whose senescent cells are capable of being induced to undergo apoptosis;

preferably, the animal is a mammal comprising: a rodent; preferably, the rodent comprises a mouse or a rat (etc.).

In a preferred embodiment, the resulting animal is further capable of producing offspring capable of localizing, tracking or inducing apoptosis in senescent cells.

In another aspect of the invention, there is provided the use of said construct, said cell or an animal obtained by the method described above for the study of cellular senescence (preferably, not for diagnostic or therapeutic purposes), or for the preparation of a model for the study of cellular senescence.

In another preferred example, the model includes: an animal model or a cell (cell culture) model.

In another aspect of the invention, a kit for conducting aging studies is provided, comprising: a construct as described in any of the preceding; or a cell as described previously.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, schematic diagram of mouse construction strategy.

FIG. 2, plasmid map of homologous recombination vector.

FIG. 3 shows the electrophoresis chart of enzyme digestion identification of homologous recombination vector. 1: according to the EcoRI enzyme digestion identification result, the theoretical band sizes are 7548bp, 5363bp, 2240bp and 1891 bp; m: 1kb DNA ladder.

FIG. 4 shows PCR identification of electrophoresis of homologous recombination-positive ES cells. The number: an ES clone number; WT: a wild-type control; m: 1kb DNA marker.

FIG. 5 shows the results of luminescence detection of positive AkaLuc after aging induction with D-galactose for ES-positive clones.

FIG. 6, the electrophoresis verification chart of the PCR identification product of mouse generation F1.

FIG. 7 SA- β -Gal staining of aged skin fibroblasts of F2 generation mice and WT mice (upper panel); and the results of measurement of the mRNA expression levels of intracellular p21, Akalumin, tdTomato, DTR and p16 (lower panel).

FIG. 8 and H₂O₂tdTomato fluorescence and fluorescence intensity detection for aging induction.

FIG. 9, DTR expression assay in P21-ATD mouse cells.

FIG. 10 shows the results of luminescence measured by an IVIS in vivo optical imaging system after the skin fibroblasts of WT mice and P21-ATD mice are inoculated on a black-wall and bottom-penetrating 96-well plate for culture.

Figure 11, DT diphtheria toxin effectively induced apoptosis in aged mouse skin fibroblasts.

Detailed Description

The inventor of the invention has intensively studied and discloses a construct integrating the positioning, tracing, sorting, inducing apoptosis and/or eliminating senescent cells, and a cell or an animal model modified by the construct. The construct, which functions by targeting the P21 gene in the genome and inserting it in its proper location, contains a reporter gene and a diphtheria toxin receptor gene. The cell or animal model obtained by the method of the invention has multiple functions and can be used for researching aging and aging-related diseases.

Term(s) for

As used herein, a "construct" refers to a single-or double-stranded DNA molecule that has been artificially intervened to contain DNA segments that are combined and arranged according to sequences that do not occur in nature. The "construct" includes "(expression) vectors".

As used herein, "element" refers to a functional nucleic acid sequence, and in a preferred form of the invention, the "element" is systematically constructed to form a construct for gene targeting. The sequences of the "elements" may be those provided in the present invention, including variations thereof, provided that such variations substantially retain the function of the "elements".

As used herein, "homology arms" refer to DNA sequences homologous to a selected gene targeting site in the genome for recombination of a vector with the target site. After the gene targeting site is determined, the design of the homology arms can be made based on the sequence in the vicinity of the targeting site.

As used herein, the terms "operably linked", "operably linked" or "operably linked" refer to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed in a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the "animal" is an animal in which the P21cip1 gene or its homologous gene is present; preferably the animal is one which does not itself express Diphtheria Toxin Receptor (DTR). In some preferred embodiments, the animal comprises a rodent, such as a mouse or a rat.

As used herein, unless otherwise specified, the "cell" is a cell in which the P21cip1 gene or a homologous gene thereof is present; preferably the cell is one which does not itself express Diphtheria Toxin Receptor (DTR). The cells can be germ cells, embryonic stem cells and somatic cells; the cells may also be malignant/benign proliferating cells, such as tumors.

As used herein, an "embryonic stem cell" is a human or non-human mammalian embryonic stem cell. For example, embryonic stem cells that are non-human mammals, such as embryonic stem cells that are murine (e.g., mouse). The "embryonic stem cells" may be commercial or established lines of cells.

P21cip1 (abbreviation P21)

The present inventors have conducted a great deal of research work in order to achieve positioning, tracking, sorting, etc. of senescent cells and to establish good cell or animal models. By comparison and analysis, the present inventors determined that the P21 gene was targeted for cellular genome engineering. The inventor finds that the gene targeting construct is established, targets the 3 rd exon of the P21 gene, and is inserted into a construct at a specific site, so that the positioning, tracing and sorting of the senescent cells can be effectively realized, and meanwhile, the apoptosis of the senescent cells can be induced.

The human P21(P21Cip1(CDNK1A)) gene maps to the short arm of chromosome 6, and has a DNA length of 85kb and 3 exons, and a cDNA length of about 2.1 kb.

The P21 gene is conserved in animals, and the murine P21 gene is highly homologous to the human P21 gene. Transcript number Ensembl of murine P21 gene: p21-205(ENSMUST 00000233296.1.

It should be understood that "P21" of the present invention also includes various variants and homologues of the naturally occurring P21 Gene, and the specific variants can be obtained by query from the Gene database of NCBI, the Ensemble genome browser, the UCSC genome database, and uniport. Representative examples include: nucleotide sequence of P21 protein which is identical with wild type due to codon degeneracy, and nucleotide sequence of conservative variant polypeptide of wild type P21 protein.

P21 is involved in various functional activities of cells, its basic biological action includes the regulation of cell cycle, and it is a cell cycle inhibitor, its regulation includes P53 dependent regulation and P53 independent regulation. Furthermore, P21 has a correlation with the development of tumors, and is also considered to be an anti-cancer gene. Although P21 has been functionally studied in the prior art, the art has not successfully utilized it to achieve senescent cell localization, tracking, sorting, and/or induction of senescent apoptosis in view of the complexity of its function exerted within the cell.

Construct

The present invention provides a construct for gene targeting that targets the P21 gene, particularly exon 3 thereof (in exon 3), and comprises a reporter gene and a diphtheria toxin receptor gene; the constructs are capable of engineering the genome of a cell or animal, performing senescent cell localization, tracking, sorting, inducing apoptosis of senescent cells, and/or eliminating senescent cells in a cell or animal body. In a particularly preferred form of the invention, the construct is targeted for insertion after base 47 of exon 3.

In the invention, tracing and sorting are carried out through the reporter gene. This can be accomplished using a variety of reporter genes, including but not limited to luciferase gene, tdTomato, green fluorescent protein gene, enhanced green fluorescent protein coding gene, red fluorescent protein gene, beta-galactosidase coding gene, and the like. In a particularly preferred embodiment of the present invention, the reporter gene includes: luciferase gene and tdTomato; more preferably, the luciferase is AkaLuc.

The inventors have compared various reporter genes and found that AkaLuc is relatively optimal when used in the present invention, being able to work well and express well in conjunction with other elements of the constructs of the invention. The in vivo bioluminescence imaging (BLI) technique allows for the imaging and tracking of biological behavior in vivo in living tissues or small animals under non-invasive conditions, the most widely used fluorescein being D-luciferin (substrate for luciferase). However, D-luciferin, when applied to the technical solution of the present invention, is relatively inferior to AkaLuc in its effect.

In a preferred embodiment of the present invention, other fluorescent genes are also used as a reporter gene, and preferably tdTomato is used. Based on the inventors' comparison of fluorescent proteins of various spectral types, tdTomato was found to be relatively optimal, being able to fit other elements of the constructs of the invention, express well and function, with higher brightness than red and green fluorescent proteins. It is an orange derivative of the original fruit protein. tdTomato is derived from an intermediate designated dimer2, which is produced during the disintegration of tetrameric DsRed protein. the existence of the tdTomato gene can realize the sorting and tissue positioning of the senescent cells.

In the present invention, a gene encoding Diphtheria Toxin Receptor (DTR) is constructed on the construct to express DTR in cells or animals, which can be well expressed and function in cooperation with other elements of the construct of the present invention. DTR is not present in rodents, and when rodents are injected with large amounts of Diphtheria Toxin (DT), the safety of the animals themselves is not greatly affected. When diphtheria toxin is injected into animals, after diphtheria toxin is combined with Diphtheria Toxin Receptor (DTR) on aged cells positive to p21, the ADP ribosylation activity is exerted to inhibit cell protein synthesis and induce apoptosis. The inventor finds that the killing system based on DTR-DT has good safety, and has little influence on normal cells around the aged cells while the target aged cells play a killing role.

As a preferred mode of the invention, the construct comprises the following operably linked elements in the order from 5 'to 3': AkaLuc, tdTomato, diphtheria toxin receptor gene.

In a more preferred embodiment of the present invention, the 5' end of AkaLuc, between AkaLuc and tdTomato, and between tdTomato and diphtheria toxin receptor genes, further comprises a ribosome skipping sequence (2A) (abbreviated as 2A) sequence, which is a core sequence from foot-and-mouth disease virus, has a "self-splicing" function, and is used for co-expression of upstream and downstream genes.

In a more preferred embodiment of the present invention, the diphtheria toxin receptor gene further includes a terminator at the 3' end thereof; preferably the terminator is poly a.

In a more preferred form of the invention, the construct further comprises an FLP recognition site (FRT); preferably, the FLP recognition sites are 2 and a positive selection marker gene is also included between them. The positive selection marker gene is, for example, a Neo gene.

In a more preferred form of the invention, the construct further comprises a herpes simplex virus thymidine kinase negative selection gene, in coordination with the gene targeting selection of the construct. In a specific example of the present invention, a MC1-TK-polyA negative selection marker was established.

In a more preferred form of the invention, both ends of the construct, preferably upstream of the AkaLuc and downstream of the terminator; more preferably at the 5 'end of 2A and 3' end of the positive selection marker gene, and further comprising 5 'and 3' homology arm sequences, respectively. In a specific embodiment of the present invention, a 5 'homology arm comprising 5.3kb and a 3' homology arm comprising 1.3kb is used, which allows the construct to be integrated into the genome at the appropriate position after transfer into a cell, allowing for correct gene targeting.

In the embodiment of the invention, a 2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt expression frame is knocked in a site-specific manner at a stop codon of a P21 gene by using a cell targeting mode through a homologous recombination principle.

Based on the information disclosed in the present invention, those skilled in the art can make appropriate changes on the basis of the constructs provided by the present invention or elements thereof, and such changes should be included in the present invention. Variants or codon optimised versions of the elements of the invention are also encompassed by the invention.

The full-length nucleotide sequence of the gene to which each element of the present invention is directed or a fragment thereof can be obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates.

Restriction sites may also be included between elements in the construct to facilitate organic ligation of the elements.

Typically, the construct is located on an expression vector. Thus, the invention also includes a vector comprising the construct. Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

Vectors containing the appropriate polynucleotide sequences described above, together with appropriate promoter or control sequences, may be used to transform host cells in which gene targeting is desired. Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art, such as microinjection, electroporation, and the like.

After the construct constructed by the invention transfects animal embryonic stem cells, the construct can be well inserted in a targeting way at a specific position of a P21 gene of a target genome, and each element has stable expression and ideal activity.

The construct constructed by the inventor integrates multiple functions of single aging cell tracing, fluorescence positioning, fluorescence sorting, suicide induction of aging cell apoptosis and the like, can be applied to various animal disease models, and has extremely excellent technical effects.

Preparation of cells or animal models

The inventor designs a vector integrating tracing, sorting and clearing effects on senescent cells with positive p21 expression. According to the invention, the constructs can be used for the production of cells or animal models.

In the present invention, there is provided a method for preparing a cell using the construct, comprising: transfecting the construct into a cell to obtain a cell with the exogenous construct integrated in the genome.

In the present invention, methods of making animal models are also provided. The method comprises the following steps: introducing said construct into a germ cell or embryonic stem cell to obtain a germ cell or embryonic stem cell having the genome into which said construct is integrated; using the germ cells or embryonic stem cells to produce an animal body, wherein the animal body can be subjected to senescent cell tracing or senescent cells of the animal body can be induced to undergo apoptosis; preferably, the animal is a mammal comprising: a rodent; preferably, the rodent comprises a mouse or a rat (etc.).

The cell model includes mammalian cell models or other eukaryotic cell models, such as human cells or mouse cells. Such animals include, but are not limited to: rodent (including rat, mouse, hamster, etc.) and other model animals, in which the P21 gene is conserved. Preferably, the animal is a rodent.

In a specific example of the present invention, a heterozygote mouse of the P21 gene by site-directed knock-in 2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt was obtained. More specifically, the embodiment of the invention establishes a homologous recombination vector plasmid P21- (2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt-pA) 1; after the carrier is linearized, JM8A3 ES cells are electrotransfected; obtaining positive clone of correct homologous recombination through long-fragment PCR identification; amplifying the positive ES cell clone, and injecting the amplified positive ES cell clone into a blastocyst of a C57BL/6J mouse to obtain a chimeric mouse; a high proportion of chimeric mice were mated with Flp mice to obtain positive F1 generation de-Neo-heterozygous mice.

The method of the present invention can track the distribution, type, terminal differentiation of senescent cells very effectively.

Reagent kit

Based on the present disclosure, the present invention also provides a kit for conducting aging studies, comprising: the constructs of the invention; or a cell incorporating a construct of the invention.

The kit may further include instructions for performing the methods of the invention (including methods of creating constructs, creating engineered cells or animal models) for use by one of skill in the art.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 construction of the constructs

The present inventors have conducted extensive research works on p21 gene, and have conducted genetic modification to animals by gene targeting. After repeated studies, the present inventors determined a suitable targeting site for targeting, targeting a specific site on exon 3 of p21 gene.

The construction strategy of the genetically modified animal is shown in figure 1, and a foreign construct is inserted into the position after the 47 th base in exon 3 of a wild animal through a targeting plasmid, so that the foreign construct is integrated into the genome of the animal. The exogenous constructs were as follows:

2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt。

based on the construction strategy, the specific construction method is as follows:

1. construction of targeting plasmid (homologous recombination vector plasmid)

A homologous recombination vector plasmid P21- (2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt)1 as shown in FIG. 2 was constructed. The elements of the homologous recombination vector plasmid are shown in Table 1.

TABLE 1

Component	Name (R)
		Rep	Replication initiation site
Amp	Ampicillin resistance selection gene
		5’arm	5' homology arm
PGK	PGK promoter
		Neo	Neo positive selection marker gene
3’arm	3' homology arm
		HSV-TK	Herpes simplex virus thymidine kinase negative screening gene

When the plasmid is constructed, firstly, a vector P21-5arm +3arm is established, and the establishment method comprises the following steps: amplifying by using a genome of a wild mouse as a template and using primers P21-5F and P21-3R to obtain a 5arm amplification product; obtaining the target gene P21-5arm +3 arm.

P21-5F：CGCGGTCGACAAGCTtacagagtctgaactccaggaccac(SEQ ID NO:8)；

P21-3R：cgactctagaggatccgagaagtatttattgagcaccagc(SEQ ID NO:9)。

Secondly, PCR amplification is carried out by taking the primers P21-3F and P21-5R and the constructed vector P21-5arm +3arm as a template to obtain a target fragment P21-3F 5R.

p21-3F：agcttcgtggcagggaacaataggtgatggtacttgaacctaaagccttgaagtgcccacgggagc cccg ccctct(SEQ ID NO:10)；

P21-5R：gggttttctcttgcagaagaccaat(SEQ ID NO:11)。

The target fragment 2A-Akaluc is obtained by PCR amplification by using primers P21-Aka-F and P21-Aka-R and P21-3F5R as a template.

P21-Aka-F：tgcaagagaaaacccggaagcggagctactaacttcagcc(SEQ ID NO:12)；

P21-Aka-R：cagcctgcttcagcaggctgaagttagtagctccgctacccacggcgatcttgccgtccttcttg(SEQ ID NO:13)。

The target fragment 2A-tdTomato-2A-DTR is obtained by PCR amplification by using primers P21-td-F and P21-td-R and P21-3F5R as a template.

P21-td-F：tgctgaagcaggctggcgacgtggaggagaaccctggtcctatggtgagcaagggcgaggaggt ca(SEQ ID NO:14)；

P21-td-R：tcagtgggaattagtcatgcccaac(SEQ ID NO:15)。

The target fragment pA-Frt-Neo-Frt-pA is obtained by PCR amplification by using primers P21-Neo-F and P21-Neo-R and P21-3F5R as a template.

P21-neo-F：actaattcccactgacctccccgcatcgataccgtcgatc(SEQ ID NO:16)；

P21-neo-R：ccctgccacgaagcttggatccattatgtacctgactgat(SEQ ID NO:17)。

By passing

The HD Cloning Kit enzyme recombines the obtained target fragments 2A-Akalc, 2A-tdTomato-2A-DTR, pA-Frt-Neo-Frt-pA and P21-3F5R to obtain the vector.

The electrophoresis chart of the homologous recombination vector for enzyme digestion identification is shown in figure 3, and the theoretical band sizes are 7548bp, 5363bp, 2240bp and 1891 bp. According to the results in the figure, the recombinant plasmid P21- (2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt)1 with correct construction was obtained.

Example 2 obtaining and identifying of homologous recombinant ES cells

The homologous recombination vector plasmid P21- (2A-AkaLuc-2A-tdTomato-2A-DTR-pA-Frt-Neo-Frt-pA)1 is linearized.

Embryonic stem cell targeting was performed using JM8A3 ES cells (Shanghai south model Bioresearch center). The aforementioned linearized vector was introduced into embryonic stem cells by microinjection.

Targeting via embryonic stem cells (ES), the inventors obtained a series of resistant ES cell clones. After the genome DNA of the resistant ES cell clone is extracted, the homologous recombination positive clone is screened in a long-fragment PCR mode.

And (3) identifying the ES cells: carrying out PCR amplification by using the 5 'or 3' homology arm detection primer pair of tables 2-3; 5' arm homologous recombination positive clone should amplify 5.5kb fragment, negative clone should have no product; the 3' arm homologous recombination positive clone can amplify 1.6kb fragment, and the negative clone can amplify 7.0kb fragment.

TABLE 2

Primer and method for producing the same	Sequence (5 '→ 3')	SEQ ID NO:
			I (Forward)	aaggcttcgtttgttggagtgtg	18
II (reverse)	gcgcttcatggctttgtg	19

TABLE 3

Primer and method for producing the same	Sequence (5 '→ 3')	SEQ ID NO:
			III (Forward)	agcttcgtggcagggaacaatag	20
IV (reverse)	gggtgcaaacagcacaagtagcc	21

The positive clones obtained by PCR were confirmed by sequencing to obtain 8 positive ES cell clones with correct homologous recombination, as shown in FIG. 4. Double-arm homologous recombination positive ES clones were designated A2, A7, A9, B8, C2, D2, D4, D11.

Meanwhile, the present inventors performed D-galactose-induced senescence on the corresponding ES positive clones, followed by luminescence detection of positive AKaluc, as shown in FIG. 5. As can be seen from FIG. 5, multiple positive ES cell clones were able to measure fluorescence higher than WT, with A2 fluorescence being most desirable and D11 being the next.

Example 3 preparation of chimera Male mouse and F1 Generation mouse

And (3) cloning the obtained homologous recombinant positive ES cells, amplifying, injecting into C57BL/6J mouse blastocysts, and performing embryo transplantation to obtain the high chimeric male mouse.

The highly chimeric mice were mated with Flp mice (Shanghai south model biology research center) and bred to obtain F1 generation mice, which were genotyped by PCR identification and sequencing. The results of the identification are shown in FIG. 6.

Therefore, the inventors succeeded in obtaining a P21 genetically engineered mouse model (de-Neo-heterozygous mouse), which the inventors named as P21-ATD.

Example 4 identification of senescent cells

Replacing the obtained F2 with new oneAfter 3 days old rat tails of P21-ATD positive mice and WT mice were sterilized with 75% ethanol, the rat tails were cut into pieces and placed in a 6cm petri dish, 500ul of DMEM medium was added to the petri dish, and the volume of the medium was made up to 3ml after 24 hours. The mouse skin fibroblast count reached 10% after about 3 days and 70% after 1 week, using 200. mu.M H, respectively₂O₂Inducing the fibroblast senescence of P21-ATD positive mice and WT mice for 1h, and then replacing a DMEM normal culture medium for 3 days. The medium was discarded and washed 3 times with PBS, fixed for 15min with the Byunnan SA-beta-Gal senescent cell staining kit fixative and washed 3 times with PBS. 2ml of working solution (beta-galactose C: beta-galactose A: beta-galactose B: X-gal primer: 930: 10: 10: 50) is added dropwise, and the mixture is incubated for 15 hours in a bacterial incubator at 37 ℃ in a dark place, washed with PBS for 3 times and observed under the microscope. The results are shown in the upper panel of FIG. 7, which is similar to that of P21-ATD mouse fibroblast cell non-H₂O₂Treatment group comparison, H₂O₂The staining of the treated group with beta-galactosidase was evident, demonstrating H₂O₂The effect of inducing the mouse fibroblast to age is good.

Without addition of H₂O₂Treated WT mouse fibroblasts with H₂O₂Aging-inducing fibroblast cells without addition of H₂O₂Treated P21-ATD mouse fibroblasts with H₂O₂Adding 1ml of TRIzol lysate into aging-inducing fibroblasts, then adding 200 μ L of chloroform, layering the solution, wherein RNA is in an aqueous layer, the middle layer is DNA, and the lower layer is protein. 200. mu.L of the aqueous phase was removed, 500. mu.L of isopropanol was added, and centrifugation was carried out at 12000rpm for 15 min. Discarding supernatant, adding 1ml of 75% ethanol, centrifuging at 12000rpm for 15min, repeating the above steps, air drying the precipitate, adding 20ul ddH₂And dissolving the O. 500ng of RNA was added to 2. mu.L of reverse transcriptase and appropriate amount of ddH₂And O, constructing a 10 mu L reverse transcription system, and obtaining cDNA after program PCR. Distributing all the cDNA samples to a real-time quantitative PCR reaction system, and detecting the expression conditions of the aging-related secretory phenotype genes and the related inserted genes. From the lower graph of FIG. 7, see H₂O₂Can effectively induce the mouse fibroblast to age and the expression of age-related secretory phenotype genes is increased. P21 and Ak in mouse senescent fibroblastmRNA expression levels of alumin, tdTomato, DTR, and p16 were all higher than those of non-senescent cells (NS, which did not undergo H-transition₂O₂Treated cells) were significantly improved.

Example 5 fluorescent localization

1. Experimental methods

The obtained F2-generation newborn 3-day P21-ATD positive mouse tail and newborn WT mouse tail are taken, sterilized by 75% alcohol, cut into pieces and placed in a 6cm culture dish, 500ul of DMEM medium is added into the culture dish, and the medium is supplemented to 3ml after 24 h. The mouse skin fibroblast count reached 10% after about 3 days and 70% after 1 week, using 200. mu.M H₂O₂Inducing fibroblast of P21-ATD positive mouse and WT mouse, and inducing fibroblast of P21-ATD positive mouse in another group without inducing treatment, H₂O₂The treatment group was cultured for 3 days after 1h induction by changing the normal medium of DMEM. After 3 days, fluorescence emission of the P21-ATD mouse fibroblast tdTomato under different treatment conditions is observed under a fluorescence microscope.

As a result, H can be found in FIG. 8₂O₂The tdTomato fluorescence intensity in the fibroblast cells of the induced P21-ATD mice is significantly higher than that of the non-treated group, and the WT mice are shown to be tdTomato negative.

2. DTR expression detection

Inoculating cultured P21-ATD positive P2 generation rat tail fibroblast and WT rat tail fibroblast on cell slide prepared from glass slide, culturing overnight, and culturing with 200 μ M H₂O₂Inducing for 1h, replacing normal complete culture medium, continuing culturing for 4d, then abandoning the culture medium, adding PFA for fixing for 30min, and washing with PBS for 3 times. Blocking with 3% BSA-PBS for 1h, washing 3 times with PBS. Permeabilize with 0.25% TritonX-100-3% BSA-PBS for 30min, and wash with PBS 3 times. Rabbit anti-HBEGF/DTR (Abcam, ab92620) was diluted with 3% BSA-PBS to a final concentration of 5. mu.g/mL, incubated overnight at 4 ℃ and washed 3 times with PBS. Goat anti-rabbit Alexa Fluor 488(1:1000) was diluted with 3% BSA-PBS and mixed with 1:1000 diluted DAPI, incubated for 1h at room temperature and washed 3 times with PBS. And (5) drying and performing microscopic examination.

As a result, as shown in FIG. 9, DTR was highly expressed in the P21-ATD mouse fibroblasts of the senescence-induced group, as compared with the WT mouse fibroblasts of the senescence-induced group.

3. Akaluc expression assay

Inoculating cultured P21-ATD positive P2 generation rat tail fibroblast and WT rat tail fibroblast into black wall and transparent bottom 96-well plate, culturing overnight, and culturing with 200 μ M H₂O₂Inducing for 1h, replacing normal complete DMEM medium to continue culturing for 5 d.

And (3) IVIS detection: the medium was discarded, 100uL of 250. mu.M Akalumine-HCl was added, and 180Sec was exposed.

The results are shown in FIG. 10, which shows that Akaluc is highly expressed in P21-ATD mouse fibroblasts after being induced to senescence.

Example 6 suicide Induction of apoptosis in senescent cells

Dermal fibroblasts from WT mice and P21-ATD mice were seeded into 6cm dishes and 200. mu. M H was added when grown to 70% confluence₂O₂Induction was carried out for 1h, after which time the culture was continued for 2d by changing the normal complete DMEM medium, after which 50ng/ml diphtheria toxin (DT, Sigma) was added. After 2 days, a significant reduction in the number of skin fibroblasts from P21-ATD positive mice was found compared to wild-type skin fibroblasts.

Tunel fluorescence detection is carried out by using a Roche apoptosis fluorescence detection kit: after the fibroblasts were washed 3 times with PBS, the cells were fixed with 4% PFA for 15min, washed 3 times with PBS, and then Tunel working solution (solution a: solution B: 50: 450) was added, and incubated at 37 ℃ for 60min in the dark. Washing with PBS for 3 times, adding DAPI (1:1000) dropwise for 5min, and performing microscopic examination after washing.

Results as shown in figure 11, Diphtheria Toxin (DT) was very effective in inducing apoptosis in aged skin fibroblasts.

Sequence information

5' homology arm sequence (SEQ ID NO: 1):

tacagagtctgaactccaggaccacggatggcagccactcttccagtccttggagaccgggtccgagcgatggctggcggggatcggctaccgggagctcgcttctggttgccaatgtttctagggattccaggtgcggctcgcccaaagcgtgagaatgaagctcacggtgccccgagatgggtcctgggtggcggttgtgacaagcttcggcgagtctatctagagcttagcgcagagcggttctccgatcccatgggtgccgccggatccccggtccttgtgaaccaagtgccctgtttcgcggtagccaccaccccactgggctctgaggtcagcgcgagctgtgtcctccgccctcgggatcgtgcctgggcacgtcctagaggacaggcgaggaggtgactcattgtgacaaggagaccccgggcactggattgagaccagaatcgcgccacaactggggatgggtcagtggcagcccagaagcccctttccctgcccgggtacagcctcgttcctgccccgtcccctcccccgcgcggcacagtgacctatttggcgggcacagtatgttcccagggaacccgggacacggggaggtccaggacgcggtgtccggtccccgctcggcggcgcgccctcggggacagggagtctgggcgggtcctgtccaccctgccagcccgggaagcgcaaagggtcccacccaccgcgcccaccacgacccggaccagtgaccgtggggcgcgagaggagcccgcactgtagagcggccggtatcctcgcggagtgaggcttacgggtgccgtacatcagacgccccggggctggggccctcgacccagtccacacccagtgtaggaaggtgaccaggctgagtgcgcccactagggcacatccattccactcggaacctccggggacaagggctacacttgcaccctggtcgtgcgggaaatactttgggcttgggtttggcatttgggggcgcgctggcagcttcctctgtcccctacgtcgcgtttcagagaggacactcaggcggttttttgttgtcctcgccctcatctatttttattttccagggatctgactcatcgcgtgctttgggcgtggagatcaaggtggagggggccagagctaagagcactttctttgctctcgagtgtccgtgcgtgcgtgtgtgcgtgcgtgcgtatgtgtgtgtgtgtgtgtgtctgtgtacgtgcgtgtgtgtgtgtgtgtgtatgcgtgcgcgcgcgcgcgctcagtctggtttcccaacataggcgggaatttaggaagcctgggctcatcgtgacgtgttttgtggcccggggtccccctcctttcctctcctctctccaccccagggtgacgcgcagctccggtgcccaagcagttttggcgggcgggcagcgccgggcaggaaactgactcaccactccttgctcggcctacgcgccgcctaccgagcgccatttcctgagtgcacagcgccccctaccggcatcagtgaggactgcaaggccggctaggcgcgggtggcaccacgggcttctgccacccccaccgcggcttaaagggaattaaaatatgcctgatggccagatacaagtgattttagcagggtgtgctagcggactattagggaggcgtggcccttgagagacaaggtggagatcgtgcccctcttgggttcctggtcttatcttcagctatgaaaaactggggactatccagccttagccgagtccattggtgtcttcccttccttcctctggcccctgtgagctaccgtctggggaagggcaccaaacttgtttttcggctcagtttgtctggggaaatgagccaatctatatttctatatctatatagatctctctctctgtctctgtgtgtgtgtgaaagagagacagagacagagagacaagcttacaaaatggccccagctaccagtactatatagacaggctggcccagtggcctggaactcataaggatcacgtgcctctggtggagtgctgggattaaagactctccccataacatggagctcagtctatatttaataaaaaagcttttccagctgattttaatggagtcctctggtcatgcccctcaaggtgagaagacgagagaatgatcaatactagaccaaaagggtctgaattcaaaccccgcttgtgagccctgtgcctttggacacacattctgaacctggattagctctgccgtaaaagaatgtgatattgccatctattaacgatagcttttggtggcttacaactgcaatcccagcacactagaggcagaggcaggaggatgactgagttgtactaaaggatgtgaataggccccaagctctacctgtaggcagaggccatctgaggtcctttaggcctctgaggtctgcaccttgagaatccttttgcgggcagtgggaatccttgttagacctgtgtttcggttggttggtgtttagccaggctctcactatgcacaccaggatggccttcaactcacagacattttgcctctgcctcccgagtgctgtgttttgggaattggctgttttacttatttatggaagccacaatgctggccacgttccctatgtagtagccaaggtagaccctgaacttctttccctacctctgcctccagcgattacaggtgtgtgccgccacacccagttttatagagagctccttgcttagcacaaggcccaggctttaccatcggggccccagacgcttcatctcttctggcttcctaccttgaatcatagtctgggtacccccttctctcttccagctgtgggcaaactcaccctgaggtcttaaggagctgggcgtgtgtatgtgtgtggtagtgtatgtggtaagagcccagggggtcttgcctcaggaagccactggggctcaccttgcaagctcaaaggccctccttagaagctgcatcccctagtcttggagtcaggatgggccactcaggtatccctctttcctggagaggagccaggtaaaccaacacctttaacttttttttttttttcagtgcctgtaaaacgggttccttgggcacaggctataggcacgtctggggctgggaggtgtctagactccagattacctgatctgcctggcaggatgtggctcagcctgggagagagccttaaccccgctccaccccttcagactctcctctcccccaggctactgcctccccccaggctgctgcctccccccaggctactgcctccccccaggctgctgcctcctcccaggctactgcctcctcccaggctgctgcctcctcccaggctactgcctgccagcgtcctttggaaaggcctccagcctggagcacctgtcagtgacagtgggagggagggaggggcagggaggaggctctgtcagaattaggggcagagaggcacgctcatggcttctgtttctcaggattgcctatgttaacttagttcattctcaaatagggagcccagagatgttagggtacttattcggggtcacccagcaaagccttgattctgatctgggcagtctagctccggcattctcgcgcctctctcccagccaccatgccagcctcgcgagtatgctgccacaaccacactggctaagaaacagaggctggagacatggagtcactttttaaaactggtgccaagtagcagcactaatgctatacagtttatgtgtagtatcccaaagtccagggcacttttttttttttttttttttttttttttttttttttttgagacaggatgtctctgtatagccctggctgtcctggaactcactttgtagaccaggctggcctcgaactcagaaatccgcctgcctctgcctcccgagtgctgggattaaaggcgtgtgccaccacgcccggctgtccagggtacttttgattggcctgatggagttaatcaccaagacagcagggtagggagaccactggacctagcaattcacacgtatttgggatgttcacacccatgaagaacacgttagcacattgattttggctaatagaattcctggggtaaacaggacggtgactcctacttctgtggacatcacccgtgaccttggggtgcagggctggctgaactcaacacccaccttagtctcatggtgtggtggaaaagcacctgcaaggaccagagggagcctgaagactgtgatggggtagtttccatagtgacccgggtccttcttgtgtttcagccacaggcaccatgtccaatcctggtgatgtccgacctgttccgcacaggagcaaagtgtgccgttgtctcttcggtcccgtggacagtgagcagttgcgccgtgattgcgatgcgctcatggcgggctgtctccaggaggcccgagaacggtggaactttgacttcgtcacggagacgccgctggagggcaacttcgtctgggagcgcgttcggagcctagggctgcccaaggtctacctgagccctgggtcccgcagccgtgacgacctgggaggggacaagaggcccagtacttcctctgccctgctgcaggggccagctccggaggaccacgtggccttgtcgctgtcttgcactctggtgtctgagcggcctgaagattccccgggtgggcccggaacatctcagggccgaaaacggaggcagaccagcctgacaggtaaggacaggagcagagaaggagaaagatcctgcaagaggcctggagaggagaggccaccatttgaggatggcctttacagagaacattccagcccttccccaccaccaagccattccataggcgtgggacctcgtggggctcagaggaacagttgatgttcatatgatccaggcatttttctctgcagtgaccgaaatgcccaggatagtgtggtgattggcagtagagctctaagaagggagccgggctgaagagatggctcagcagatgagggcacttactcttgctgagggcctgattcccagcaccggaaatgacaacttcctataactaactctgggcgttgggggatctaccctctctagagccctgtccctctgaccaggaggtgttgtgccctgtggctgtggcttttccccacgatgagccacatgtcccttagactctggggaatgatgtccttccccttggcatctggcctgacatctgttctctctccacagatttctatcactccaagcgcagattggtcttctgcaagagaaaacccggaagcggagctactaacttcagcctgctgaagcaggctggcgacgtggaggagaaccctggtcct

3' homology arm sequence (SEQ ID NO: 2):

agtgcccacgggagccccgccctcttctgctgtgggtcaggaggcctcttccccatcttcggccttagccctcactctgtgtgtct

taattattatttgtgttttaatttaaacgtctcctgtatatacgctgcctgccctctcccagtctccaaacttaaagttatttaaaaaaagaacaaaacaaaacaaaaaaaaccaaaacaaaacaaacctaaattagtaggacggtagggcccttagtgtgggggatttctattatgtagattattattatttaagcccctcccaacccaagctctgtgtttcctataccggaggaacagtcctactgatatcaacccatctgcatccgtttcacccaacccccctccccccattccctgcctggttccttgccacttcttacctgggggtgatcctcagacctgaatagcactttggaaaaatgagtaggactttggggtctccttgtcacctctaaggccagctaggatgacagtgaagcagtcacagcctagaacagggatggcagttaggactcaaccgtaatatcccgactcttgacattgctcagacctgtgaagacaggaatggtccccactctggatcccctttgccactcctggggagcccacctctcctgtgggtctctgccagctgcccctctattttggagggttaatctggtgatctgctgctcttttcccccaccccatacttccccttctgcaggtcggcaggaggcatatctaggcacttgccccacagctcagtggactggaagggaatgtatatgcagggtacactaagtgggattccctggtcttaccttaggcagctccagtggcaaccccctgcattgtgggtctagggtgggtccttggtggtgagacaggcctcccagagcattctatggtgtgtggtggtgggggtgggcttatctgggatggggaccccagttggggttctcagtgacttctcccatttcttagtagcagttgtacaaggagccaggccaagatggtgtcttgggggctaagggagctcacaggacactgagcaatggctgatcctttctcagtgttgaataccgtgggtgtcaaagcacttagtgggtctgactccagccccaaacatccctgtttctgtaacatcctggtctggactgtctacccttagcccgcaccccaagaacatgtattgtggctccctccctgtctccactcagattgtaagcgtctcacgagaagggacagcaccctgcattgtcccgagtcctcacacccgaccccaaagctggtgctcaataaatacttctcg

2A sequence (SEQ ID NO: 3):

ggtagcggagctactaacttcagcctgctgaagcaggctggcgacgtggaggagaaccctggtcct

AkaLuc sequence (SEQ ID NO: 4):

atggaagatgccaaaaacattaagaagggcccagcgccgttctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcgccatcgcctttaccgacgcacatattcaggtggacgttacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaggcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagctcgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcgaaaggtcctcaacgtgcaaaagaagctaccgatcatacgaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccagcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggtagtacaggattacccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggctaccagaacatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagctgcctcgctaagagcactctcatcgacaagtacgacctaagcagcttgcgcgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccgcctaccaggcatccgccagggctatggcctgacagaaacaaccaacgccgtcatgatcacccccgagggggaccgtaagcctggctcagtaggcaaggtggtgcccttcttcgaggctaaggtggtagacttggtcaccggtaagacactgggtgtgaaccagcgcggtgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacgaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagggcatcctgctgcaacacccctacatcttcgacgccggagtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgttggaacacggtaaaaccatgaccgagaaagagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgtttgtggatgaagtccctagaggatcgaccggcaagttagacgcccgcaagatccgcgagattctcactaaggccaagaaggacggcaagatcgccgtg

tdTomato sequence (SEQ ID NO: 5):

atggtgagcaagggcgaggaggtcatcaaagagttcatgcgcttcaaggtgcgcatggagggctccatgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggcggccccctgcccttcgcctgggacatcctgtccccccagttcatgtacggctccaaggcgtacgtgaagcaccccgccgacatccccgattacaagaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggtctggtgaccgtgacccaggactcctccctgcaggacggcacgctgatctacaaggtgaagatgcgcggcaccaacttcccccccgacggccccgtaatgcagaagaagaccatgggctgggaggcctccaccgagcgcctgtacccccgcgacggcgtgctgaagggcgagatccaccaggccctgaagctgaaggacggcggccactacctggtggagttcaagaccatctacatggccaagaagcccgtgcaactgcccggctactactacgtggacaccaagctggacatcacctcccacaacgaggactacaccatcgtggaacagtacgagcgctccgagggccgccaccacctgttcctggggcatggcaccggcagcaccggcagcggcagctccggcaccgcctcctccgaggacaacaacatggccgtcatcaaagagttcatgcgcttcaaggtgcgcatggagggctccatgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggcggccccctgcccttcgcctgggacatcctgtccccccagttcatgtacggctccaaggcgtacgtgaagcaccccgccgacatccccgattacaagaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggtctggtgaccgtgacccaggactcctccctgcaggacggcacgctgatctacaaggtgaagatgcgcggcaccaacttcccccccgacggccccgtaatgcagaagaagaccatgggctgggaggcctccaccgagcgcctgtacccccgcgacggcgtgctgaagggcgagatccaccaggccctgaagctgaaggacggcggccactacctggtggagttcaagaccatctacatggccaagaagcccgtgcaactgcccggctactactacgtggacaccaagctggacatcacctcccacaacgaggactacaccatcgtggaacagtacgagcgctccgagggccgccaccacctgttcctgtacggcatggacgagctgtacaag

diphtheria Toxin Receptor (DTR) sequence (SEQ ID NO: 6):

atgaagctgctgccgtcggtggtgctgaagctccttctggctgcagttctttcggcactggtgactggcgagagcctggagcagcttcggagagggctagctgctggaaccagcaacccggacccttccactggatctacggaccagctgctacgcctaggaggcggccgggaccggaaagtccgtgacttgcaagaggcagatctggaccttttgagagtcactttatcctccaagccacaagcactggccacaccaagcaaggaggagcacgggaaaagaaagaagaaaggcaagggactagggaagaagagggacccatgtcttcggaaatacaaggacttctgcatccacggagaatgcaaatatgtgaaggagctccgggctccctcctgcatctgccacccaggttaccatggagagaggtgtcatgggctgagcctcccagtggaaaatcgcttatatacctatgaccatacaactatcctggctgtggtggccgtggtgctgtcctctgtctgtctgctggtcatcgtggggcttctcatgtttaggtaccataggagaggtggttatgatgtggaaaacgaagagaaagtgaagttgggcatgactaattcccactga

the recombinase recognition site FRT sequence is (SEQ ID NO: 7):

gaagttcctattctctagaaagtataggaacttc

the Neo sequence: candida Gene symbol LOC 110847127.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai City eastern Hospital (affiliated eastern Hospital of Tongji university)

<120> a construct and its use in animal senescent cell tracing and senescent cell clearing

<130> 201742

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5322

<212> DNA

<213> mouse (Mus musculus)

<400> 1

tacagagtct gaactccagg accacggatg gcagccactc ttccagtcct tggagaccgg 60

gtccgagcga tggctggcgg ggatcggcta ccgggagctc gcttctggtt gccaatgttt 120

ctagggattc caggtgcggc tcgcccaaag cgtgagaatg aagctcacgg tgccccgaga 180

tgggtcctgg gtggcggttg tgacaagctt cggcgagtct atctagagct tagcgcagag 240

cggttctccg atcccatggg tgccgccgga tccccggtcc ttgtgaacca agtgccctgt 300

ttcgcggtag ccaccacccc actgggctct gaggtcagcg cgagctgtgt cctccgccct 360

cgggatcgtg cctgggcacg tcctagagga caggcgagga ggtgactcat tgtgacaagg 420

agaccccggg cactggattg agaccagaat cgcgccacaa ctggggatgg gtcagtggca 480

gcccagaagc ccctttccct gcccgggtac agcctcgttc ctgccccgtc ccctcccccg 540

cgcggcacag tgacctattt ggcgggcaca gtatgttccc agggaacccg ggacacgggg 600

aggtccagga cgcggtgtcc ggtccccgct cggcggcgcg ccctcgggga cagggagtct 660

gggcgggtcc tgtccaccct gccagcccgg gaagcgcaaa gggtcccacc caccgcgccc 720

accacgaccc ggaccagtga ccgtggggcg cgagaggagc ccgcactgta gagcggccgg 780

tatcctcgcg gagtgaggct tacgggtgcc gtacatcaga cgccccgggg ctggggccct 840

cgacccagtc cacacccagt gtaggaaggt gaccaggctg agtgcgccca ctagggcaca 900

tccattccac tcggaacctc cggggacaag ggctacactt gcaccctggt cgtgcgggaa 960

atactttggg cttgggtttg gcatttgggg gcgcgctggc agcttcctct gtcccctacg 1020

tcgcgtttca gagaggacac tcaggcggtt ttttgttgtc ctcgccctca tctattttta 1080

ttttccaggg atctgactca tcgcgtgctt tgggcgtgga gatcaaggtg gagggggcca 1140

gagctaagag cactttcttt gctctcgagt gtccgtgcgt gcgtgtgtgc gtgcgtgcgt 1200

atgtgtgtgt gtgtgtgtgt ctgtgtacgt gcgtgtgtgt gtgtgtgtgt atgcgtgcgc 1260

gcgcgcgcgc tcagtctggt ttcccaacat aggcgggaat ttaggaagcc tgggctcatc 1320

gtgacgtgtt ttgtggcccg gggtccccct cctttcctct cctctctcca ccccagggtg 1380

acgcgcagct ccggtgccca agcagttttg gcgggcgggc agcgccgggc aggaaactga 1440

ctcaccactc cttgctcggc ctacgcgccg cctaccgagc gccatttcct gagtgcacag 1500

cgccccctac cggcatcagt gaggactgca aggccggcta ggcgcgggtg gcaccacggg 1560

cttctgccac ccccaccgcg gcttaaaggg aattaaaata tgcctgatgg ccagatacaa 1620

gtgattttag cagggtgtgc tagcggacta ttagggaggc gtggcccttg agagacaagg 1680

tggagatcgt gcccctcttg ggttcctggt cttatcttca gctatgaaaa actggggact 1740

atccagcctt agccgagtcc attggtgtct tcccttcctt cctctggccc ctgtgagcta 1800

ccgtctgggg aagggcacca aacttgtttt tcggctcagt ttgtctgggg aaatgagcca 1860

atctatattt ctatatctat atagatctct ctctctgtct ctgtgtgtgt gtgaaagaga 1920

gacagagaca gagagacaag cttacaaaat ggccccagct accagtacta tatagacagg 1980

ctggcccagt ggcctggaac tcataaggat cacgtgcctc tggtggagtg ctgggattaa 2040

agactctccc cataacatgg agctcagtct atatttaata aaaaagcttt tccagctgat 2100

tttaatggag tcctctggtc atgcccctca aggtgagaag acgagagaat gatcaatact 2160

agaccaaaag ggtctgaatt caaaccccgc ttgtgagccc tgtgcctttg gacacacatt 2220

ctgaacctgg attagctctg ccgtaaaaga atgtgatatt gccatctatt aacgatagct 2280

tttggtggct tacaactgca atcccagcac actagaggca gaggcaggag gatgactgag 2340

ttgtactaaa ggatgtgaat aggccccaag ctctacctgt aggcagaggc catctgaggt 2400

cctttaggcc tctgaggtct gcaccttgag aatccttttg cgggcagtgg gaatccttgt 2460

tagacctgtg tttcggttgg ttggtgttta gccaggctct cactatgcac accaggatgg 2520

ccttcaactc acagacattt tgcctctgcc tcccgagtgc tgtgttttgg gaattggctg 2580

ttttacttat ttatggaagc cacaatgctg gccacgttcc ctatgtagta gccaaggtag 2640

accctgaact tctttcccta cctctgcctc cagcgattac aggtgtgtgc cgccacaccc 2700

agttttatag agagctcctt gcttagcaca aggcccaggc tttaccatcg gggccccaga 2760

cgcttcatct cttctggctt cctaccttga atcatagtct gggtaccccc ttctctcttc 2820

cagctgtggg caaactcacc ctgaggtctt aaggagctgg gcgtgtgtat gtgtgtggta 2880

gtgtatgtgg taagagccca gggggtcttg cctcaggaag ccactggggc tcaccttgca 2940

agctcaaagg ccctccttag aagctgcatc ccctagtctt ggagtcagga tgggccactc 3000

aggtatccct ctttcctgga gaggagccag gtaaaccaac acctttaact tttttttttt 3060

tttcagtgcc tgtaaaacgg gttccttggg cacaggctat aggcacgtct ggggctggga 3120

ggtgtctaga ctccagatta cctgatctgc ctggcaggat gtggctcagc ctgggagaga 3180

gccttaaccc cgctccaccc cttcagactc tcctctcccc caggctactg cctcccccca 3240

ggctgctgcc tccccccagg ctactgcctc cccccaggct gctgcctcct cccaggctac 3300

tgcctcctcc caggctgctg cctcctccca ggctactgcc tgccagcgtc ctttggaaag 3360

gcctccagcc tggagcacct gtcagtgaca gtgggaggga gggaggggca gggaggaggc 3420

tctgtcagaa ttaggggcag agaggcacgc tcatggcttc tgtttctcag gattgcctat 3480

gttaacttag ttcattctca aatagggagc ccagagatgt tagggtactt attcggggtc 3540

acccagcaaa gccttgattc tgatctgggc agtctagctc cggcattctc gcgcctctct 3600

cccagccacc atgccagcct cgcgagtatg ctgccacaac cacactggct aagaaacaga 3660

ggctggagac atggagtcac tttttaaaac tggtgccaag tagcagcact aatgctatac 3720

agtttatgtg tagtatccca aagtccaggg cacttttttt tttttttttt tttttttttt 3780

tttttttttt tgagacagga tgtctctgta tagccctggc tgtcctggaa ctcactttgt 3840

agaccaggct ggcctcgaac tcagaaatcc gcctgcctct gcctcccgag tgctgggatt 3900

aaaggcgtgt gccaccacgc ccggctgtcc agggtacttt tgattggcct gatggagtta 3960

atcaccaaga cagcagggta gggagaccac tggacctagc aattcacacg tatttgggat 4020

gttcacaccc atgaagaaca cgttagcaca ttgattttgg ctaatagaat tcctggggta 4080

aacaggacgg tgactcctac ttctgtggac atcacccgtg accttggggt gcagggctgg 4140

ctgaactcaa cacccacctt agtctcatgg tgtggtggaa aagcacctgc aaggaccaga 4200

gggagcctga agactgtgat ggggtagttt ccatagtgac ccgggtcctt cttgtgtttc 4260

agccacaggc accatgtcca atcctggtga tgtccgacct gttccgcaca ggagcaaagt 4320

gtgccgttgt ctcttcggtc ccgtggacag tgagcagttg cgccgtgatt gcgatgcgct 4380

catggcgggc tgtctccagg aggcccgaga acggtggaac tttgacttcg tcacggagac 4440

gccgctggag ggcaacttcg tctgggagcg cgttcggagc ctagggctgc ccaaggtcta 4500

cctgagccct gggtcccgca gccgtgacga cctgggaggg gacaagaggc ccagtacttc 4560

ctctgccctg ctgcaggggc cagctccgga ggaccacgtg gccttgtcgc tgtcttgcac 4620

tctggtgtct gagcggcctg aagattcccc gggtgggccc ggaacatctc agggccgaaa 4680

acggaggcag accagcctga caggtaagga caggagcaga gaaggagaaa gatcctgcaa 4740

gaggcctgga gaggagaggc caccatttga ggatggcctt tacagagaac attccagccc 4800

ttccccacca ccaagccatt ccataggcgt gggacctcgt ggggctcaga ggaacagttg 4860

atgttcatat gatccaggca tttttctctg cagtgaccga aatgcccagg atagtgtggt 4920

gattggcagt agagctctaa gaagggagcc gggctgaaga gatggctcag cagatgaggg 4980

cacttactct tgctgagggc ctgattccca gcaccggaaa tgacaacttc ctataactaa 5040

ctctgggcgt tgggggatct accctctcta gagccctgtc cctctgacca ggaggtgttg 5100

tgccctgtgg ctgtggcttt tccccacgat gagccacatg tcccttagac tctggggaat 5160

gatgtccttc cccttggcat ctggcctgac atctgttctc tctccacaga tttctatcac 5220

tccaagcgca gattggtctt ctgcaagaga aaacccggaa gcggagctac taacttcagc 5280

ctgctgaagc aggctggcga cgtggaggag aaccctggtc ct 5322

<210> 2

<211> 1323

<212> DNA

<213> mouse (Mus musculus)

<400> 2

agtgcccacg ggagccccgc cctcttctgc tgtgggtcag gaggcctctt ccccatcttc 60

ggccttagcc ctcactctgt gtgtcttaat tattatttgt gttttaattt aaacgtctcc 120

tgtatatacg ctgcctgccc tctcccagtc tccaaactta aagttattta aaaaaagaac 180

aaaacaaaac aaaaaaaacc aaaacaaaac aaacctaaat tagtaggacg gtagggccct 240

tagtgtgggg gatttctatt atgtagatta ttattattta agcccctccc aacccaagct 300

ctgtgtttcc tataccggag gaacagtcct actgatatca acccatctgc atccgtttca 360

cccaaccccc ctccccccat tccctgcctg gttccttgcc acttcttacc tgggggtgat 420

cctcagacct gaatagcact ttggaaaaat gagtaggact ttggggtctc cttgtcacct 480

ctaaggccag ctaggatgac agtgaagcag tcacagccta gaacagggat ggcagttagg 540

actcaaccgt aatatcccga ctcttgacat tgctcagacc tgtgaagaca ggaatggtcc 600

ccactctgga tcccctttgc cactcctggg gagcccacct ctcctgtggg tctctgccag 660

ctgcccctct attttggagg gttaatctgg tgatctgctg ctcttttccc ccaccccata 720

cttccccttc tgcaggtcgg caggaggcat atctaggcac ttgccccaca gctcagtgga 780

ctggaaggga atgtatatgc agggtacact aagtgggatt ccctggtctt accttaggca 840

gctccagtgg caaccccctg cattgtgggt ctagggtggg tccttggtgg tgagacaggc 900

ctcccagagc attctatggt gtgtggtggt gggggtgggc ttatctggga tggggacccc 960

agttggggtt ctcagtgact tctcccattt cttagtagca gttgtacaag gagccaggcc 1020

aagatggtgt cttgggggct aagggagctc acaggacact gagcaatggc tgatcctttc 1080

tcagtgttga ataccgtggg tgtcaaagca cttagtgggt ctgactccag ccccaaacat 1140

ccctgtttct gtaacatcct ggtctggact gtctaccctt agcccgcacc ccaagaacat 1200

gtattgtggc tccctccctg tctccactca gattgtaagc gtctcacgag aagggacagc 1260

accctgcatt gtcccgagtc ctcacacccg accccaaagc tggtgctcaa taaatacttc 1320

tcg 1323

<210> 3

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(66)

<223> 2A sequence

<400> 3

ggtagcggag ctactaactt cagcctgctg aagcaggctg gcgacgtgga ggagaaccct 60

ggtcct 66

<210> 4

<211> 1650

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1650)

<223> AkaLuc sequence

<400> 4

atggaagatg ccaaaaacat taagaagggc ccagcgccgt tctacccact cgaagacggg 60

accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcgccatc 120

gcctttaccg acgcacatat tcaggtggac gttacctacg ccgagtactt cgagatgagc 180

gttcggctgg cagaagctat gaggcgctat gggctgaata caaaccatcg gatcgtggtg 240

tgcagcgaga atagctcgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg 300

gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc 360

agccagccca ccgtcgtatt cgtgagcaag aaagggctgc gaaaggtcct caacgtgcaa 420

aagaagctac cgatcatacg aaagatcatc atcatggata gcaagaccga ctaccagggc 480

ttccaaagca tgtacacctt cgtgacttcc catttgccac ccagcttcaa cgagtacgac 540

ttcgtgcccg agagcttcga ccgggacaaa accatcgccc tgatcatgaa cagtagtggt 600

agtacaggat tacccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt 660

catgcccgcg accccatctt cggctaccag aacatccccg acaccgctat cctcagcgtg 720

gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt 780

cgggtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat 840

aagattcaat ctgccctgct ggtgcccaca ctatttagct gcctcgctaa gagcactctc 900

atcgacaagt acgacctaag cagcttgcgc gagatcgcca gcggcggggc gccgctcagc 960

aaggaggtag gtgaggccgt ggccaaacgc ttccgcctac caggcatccg ccagggctat 1020

ggcctgacag aaacaaccaa cgccgtcatg atcacccccg agggggaccg taagcctggc 1080

tcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tagacttggt caccggtaag 1140

acactgggtg tgaaccagcg cggtgagctg tgcgtccgtg gccccatgat catgagcggc 1200

tacgttaaca accccgaggc tacgaacgct ctcatcgaca aggacggctg gctgcacagc 1260

ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320

ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagggcat cctgctgcaa 1380

cacccctaca tcttcgacgc cggagtcgcc ggcctgcccg acgacgatgc cggcgagctg 1440

cccgccgcag tcgtcgtgtt ggaacacggt aaaaccatga ccgagaaaga gatcgtggac 1500

tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gtttgtggat 1560

gaagtcccta gaggatcgac cggcaagtta gacgcccgca agatccgcga gattctcact 1620

aaggccaaga aggacggcaa gatcgccgtg 1650

<210> 5

<211> 1428

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1428)

<223> tdTomato sequence

<400> 5

atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag 60

ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120

ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180

atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc 240

cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300

gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc 360

tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag 420

accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480

gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc 540

atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg 600

gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc 660

cgccaccacc tgttcctggg gcatggcacc ggcagcaccg gcagcggcag ctccggcacc 720

gcctcctccg aggacaacaa catggccgtc atcaaagagt tcatgcgctt caaggtgcgc 780

atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 840

tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc 900

tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc 960

gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 1020

aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg 1080

ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag 1140

aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 1200

aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 1260

aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc 1320

aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc 1380

gagggccgcc accacctgtt cctgtacggc atggacgagc tgtacaag 1428

<210> 6

<211> 627

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(627)

<223> Diphtheria Toxin Receptor (DTR) sequences

<400> 6

atgaagctgc tgccgtcggt ggtgctgaag ctccttctgg ctgcagttct ttcggcactg 60

gtgactggcg agagcctgga gcagcttcgg agagggctag ctgctggaac cagcaacccg 120

gacccttcca ctggatctac ggaccagctg ctacgcctag gaggcggccg ggaccggaaa 180

gtccgtgact tgcaagaggc agatctggac cttttgagag tcactttatc ctccaagcca 240

caagcactgg ccacaccaag caaggaggag cacgggaaaa gaaagaagaa aggcaaggga 300

ctagggaaga agagggaccc atgtcttcgg aaatacaagg acttctgcat ccacggagaa 360

tgcaaatatg tgaaggagct ccgggctccc tcctgcatct gccacccagg ttaccatgga 420

gagaggtgtc atgggctgag cctcccagtg gaaaatcgct tatataccta tgaccataca 480

actatcctgg ctgtggtggc cgtggtgctg tcctctgtct gtctgctggt catcgtgggg 540

cttctcatgt ttaggtacca taggagaggt ggttatgatg tggaaaacga agagaaagtg 600

aagttgggca tgactaattc ccactga 627

<210> 7

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(34)

<223> recombinase recognition site FRT sequence

<400> 7

gaagttccta ttctctagaa agtataggaa cttc 34

<210> 8

<211> 40

<212> DNA

<213> primers (Primer)

<400> 8

cgcggtcgac aagcttacag agtctgaact ccaggaccac 40

<210> 9

<211> 40

<212> DNA

<213> primers (Primer)

<400> 9

cgactctaga ggatccgaga agtatttatt gagcaccagc 40

<210> 10

<211> 76

<212> DNA

<213> primers (Primer)

<400> 10

agcttcgtgg cagggaacaa taggtgatgg tacttgaacc taaagccttg aagtgcccac 60

gggagccccg ccctct 76

<210> 11

<211> 25

<212> DNA

<213> primers (Primer)

<400> 11

gggttttctc ttgcagaaga ccaat 25

<210> 12

<211> 40

<212> DNA

<213> primers (Primer)

<400> 12

tgcaagagaa aacccggaag cggagctact aacttcagcc 40

<210> 13

<211> 65

<212> DNA

<213> primers (Primer)

<400> 13

cagcctgctt cagcaggctg aagttagtag ctccgctacc cacggcgatc ttgccgtcct 60

tcttg 65

<210> 14

<211> 66

<212> DNA

<213> primers (Primer)

<400> 14

tgctgaagca ggctggcgac gtggaggaga accctggtcc tatggtgagc aagggcgagg 60

aggtca 66

<210> 15

<211> 25

<212> DNA

<213> primers (Primer)

<400> 15

tcagtgggaa ttagtcatgc ccaac 25

<210> 16

<211> 40

<212> DNA

<213> primers (Primer)

<400> 16

actaattccc actgacctcc ccgcatcgat accgtcgatc 40

<210> 17

<211> 40

<212> DNA

<213> primers (Primer)

<400> 17

ccctgccacg aagcttggat ccattatgta cctgactgat 40

<210> 18

<211> 23

<212> DNA

<213> primers (Primer)

<400> 18

aaggcttcgt ttgttggagt gtg 23

<210> 19

<211> 18

<212> DNA

<213> primers (Primer)

<400> 19

gcgcttcatg gctttgtg 18

<210> 20

<211> 23

<212> DNA

<213> primers (Primer)

<400> 20

agcttcgtgg cagggaacaa tag 23

<210> 21

<211> 23

<212> DNA

<213> primers (Primer)

<400> 21

gggtgcaaac agcacaagta gcc 23

Claims (10)

1. An isolated construct, wherein the construct is targeted to the P21 gene and comprises a reporter gene and a diphtheria toxin receptor gene; the constructs are capable of engineering the genome of a cell or animal, performing senescent cell localization, tracking, sorting, inducing apoptosis of senescent cells, and/or eliminating senescent cells in a cell or animal body.

2. The construct of claim 1, wherein the construct targets exon 3 of the P21 gene; preferably after the 47 th base of exon 3.

3. The construct of claim 1, wherein the reporter gene comprises a reporter gene selected from the group consisting of: luciferase gene, tdTomato, green fluorescent protein gene, enhanced green fluorescent protein coding gene, red fluorescent protein gene, beta-galactosidase coding gene and the like; preferably, the reporter gene comprises: luciferase gene and tdTomato; more preferably, the luciferase is AkaLuc; more preferably, the construct comprises the following operably linked elements in order from 5 'to 3': AkaLuc, tdTomato, diphtheria toxin receptor gene.

4. The construct of claim 3, wherein the 5' end of AkaLuc, between AkaLuc and tdTomato, and between tdTomato and diphtheria toxin receptor gene, further comprises a 2A sequence, P2A, and/or T2A.

3' end of diphtheria toxin receptor gene, and terminator; preferably the terminator is poly a;

the construct further comprises an FLP recognition site; preferably, the FLP recognition sites are 2 and further include a positive selection marker gene therebetween: PGK-Neo and/or DDSDC-Neo;

the construct further comprises a herpes simplex virus thymidine kinase negative selection gene; and/or

Both ends of the construct, preferably, the upstream of the AkaLuc and the downstream of the terminator, further comprise a 5 'homology arm sequence and a 3' homology arm sequence, respectively.

5. Use of the construct of any one of claims 1 to 4 for targeting the P21 gene, modifying the genome of a cell or animal, performing senescent cell localization, tracking, sorting, inducing apoptosis and/or eliminating senescent cells in a cell or animal body; preferably, the construct targets exon 3 of the P21 gene.

6. A cell having integrated into its genome an exogenous construct according to any one of claims 1 to 4; preferably, the construct is integrated into the P21 gene of the cell, more preferably into exon 3 of the P21 gene; preferably, the cell comprises: embryonic stem cells, somatic cells, germ cells, or tumor cells; preferably, the germ cells include fertilized eggs, oocytes, sperm; preferably, the somatic cells comprise: fibroblasts, epithelial cells, blood cells, cells of tissue or organ origin.

7. A method of localizing, tracking, sorting, inducing apoptosis and/or eliminating senescent cells, the method comprising:

(1) transfecting a test cell with a construct according to any one of claims 1 to 4 to obtain a cell having the exogenous construct integrated into its genome;

(2) observing the expression of the reporter gene in the cell, thereby locating, tracking, sorting senescent cells, inducing apoptosis of the senescent cells and/or eliminating senescent cells;

preferably, the test cells comprise: embryonic stem cells, somatic cells, germ cells, or tumor cells; preferably, the germ cells include fertilized eggs, oocytes, sperm; preferably, the somatic cells comprise: fibroblasts, epithelial cells, blood cells, cells of tissue or organ origin.

8. A method of preparing an animal in which senescent cells can be localized, traced, induced to undergo apoptosis and/or eliminated, comprising:

(1) introducing the construct of any one of claims 1 to 4 into an embryonic stem cell or germ cell to obtain an embryonic stem cell or germ cell having the construct integrated into its genome;

(2) using said embryonic stem or germ cells to produce an animal body that is capable of undergoing senescent cell tracking, or whose senescent cells are capable of being induced to undergo apoptosis;

preferably, the animal is a mammal comprising: a rodent; preferably, the rodent comprises a mouse or a rat.

9. Use of the construct of any one of claims 1 to 4, the cell of claim 6 or the animal produced by the method of claim 8 for studying cellular senescence or for producing a model for studying cellular senescence; preferably, the model comprises: an animal model or a cellular model.

10. A kit for conducting a senescence study comprising: the construct of any one of claims 1 to 4; or the cell of claim 6.

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