CN108588028B

CN108588028B - CIC cell model of targeting CDKN2A and preparation method thereof

Info

Publication number: CN108588028B
Application number: CN201810385867.1A
Authority: CN
Inventors: 孙强; 梁剑青; 牛祖彪; 黄红艳
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2021-11-09
Anticipated expiration: 2038-04-26
Also published as: CN108588028A

Abstract

本发明公开了一种靶向CDKN2A的cell‑in‑cell(CIC)细胞模型及其制备方法。本发明所提供的制备cell‑in‑cell相关细胞模型的方法，是通过调控靶细胞中CDKN2A基因的表达来制备得到cell‑in‑cell相关细胞模型。抑制靶细胞中CDKN2A基因的表达可制备诱导cell‑in‑cell细胞模型；使靶细胞中过表达CDKN2A基因可制备抑制cell‑in‑cell细胞模型。利用本发明所提供的这种通过干预CDKN2A基因表达促进CIC结构形成的方法。针对CDKN2A表达完整的肿瘤细胞，通过该方法有望促使CIC介导的胞内死亡，实现杀死肿瘤细胞、抑制肿瘤生长的目的。The invention discloses a cell-in-cell (CIC) cell model targeting CDKN2A and a preparation method thereof. The method for preparing a cell-in-cell related cell model provided by the present invention is to prepare a cell-in-cell related cell model by regulating the expression of CDKN2A gene in target cells. Inhibiting the expression of CDKN2A gene in target cells can create an induced cell-in-cell cell model; overexpressing CDKN2A gene in target cells can create a cell-in-cell cell model of inhibiting. The method for promoting the formation of CIC structure by interfering with CDKN2A gene expression provided by the present invention is utilized. For tumor cells with complete expression of CDKN2A, this method is expected to promote CIC-mediated intracellular death to achieve the purpose of killing tumor cells and inhibiting tumor growth.

Description

CIC cell model of targeting CDKN2A and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a cell-in-cell (CIC) cell model of a target CDKN2A and a preparation method thereof.

Background

Cell-in-Cell (CIC) structure refers to a unique Cell-stack-like structure formed by one or more living cells located inside another Cell. The CIC phenomenon is widespread among species, including the lower organisms amoeba, to the metazoa caenorhabditis elegans and in mammalian systems. According to the different types of cells forming CIC structure, the CIC structure can be divided into homogeneous CIC, such as CIC structure formed between tumor or epithelial cells with the same source; and heterogeneous CIC, CIC structures formed between cells of different origin.

At present, tumors are the most active field for the research on the structural biological significance of homogeneous CIC. Clinical tissue sample research reports that the CIC phenomenon is commonly found in human tumor tissues, such as breast cancer, colon cancer, cervical cancer, prostate cancer, liver cancer and the like, but is rarely found in normal tissues. Since the formation of homogeneous CIC structure mediates the death of internal cells, it is believed by the scholars that homogeneous CIC is essentially a cell death mechanism that can eliminate cells that escape the extracellular matrix or maliciously transformed cells, and it is seen that the formation of CIC structure between tumor cells is beneficial to killing tumor cells and limiting tumor growth.

The existing CIC research has the defects that a cell model regulated by a specific gene is lacked, an in-vitro research system is difficult to obtain, and the target specific gene cannot be realized.

Disclosure of Invention

The inventor of the invention discovers for the first time that the reduction of the expression of the cancer suppressor gene CDKN2A in cells can promote the formation of a CIC structure, further mediate the death of internal cells and achieve the purpose of killing the cells. The model is applicable to both non-tumor cell lines and tumor cell lines.

In a first aspect, the invention claims a method of preparing a cell-in-cell related cell model.

The method for preparing the cell-in-cell related cell model provided by the invention is used for preparing the cell-in-cell related cell model by regulating the expression of the CDKN2A gene in the target cell.

Wherein the target cell may be a tumor cell or a non-tumor cell. The target cell may be a human cell.

Further, the method may be method a or method B as follows:

the method A comprises the following steps: a method of preparing an induced cell-in-cell model may comprise the steps of: inhibiting the expression of the CDKN2A gene in the target cell.

The method B comprises the following steps: the method is a method for preparing a cell-in-cell inhibition cell model, and can comprise the following steps: overexpressing the CDKN2A gene in the target cells.

Further, in the method a, the target cell may be a cell in which the CDKN2A gene is efficiently expressed (e.g., expression of a protein encoded by the CDKN2A gene can be detected by Western blot). In method B, the target cell may be a cell in which CDKN2A gene is expressed or has a loss of function (e.g., expression of a protein encoded by CDKN2A gene cannot be detected by Western blot).

In a particular embodiment of the invention, in said method a, said target cell is in particular a HEK293 cell (normal human embryonic kidney cell). In said method B, said target cells are specifically MCF10A cells (non-transformed mammary epithelial cell line) or MCF7 cells (mammary cancer cell line).

Still further, in the method a, the "inhibiting the expression of the CDKN2A gene in the target cell" can be achieved by any technical means capable of achieving the purpose, such as specifically cleaving the CDKN2A gene by a sequence-specific nuclease (e.g., CRISPR/Cas9 nuclease), thereby reducing the expression thereof in the target cell; or protein function by inhibiting the expression of the CDKN2A gene by a compound, such as a CDKN2A inhibitor.

In a particular embodiment of the invention, in said method a, inhibiting the expression of the CDKN2A gene in said target cell is achieved in particular by CRISPR/Cas9 technology. In particular to the coincidence of 5' -GN in the CDKN2A gene sequence₁₉-a fragment of the regular NGG-3' sequence arrangement is the target sequence; n represents any one of A, G, C and T, N₁₉Represents 19 consecutive deoxyribonucleosides. If no match 5' -GN can be found within the target site range₁₉Sequence of-NGG-3 'alignment rule, then look for 5' -N₂₀NGG-3 'with G, i.e. 5' -G-N, added before the sgRNA₂₀-NGG-3'. Correspondingly, the two complementary sgRNA oligos corresponding to the target sequence are specifically SEQ ID No.1 and SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, or SEQ ID No.5 and SEQ ID No. 6.

Further, in the method B, the overexpression of the CDKN2A gene in the target cell is achieved by: overexpressing in the target cell at least one of four transcripts of the CDKN2A gene, the four transcripts of the CDKN2A gene being p16INK4a, p16 γ, p12, and p14 ARF.

In a particular embodiment of the invention, the overexpression of the transcript p16INK4a of the CDKN2A gene in the target cell is achieved in particular by introducing into the target cell a recombinant expression vector containing the gene coding for the transcript p16INK4 a; overexpressing the transcript p16 γ of the CDKN2A gene in the target cell, in particular by introducing into the target cell a recombinant expression vector containing the gene encoding the transcript p16 γ; overexpressing the transcript p12 of the CDKN2A gene in the target cell, in particular by introducing into the target cell a recombinant expression vector containing the gene encoding the transcript p 12; the overexpression of the transcript p14ARF of the CDKN2A gene in the target cell is specifically achieved by introducing a recombinant expression vector containing a gene coding for the transcript p14ARF into the target cell.

The recombinant expression vector is constructed by taking a retrovirus vector pQCXIP-EGFP-N1 as a framework and constructing a CDKN2A overexpression vector. Four transcripts of the CDKN2A gene were inserted between EcoRI and MfeI of the retroviral vector pQCXIP-EGFP-N1, respectively, to give 4 recombinant expression vectors.

In the present invention, the nucleotide sequence of the CDKN2A gene is as shown in GenBank: bit 21967752 and 21995043 of NC _000009.12 (completion, 2018-3-26). The nucleotide sequence of the coding gene of the transcript p16INK4a is specifically shown as SEQ ID No. 7. The nucleotide sequence of the coding gene of the transcript p16 gamma is specifically shown as SEQ ID No. 8. The nucleotide sequence of the coding gene of the transcript p12 is specifically shown as SEQ ID No. 9. The nucleotide sequence of the coding gene of the transcript p14ARF is specifically shown as SEQ ID No. 10.

In the present invention, the type of the cell-in-cell is specifically a homogeneous cell-in-cell (i.e., a cell-in-cell structure formed between homogeneous cells).

In a second aspect, the invention claims a cell model prepared by the method described above.

In a third aspect, the invention claims the use of the cell model in any of:

(A1) evaluating the influence of the substance to be tested (such as specific compound, extract, small molecule, polypeptide, protein, gene, therapeutic cell, etc.) on the disease process such as tumor growth by the cell model;

(A2) targeting a test agent (e.g., a particular compound, extract, small molecule, polypeptide, protein, gene, therapeutic cell, etc.) to the cell model to evaluate the biological safety of the test agent;

(A3) preparing a tumor treatment drug by using the cell model; for example, the cell-in-cell can be used for establishing a new disease (such as tumor) treatment means, including cells, nucleic acid, protein, small molecules and the like which can be used for treatment;

(A4) establishing a new animal model by or with the help of said cell model.

Experiments prove that the normal human embryonic kidney cell HEK293 (the cell effectively expresses CDKN2A) is used, a CRISPR/Cas9 system is adopted, and the expression of endogenous CDKN2A is knocked down by designing sgRNA, so that the formation of a CIC structure can be promoted. The CIC-inhibited cell model was obtained by re-expressing CDKN2A by retroviral vector transfection using a non-transformed mammary epithelial cell line MCF10A deficient in CDKN2A expression and a mammary cancer cell line MCF 7. The method for promoting the formation of the CIC structure by interfering the expression of the CDKN2A gene is utilized. Aiming at the tumor cells with effective CDKN2A expression, CIC mediated intracellular death is hopeful to be promoted by the method, and the purposes of killing the tumor cells and inhibiting the growth of the tumor are achieved.

Drawings

FIG. 1 shows the expression level of CDKN2A in each cell line measured by Western blot.

FIG. 2 is a schematic immunofluorescence of a cell-in-cell structure.

FIG. 3 shows that the targeted knock-down of CDKN2A gene promotes the formation of cell-in-cell structure. A: detecting the knocking efficiency of CDKN2A-sgRNA of three different targets by Western blot; b: cell-in-cell formation rate. P < 0.01; c: sequencing results of the pCS-CDKN2A-sgRNA expression plasmid.

FIG. 4 shows that over-expression of CDKN2A inhibits cell-in-cell structure formation from four different transcripts. A: non-transformed mammary epithelial cell line MCF 10A; b: the breast cancer cell line MCF 7. P < 0.01.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

HEK293 cells: ATCC cell bank, # CRL-1573.

cas9 working system plasmid precut pCS (puro): wu heyi doctor, a institute of bioengineering, purchased from beijing baiosai picture company.

Retroviral vector pQCXIP-EGFP-N1: described in "Wang M, Ning X, Chen A, Huang H, Ni C, Zhou C, et al. Impatient formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression. scientific reports. 2015; 5:12223 ", publicly available from the applicant, can only be used in the duplication of experiments.

pCMV-VSV-G plasmid: addgene, # 8454.

gag/pol plasmid: addgene, # 14887.

293FT cells: beina biological cell bank, BNCC 339263.

MCF10A cells: described in "Wang M, Ning X, Chen A, Huang H, Ni C, Zhou C, et al. Impatient formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression. scientific reports. 2015; 5:12223 ", publicly available from the applicant, can only be used in the duplication of experiments.

MCF7 cells: described in "Wang M, Ning X, Chen A, Huang H, Ni C, Zhou C, et al. Impatient formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression. scientific reports. 2015; 5:12223 ", publicly available from the applicant, can only be used in the duplication of experiments.

Example 1 establishment of induced CIC cell model

The target cell in this example is HEK293 cell, Western blot can detect expression of CDKN2A gene-encoded protein in HEK293 cell (fig. 1), and the specific detection method is described in Western blot in step one 6 of this example.

Establishment of HEK293/CDKN2A-cas9 knockdown cell line

1. Design of sgRNA oligo

According to the principle of adding PAM sequence after the target sequence, 3 CDKN2A-sgRNA were designed by using CDKN2A gene (shown in position 21967752-21995043 of GenBank: NC-000009.12, completion, 2018-3-26) as the target sequence as follows:

sgRNA-1：5’-caccGCACCGAATAGTTACGGTCGG-3’(SEQ ID No.1)；

5’-aaacCCGACCGTAACTATTCGGTGC-3’(SEQ ID No.2)。

sgRNA-2：5’-caccGACCGTAACTATTCGGTGCGT-3’(SEQ ID No.3)；

5’-aaacACGCACCGAATAGTTACGGTC-3’(SEQ ID No.4)。

sgRNA-3：5’-caccGTGGGCCATCGCGATGTCGCA-3’(SEQ ID No.5)；

5’-aaacTGCGACATCGCGATGGCCCAC-3’(SEQ ID No.6)。

2. construction of plasmid expressing CDKN2A-sgRNA

After primer synthesis, the sgRNA oligo was applied with ddH₂Dissolving O, wherein the final concentration is 100 mu M, mixing 15 mu L of each of the two complementary sgRNA oligos, putting the mixture into a boiling water bath, boiling for 5min, and naturally cooling to room temperature to finish annealing; then digesting a plasmid precut pCS (puro) of a cas9 working system by Bbs I, linearizing the plasmid, connecting the digested vector with the annealed sgRNA to obtain a plasmid pCS-CDKN2A-sgRNA for expressing CDKN2A-sgRNA, and verifying the construction by sequencing.

3. Plating cells

HEK293 cells were first plated at 1X 10 per well⁶The density of each cell was uniformly plated on a six-well plate previously embedded with collagen (collagen I), supplemented with DMEM (containing 10% FBS,% representing the volume percentage) to 2mL, and placed in an incubator to be cultured for 16 hours, followed by transfection experiments.

4. Liposome transfection system

Solution A: mixing 0.4 μ g of target plasmid pCS-CDKN2A-sgRNA and 100 μ L of Opti-MEM, standing at room temperature for 5 min; and B, liquid B: gently inverting and mixing the transfection reagent Lipo 2000, diluting 2.5 mu L Lipo 2000 into 100 mu L Opti-MEM, gently inverting and mixing, and standing at room temperature for 5 min; and C, liquid C: mixing solution A and solution B, mixing, and standing at room temperature for 30 min; the transfection mixture D was obtained by mixing 800. mu.L of Opti-MEM with 200. mu.L of solution C.

5. Transfected cells

The HEK293 original medium was discarded and 1mL of the mixed solution D was gently added along the inner wall of the well plate. After 5-6h of culture, the transfection solution is discarded, and the medium is replaced by a normal medium containing 10% (volume percentage content) FBS for continuous culture.

6. Pressure screening

Cells were pressure-screened for 5 days at 24h transfection using puromycin at a final concentration of 1 μ g/m to obtain a HEK293/CDKN2A-cas9 knockdown cell line. And detecting the knocking efficiency of CDKN2A-sgRNA of three different targets by Western blot.

Western blot detection steps are as follows: extracting target cell protein, adding cell lysate into ice, standing for 30min, shaking for 2-3 times by using a vortex oscillator to fully lyse cells, centrifuging at 4 ℃ for 20min at 12,000r/min, collecting supernatant, and quantifying the protein. The extracted protein is denatured for 5min at 95 ℃, 15% SDS-PAGE gel is adopted, the sample loading amount is 10 mu g, 100V is converted into a membrane for 40min, 5% BSA is sealed for 1h at room temperature, membrane cutting is carried out according to the molecular weight of the protein, an anti-E-cadherin antibody (BD; #610182), an anti-beta-Tubulin antibody (CWBIO; # CW0256) and an anti-p16INK4a antibody (BOSTER; # BM1592) are respectively added, the incubation is carried out at 4 ℃ overnight, TBST is washed for 3 times multiplied by 10min, a secondary antibody marked by horseradish peroxidase is added for incubation for 1h at room temperature, TBST is washed for 3 times multiplied by 10min, and finally a target band is detected by a chemiluminescence method. To ensure consistent loading, the Tubulin band was used as an internal control.

Second, Cell-in-Cell formation experiment

1. Preparing soft agar

A0.5% (0.5g/100mL) soft agar solution was prepared using pre-warmed PBS buffer, quickly added to a six-well cell culture plate at 1 mL/well, and the plate was gently shaken to evenly spread the soft agar solution on the bottom of the plate. Standing in room temperature water for several hours until it solidifies.

2. Cell suspension culture

Cells were digested into single cell suspensions using pancreatin, counting 2X 10 per well⁵The individual cells were plated on solidified soft agar, the medium was filled to 2 mL/well, and the cells were placed in a cell incubator for suspension culture for 13 hours.

3. Cell swinging sheet

After suspension culture is carried out for 13h, the phenomenon of cell suspension agglomeration can be observed under a microscope, the cell suspension is slightly sucked out, the cell suspension is placed in a centrifuge tube and centrifuged for 4min at 800rpm, the supernatant is discarded, 1mL of PBS is used for resuspension, the cell suspension is blown for 10-20 times by a gun to form single cell suspension, 200 mu L of the single cell suspension is rapidly taken and added into a flail machine, and the cells are flail for 4min at the rotating speed of 500rpm to be flail onto a glass slide.

4. Tabletting

Fixing the thrown slices with 4% paraformaldehyde at room temperature for 10-15min, washing with PBS for 2 times, 5min each time, slightly wiping the water on the slide, penetrating the membrane with 0.2% Triton X-100/PBS for 4min, and washing with PBS for 2 times × 5 min; blocking for 1h at room temperature by using 5% bovine serum albumin; adding an anti-E-cadherin antibody (1: 400 dilution; BD; #610182) after the sealing, and incubating overnight at 4 ℃ in a wet box; washing with PBS for 3 times × 10min the next day; adding a fluorescein-labeled secondary antibody (diluted 1: 500), and incubating for 1h at room temperature in a dry box; PBS was washed 3 times × 10min, and the slides were preserved with DAPI-containing slides, observed with a rotary disk laser confocal microscope (Perkin Elmer) and recorded by photography.

5. Cell-in-Cell statistics

And (3) observing the cell morphology and the cell-in-cell formation condition under a fluorescence microscope, wherein four uniform visual fields are randomly selected from each slide, and at least 200 cells (not including drilled cells) are counted in each visual field. Cells were identified by DAPI labeling of the nuclei, and the structure completely encapsulated by the outer cells was designated as 1 cell-in-cell structure.

cell-in-cell (%) — the average of the number of cells forming cell-in-cell/total number of cells × 100 ± SD.

The Cell-in-Cell structure is shown in FIG. 2, blue fluorescence indicates Cell nucleus, and red fluorescence marks Cell membrane protein E-cadherin for distinguishing boundaries between cells. Statistical results show that the sgRNA knockdown of the CDKN2A gene can significantly promote the formation of a cell-in-cell structure (fig. 3).

Example 2 establishment of CIC-inhibited cell model

Target cells in this example are MCF10A cells and MCF7 cells, Western blot cannot detect expression of CDKN2A gene-encoded protein in MCF10A cells and MCF7 cells (fig. 1), and the specific detection method refers to Western blot in step one 6 of this example.

First, establishment of MCF10A/CDKN2A overexpression cell line

A retroviral vector pQCXIP-EGFP-N1 is used as a framework to construct a CDKN2A overexpression vector, which comprises four different transcripts thereof, namely p16INK4a, p16 gamma, p12 and p14 ARF. The nucleotide sequence of the coding gene of the transcript p16INK4a is specifically shown as SEQ ID No. 7. The nucleotide sequence of the coding gene of the transcript p16 gamma is specifically shown as SEQ ID No. 8. The nucleotide sequence of the coding gene of the transcript p12 is specifically shown as SEQ ID No. 9. The nucleotide sequence of the coding gene of the transcript p14ARF is specifically shown as SEQ ID No. 10. The retroviral vector pQCXIP-EGFP-N1 is linearized by using restriction endonucleases EcoRI and MfeI through double enzyme digestion, then coding genes (corresponding enzyme digestion sites are added at two ends during artificial synthesis) of all transcripts are respectively inserted into the retroviral vector pQCXIP-EGFP-N1, four recombinant retroviral vectors corresponding to four transcripts are obtained, and the construction is verified to be correct through sequencing verification.

Then, the target vector is introduced into MCF10A cells by a retrovirus packaging and infection method, and the specific experimental steps are as follows: viral packaging cells 293FT were first plated at 1X 10 per well⁶Uniformly paving the cells on a six-hole plate embedded with collagen (collagen I) in advance, filling a culture medium DMEM (containing 10% FBS, wherein the volume percentage is% of the DMEM) to 2mL, putting the DMEM into an incubator, and culturing for 16h to perform a transfection experiment; the retrovirus packaging system consists of 0.4. mu.g of target vector (the four recombinant retrovirus vectors constructed above), 0.2. mu.g of pCMV-VSV-G plasmid, 0.25. mu.g of ggag/pol plasmid and lipofectin Lipo 2000; infecting the virus packaging system with cells to produce viruses, respectively collecting virus supernatants 24h, 48h and 72h after transfection, and supplementing 2mL of fresh normal culture medium after collecting the virus supernatants each time; the viral supernatant was centrifuged at 2000rpm5min, collecting supernatant, subpackaging and freezing at-80 deg.C for use.

Before infecting cells, target cells MCF10A are arranged at 1 × 10⁵Inoculating the virus into a six-hole plate, after the cells are completely adhered to the wall for 8-12 h, mixing 1mL of the virus supernatant with 500 μ l of a fresh complete culture medium, adding 2 μ l of Polybrene (the storage concentration is 10 μ g/mL), uniformly mixing, adding the mixture into the six-hole plate, infecting the cells, and after infecting for 6h, replacing the mixture with 2mL of the fresh culture medium for continuous culture; cells are subjected to pressure screening for 5 days by using puromycin with the final concentration of 2 mu g/mL 24h after infection, and cell strains stably expressing CDKN2A are obtained by taking complete cell death of blank control groups and 100% of GFP expression rate of positive control groups as judgment standards.

The MCF7/CDKN2A overexpression cell line is established by the same method as the above, and the puromycin screening concentration is 2 mu g/ml.

Second, Cell-in-Cell formation experiment

The specific procedure was the same as in step two of example 1, except that the cell suspension time was 6 hours. The re-expression of CDKN2A in the epithelial cell MCF10A or the tumor cell MCF7 can obviously inhibit the formation of a cell-in-cell structure (figure 4).

<110> military medical research institute of military science institute of people's liberation force of China

<120> CIC cell model of targeting CDKN2A and preparation method thereof

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cgccctaagc gcacattcat gtgggcattt cttgcgagcc tcgcagcctc cggaagctgt 1320

cgacttcatg acaagcattt tgtgaactag ggaagctcag gggggttact ggcttctctt 1380

gagtcacact gctagcaaat ggcagaacca aagctcaaat aaaaataaaa taattttcat 1440

tcattcactc aaaaaaaaaa aaaa 1464

<210> 9

<211> 1235

<212> DNA

<213> Artificial sequence

<400> 9

atggagccgg cggcggggag cagcatggag ccttcggctg actggctggc cacggccgcg 60

gcccggggtc gggtagagga ggtgcgggcg ctgctggagg cgggggcgct gcccaacgca 120

ccgaatagtt acggtcggag gccgatccag gtgggtagag ggtctgcagc gggagcaggg 180

gatggcgggc gactctggag gacgaagttt gcaggggaat tggaatcagg tagcgcttcg 240

attctccgga aaaaggggag gcttcctggg gagttttcag aaggggtttg taatcacaga 300

cctcctcctg gcgacgccct gggggcttgg gaagccaagg aagaggaatg aggagccacg 360

cgcgtacaga tctctcgaat gctgagaaga tctgaagggg ggaacatatt tgtattagat 420

ggaagtcatg atgatgggca gcgcccgagt ggcggagctg ctgctgctcc acggcgcgga 480

gcccaactgc gccgaccccg ccactctcac ccgacccgtg cacgacgctg cccgggaggg 540

cttcctggac acgctggtgg tgctgcaccg ggccggggcg cggctggacg tgcgcgatgc 600

ctggggccgt ctgcccgtgg acctggctga ggagctgggc catcgcgatg tcgcacggta 660

cctgcgcgcg gctgcggggg gcaccagagg cagtaaccat gcccgcatag atgccgcgga 720

aggtccctca gacatccccg attgaaagaa ccagagaggc tctgagaaac ctcgggaaac 780

ttagatcatc agtcaccgaa ggtcctacag ggccacaact gcccccgcca caacccaccc 840

cgctttcgta gttttcattt agaaaataga gcttttaaaa atgtcctgcc ttttaacgta 900

gatatatgcc ttcccccact accgtaaatg tccatttata tcatttttta tatattctta 960

taaaaatgta aaaaagaaaa acaccgcttc tgccttttca ctgtgttgga gttttctgga 1020

gtgagcactc acgccctaag cgcacattca tgtgggcatt tcttgcgagc ctcgcagcct 1080

ccggaagctg tcgacttcat gacaagcatt ttgtgaacta gggaagctca ggggggttac 1140

tggcttctct tgagtcacac tgctagcaaa tggcagaacc aaagctcaaa taaaaataaa 1200

ataattttca ttcattcact caaaaaaaaa aaaaa 1235

<210> 10

<211> 1164

<212> DNA

<213> Artificial sequence

<400> 10

cgctcaggga aggcgggtgc gcgcctgcgg ggcggagatg ggcagggggc ggtgcgtggg 60

tcccagtctg cagttaaggg ggcaggagtg gcgctgctca cctctggtgc caaagggcgg 120

cgcagcggct gccgagctcg gccctggagg cggcgagaac atggtgcgca ggttcttggt 180

gaccctccgg attcggcgcg cgtgcggccc gccgcgagtg agggttttcg tggttcacat 240

cccgcggctc acgggggagt gggcagcgcc aggggcgccc gccgctgtgg ccctcgtgct 300

gatgctactg aggagccagc gtctagggca gcagccgctt cctagaagac caggtcatga 360

tgatgggcag cgcccgagtg gcggagctgc tgctgctcca cggcgcggag cccaactgcg 420

ccgaccccgc cactctcacc cgacccgtgc acgacgctgc ccgggagggc ttcctggaca 480

cgctggtggt gctgcaccgg gccggggcgc ggctggacgt gcgcgatgcc tggggccgtc 540

tgcccgtgga cctggctgag gagctgggcc atcgcgatgt cgcacggtac ctgcgcgcgg 600

ctgcgggggg caccagaggc agtaaccatg cccgcataga tgccgcggaa ggtccctcag 660

acatccccga ttgaaagaac cagagaggct ctgagaaacc tcgggaaact tagatcatca 720

gtcaccgaag gtcctacagg gccacaactg cccccgccac aacccacccc gctttcgtag 780

ttttcattta gaaaatagag cttttaaaaa tgtcctgcct tttaacgtag atatatgcct 840

tcccccacta ccgtaaatgt ccatttatat cattttttat atattcttat aaaaatgtaa 900

aaaagaaaaa caccgcttct gccttttcac tgtgttggag ttttctggag tgagcactca 960

cgccctaagc gcacattcat gtgggcattt cttgcgagcc tcgcagcctc cggaagctgt 1020

cgacttcatg acaagcattt tgtgaactag ggaagctcag gggggttact ggcttctctt 1080

gagtcacact gctagcaaat ggcagaacca aagctcaaat aaaaataaaa taattttcat 1140

tcattcactc aaaaaaaaaa aaaa 1164

Claims

1. A method for preparing an induced cell-in-cell cell model, comprising the steps of: inhibiting the expression of CDKN2A gene in target cells; the target cells are HEK293 cells;

The type of the cell-in-cell is homogeneous cell-in-cell.

2 . The method according to claim 1 , wherein inhibiting the expression of CDKN2A gene in the target cells is achieved by CRISPR/Cas9 technology. 3 .