Construction method and application of lung cancer transformation animal model

Abstract

The application relates to the field of biotechnology, in particular to a method for constructing a lung cancer transformation animal model based on 3D organoids combined with gene editing, which comprises the following steps: culturing a normal lung epithelial organoid of a Trp53 gene deletion mouse, extracting mouse lung tissue, constructing a K-ras target gene expression vector containing the mouse lung tissue, extracting a plasmid, packaging a reverse transcription virus by adopting a calcium phosphate transfection method, infecting the organoid with the reverse transcription virus, subculturing and amplifying the successful organoid at a ratio of 1:3, preparing an organoid suspension after identification, injecting 300,000 cells in 75 muL into the left lobe of the mouse lung within 10 seconds, and then immediately suturing the incision. In the technical scheme of the application, after the mouse lung organoid is edited by the cDNA overexpression technology, the occurrence and development of human diseases can be effectively and quickly generated on the genomic and phenotypic levels, the model has stability and controllability, and the model can make up for the modeling technical bottlenecks such as hysteresis and uncontrollability of a traditional lung nodule modeling method in an animal body.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically a method for constructing a lung tuberculosis-transforming animal model based on 3D organoids combined with gene editing. Background Technology

[0002] Lung cancer is currently the leading cause of cancer-related morbidity and mortality worldwide. Early-stage lung cancer often manifests as pulmonary nodules. In recent years, with the rapid development of CT imaging and the widespread application of lung cancer screening, the global detection rate of pulmonary nodules has significantly increased. How to effectively prevent the malignant transformation of pulmonary nodules into lung cancer has become a major challenge for my country's healthcare system. Currently, clinical practice mainly focuses on follow-up observation for slowly progressing or unchanging nodules, lacking effective preventative and therapeutic drugs.

[0003] Compared to the lengthy clinical research cycle, experimental studies can answer questions about the efficacy and mechanism of action of traditional Chinese medicine formulas more quickly. Stable and reproducible animal models of pulmonary nodules, closely resembling clinicopathological characteristics, are not only key tools for studying the pathogenesis of pulmonary nodules, but also play an irreplaceable role in drug development for treating pulmonary nodules and preventing lung cancer. The most common histological type of lung cancer is non-small cell lung cancer (NSCLC), and lung adenocarcinoma is the most common type of NSCLC. Most lung adenocarcinomas develop from atypical adenomatous hyperplasia into adenocarcinoma in situ, then evolve into microinvasive adenocarcinoma, and subsequently gradually develop into invasive adenocarcinoma with an apocrine growth pattern. Therefore, animal models of lung adenocarcinoma are usually accompanied by AAH, pulmonary adenoma, hyperplasia, and other lesions, and are mostly induced by compounds or gene mutations.

[0004] However, limited basic experimental research severely restricts the improvement of pulmonary nodule diagnosis and treatment. Currently, the difficulty in pulmonary nodule experimental research lies in animal modeling. Because the etiology of pulmonary nodules is still unclear, the determination of benign or malignant nature relies on later observation, making animal modeling of pulmonary nodules extremely difficult. Current literature uses specific pathogens, such as carcinogens like urethane, Kveim antigen, Freund's complete adjuvant, Mycobacterium tuberculosis, and exogenous particles (PM2.5, silica dust, etc.). However, this modeling method is not only time-consuming, usually requiring several months or even more than a year, but may also exhibit different reactions and toxicities compared to in vivo tumors. More importantly, it cannot precisely control the pathological stage of pulmonary nodules, making it difficult to simulate the actual situation of chronic clinical disease. Therefore, there is an urgent need for a rapid, stable, controllable, and precisely localized pathological stage modeling method to fill some gaps in pulmonary nodule / early lung cancer lesion modeling and provide new modeling methods for future pulmonary nodule models. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a lung tuberculosis transformation animal model based on 3D organoids combined with gene editing.

[0006] To achieve the above objectives, the technical solution adopted by this invention is: a method for constructing a lung tuberculosis transformation animal model. This method is based on 3D organoids combined with gene editing. Specifically, it involves: culturing normal lung epithelial organoids from mice with a Trp53 gene deletion; extracting mouse lung tissue and constructing an expression vector containing the K-ras target gene, followed by plasmid extraction; packaging retroviruses using calcium phosphate transfection; infecting the organoids with viral particles; passage and amplifying the successfully gene-edited organoids at a 1:3 ratio; identifying organoids in the logarithmic growth phase using HE and IHC staining; preparing organoid suspensions; injecting 300,000 cells (75 μL each) into the left lobe of the mouse lung within 10 seconds; immediately suturing the incision; periodically monitoring the mouse's weight, behavior, and health status post-surgery; observing the size of lung nodules using MicroCT technology; and collecting mouse lung tissue at weeks 4, 6, and 10 for HE and IHC staining to assess the biological characteristics of the tumor.

[0007] Furthermore, the CDS region sequence of the K-ras target gene is shown in SEQ ID No. 1.

[0008] Furthermore, the primer sequences used to construct the expression vector are: the forward primer sequence is 5′-ATGACTGAGTATAAACTTGTGGTGG-3′, and the reverse primer sequence is 5′-TCACATAACTGTACACCTTGTCCTT-3′.

[0009] Furthermore, the process after organoid infection also includes verifying the efficiency of organoid gene editing. Cell genomes are extracted from infected lung organoids, and q-PCR is used to verify the efficiency of organoid gene editing, ensuring the success of K-ras gene editing in lung organoids.

[0010] Furthermore, all cell culture flasks used for passage amplification must be coated with matrix gel.

[0011] The pulmonary nodule carcinoma transformation animal model constructed by the method of this invention has applications in basic research such as the malignant evolution of pulmonary nodules and the exploration of the pathogenesis mechanism of lung cancer.

[0012] The beneficial technical effects of the present invention are as follows: In the technical solution of the present invention, after gene editing of mouse lung organoids using cDNA overexpression technology, it is possible to effectively and quickly generate models that closely mimic the occurrence and development of human diseases at the genomic and phenotypic levels. The models are stable and controllable, and can overcome the bottlenecks in traditional animal lung nodule modeling methods, such as lag and uncontrollability. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 This is the flowchart of Example 1.

[0015] Figure 2 Kras mRNA expression levels in lung organoids of WT, Vector, and TK mice.

[0016] Figure 3 Figure 1 shows the results of RNA transcriptome analysis of Vector and TK organoids.

[0017] Figure 4 Bright field and GFP images of normal organoid morphology of WT mouse lungs.

[0018] Figure 5 Bright field and HE images of lung organoids from WT, Vector (Trp53), and TK (Trp53 / Kras-lsl-G12D) mice on day 5.

[0019] Figure 6 Statistical chart of lung organoid numbers on day 5 in WT, Vector, and TK mice.

[0020] Figure 7 Diameter statistics of lung organoids in WT, Vector, and TK mice on day 5.

[0021] Figure 8 Percentage of hollow and solid lung organoids on day 5 in WT, Vector, and TK mice.

[0022] Figure 9 IHC and IF images of lung organoids from WT, Vector, and TK mice.

[0023] Figure 10 Statistical graph of Ki67 expression in lung organoids of WT, Vector, and TK mice on day 5.

[0024] Figure 11 Schematic diagram of intrapulmonary orthotopic injection of organoids.

[0025] Figure 12 Schematic diagram of the technical route for the C57 / B6 mouse primary or in situ lung tuberculosis transformation model based on gene editing combined with organoids.

[0026] Figure 13Representative micro-CT scans of lung tissue in the TK model at weeks 4, 6, and 10.

[0027] Figure 14 Changes in nodule size in the control group and model group over 15 weeks.

[0028] Figure 15 Representative bright-field images of lung tissue from the TK model at weeks 4, 6, and 10.

[0029] Figure 16 Representative volumetric views and GFP images of lung tissue in the TK model at weeks 4, 6, and 10.

[0030] Figure 17 Weight changes in the control group (Control) and the model group (TK) over 15 weeks.

[0031] Figure 18 Changes in net tumor weight in the control group and model group over 15 weeks.

[0032] Figure 19 Representative HE and IHC images of lung tissue in the TK model at weeks 4, 6, and 10. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] Combination Figure 1 A method for constructing an animal model of pulmonary tuberculosis transformation, the specific steps of which are as follows:

[0036] S01. Lung tissue from Trp53 gene-deleted mice was obtained, and after shearing and collagenase digestion, it was cultured using a culture medium supplemented with various cytokines and supplements and Matrigel as a growth scaffold.

[0037] One-quarter lung tissue was taken from 5-week-old Trp53 gene-deleted mice and cut into 1mm pieces. 3Granular cells were digested in 25 mL of digestion buffer (DMEM / F12 medium, Collagenase Type I, IV ratio 2:1) at 37°C in a shaker for 30 min to dissociate the cells and prepare a single-cell suspension. After centrifugation and discarding the supernatant, the cells were lysed on ice with 2 mL of erythrocyte lysis buffer for 2 min. The reaction was terminated with DMEM / F12, and the supernatant was discarded after centrifugation. Mouse lung epithelial cells were counted at a ratio of 5 × 10⁶ cells / mL. 5 Plant organoids in 48-well plates using 30 μL Matrigel per cell. Add GlutaMax, B27 supplement, N2, gastrinin I, N-Acetylcysteine, Nicotinamide, mouse recombinant egf, mouse recombinant noggin, mouse recombinant FGF 10, R-Spondin 1, 10% Wnt-3A, Y27 cytokines and supplements to each well at 150 μL, changing the medium every 3 days.

[0038] S02 dissociates organoids from the matrix gel, constructs a cDNA expression vector targeting a specific region of the K-ras gene, extracts the K-ras plasmid, and then infects the organoids.

[0039] (1) Construction of the K-ras target gene expression vector: RNA was extracted from mouse lung tissue and reverse transcribed to form a cDNA sequence. The TAIR website was opened to find the CDS sequence of the K-ras target gene, as shown in SEQ ID No. 1. The multiple cloning site was found in the final target vector book, and two restriction enzyme sites that are only in the target vector and not in the target gene fragment were found. Appropriate 5'-3' primers were added to both ends, and the selected enzyme fragments were added to the primers to design the primers.

[0040] After obtaining the primers, PCR was performed using the cDNA sequence and primers to obtain the target gene fragment. The PCR product was added to a green nucleic acid loading container (to prevent spillage during sample loading) and subjected to agarose gel electrophoresis. Subsequently, the gel was rapidly cut horizontally and vertically for recovery, and 500 μL of sol was placed in a 1.5 mL tube, melted at 65 °C, poured into an adsorption column for 2 min, labeled, centrifuged at 12000 rpm for 30 s, washed once with 600 μL of 70% ethanol, centrifuged for 2 min, the ethanol was discarded, and the column was centrifuged empty for 3 min. The product was then washed with water (65 °C, 30 μL) for 2 min, followed by centrifugation for 1 min. 4.5 μL of the recovered product was ligated into 0.5 μL of P Easy, and the recombinant product vector was obtained through molecular cloning. The recombinant product was then transformed, plated, and bacteria were picked. Genomic DNA was extracted for PCR to verify the correct colonies.

[0041] (2) Plasmid extraction: Plasmids were extracted using a small-volume plasmid extraction kit. 2 mL of logarithmic-phase bacterial culture cultured overnight in LB medium was used, and the extract was heated at 12000 rpm. -1 Centrifuge for 1 min under the specified conditions, discard the supernatant; resuspend the bacterial cells in 300 μL of Buffer S1 containing RNase-de-RNase, ensuring uniform resuscitation; add 300 μL of Buffer S2, invert 3-6 times to fully lyse the bacterial cells, forming a clear solution; add 350 μL of Buffer S3, invert 5-8 times to thoroughly mix the bacterial suspension, and centrifuge at 12000 rpm. -1 Centrifuge for 10 min under the specified conditions; transfer the supernatant to a preparation tube and centrifuge at 12000 r·min. -1 Centrifuge for 40 seconds under the specified conditions; add 600 μL of Buffer W1, and centrifuge at 12000 rpm. -1 Centrifuge for 40 seconds under the specified conditions, discard the filtrate; add 800 μL Buffer W2, incubate at 12000 r·min -1 Centrifuge for 40 seconds under the specified conditions, discard the filtrate, and repeat this step once; place the preparation tube back into the centrifuge tube and centrifuge at 12000 r·min. -1 Centrifuge for 2 min under the given conditions; place the preparation tube into a new 1.5 mL centrifuge tube, add 60-80 μL of ddH2O to the preparation tube, let stand at room temperature for 2 min, then centrifuge at 12000 rpm. -1 Centrifuge for 1 min under the specified conditions.

[0042] (3) Target cell introduction: Retroviruses were packaged using calcium phosphate transfection: 293T cells were cultured as tool cells for virus packaging. Confluent and healthy 293T cells were evenly distributed into 6-well plates and cultured in a cell culture incubator at 37°C and 5% CO2. When the cell density reached 90%, virus packaging began. The culture medium was carefully removed and replaced with fresh, preheated 293T cell culture medium. Chloroquine (100mM) was added at a ratio of 1:4000 per well (to inhibit the reaction of acidification of endocytic vesicles and fusion of lysosomes caused by the addition of exogenous substances to cells). The mixture was gently shaken and placed in a cell culture incubator for later use. Transfection reagent A was prepared according to the table below. The components of the lentivirus and retrovirus packaging reagents are as follows:

[0043]

[0044]

[0045] Take a new EP tube and add 100 μL of 2×HBS (transfection reagent B). Place solution B on a vortex mixer at approximately 1500 rpm and add solution A dropwise to solution B, mixing thoroughly. Evenly drop the mixture into each well of a six-well plate. Under a microscope, a black precipitate can be observed evenly distributed in the intercellular spaces. Return the plate to the cell culture incubator for further culture. After 10-12 hours, replace with fresh, preheated cell culture medium. Collect the culture at 18, 24, and 30 hours. After each collection, replace the supernatant with fresh, preheated 293T medium. Collect the virus and store it at 4°C for short-term use. Simultaneously, use the Vector empty vector plasmid to package retroviruses for subsequent blank control infection of gene-edited organoids.

[0046] (4) Organoid infection: Add TrypLE digestive enzyme to a 48-well plate, aspirate lung organoids into 15mL BD tubes, mix by pipetting, and place in a 37℃ water bath for digestion; after 5 minutes, pipette tip to about 20 times to fully digest the cells, and continue to digest in a 37℃ water bath for 5 minutes; after digestion, pipette tip to 10 times, and centrifuge at 1500rpm for 5 minutes.

[0047] After centrifugation, discard the supernatant, resuspend the cells in DMEM / F12, and add them to a 24-well plate. Add the virus filtered through a 0.22 μm filter, and then add Polybrene at a ratio of 1:1000. Gently shake to mix. Centrifuge the 24-well plate at 31°C and 2000 rpm for 1 hour. Remove the plate and place it in a cell culture incubator for infection for 2 hours. Transfer the infected cell suspension to a 15 mL BD tube and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, resuspend the cells in matrix gel, and seed them back into a 48-well plate. After 15 minutes, add culture medium and incubate in a cell culture incubator.

[0048] S03. Utilize Luciferace and qPCR to identify and verify the efficiency of organoid gene editing, ensuring the successful editing of Trp53 and K-ras genes in lung organoids.

[0049] Forty-eight hours post-infection, fluorescent signals in organoids could be detected using Luciferace. The luciferase was derived from a gene expressed by the Peasy vector plasmid within the organoid. Genomic DNA was extracted from infected lung organoids, and q-PCR was used to verify the organoid (TK) gene editing efficiency. Figure 2 The results showed that the expression level of Kras mRNA in TK mouse lung organoids was significantly increased, proving that the K-ras gene editing in lung organoids was successful.

[0050] After confirming successful K-ras gene overexpression, transcriptomic analysis was performed on organoids to observe the functional impact of K-ras overexpression. Library preparation and RNA-seq were conducted at Hangzhou Lianchuan Biotechnology Co., Ltd. Libraries were prepared using 30-100 ng of RNA from each culture. These libraries were sequenced on Illumina Next-Seq and Nova-Seq platforms, with each sample covering 40-60 million reads. The quality of the reads was assessed using FASTQC software.

[0051] Targeted NGS sequencing was performed using the Solid Tumor Gene List v4. DNA was extracted from FFPE tissue samples, captured using KapaRoche reagents and Integrated DNA Technology (IDT) probes, and sequenced using the Illumina Hi-Seq and Nova-Seq platforms. Multiple annotation and filtering algorithms, including COSMIC database v88, dbSNPv150, and Annovar, were used to confirm mutations. Furthermore, DNA mutations with a variant allele frequency greater than or equal to 0.25 were filtered out to exclude germline mutations. Gene set annotations were obtained in GTF format. The hisat2 index was constructed from the genome index using hisat2-build in Hisat2 version 2.1.0. After trimming with TrimGalore, the raw RNA-seq paired-end reads were aligned to the genome using hisat2. The total reads per sample ranged from 370,000 to 620,000,000, with an alignment mapping rate greater than 90%.

[0052] Next, differential gene expression among the WT, Vetor, and TK organoid groups was estimated based on the counts generated by HTSeq. We used standard DESeq2 parameters to exclude genes with no readings and genes with a p-value set to the nominal value of 1, and plotted a volcano plot of differential gene expression. Figure 3 (Left). The results showed that overexpression of the Kras gene in TK organoids altered their biological function. KEGG pathway analysis was performed using the R package clusterProfiler, and gene set enrichment analysis of the MSigDB pathway from the Broad Institute was conducted using our own algorithm. Figure 3 (Right) The differentially expressed genes are mainly closely related to tumor-related pathways, such as MAPK, PI3K / Akt, and other pathways related to tumor cell proliferation, migration, and invasion.

[0053] S04. Organoids in the logarithmic growth phase were stained with HE and IHC to confirm the pathological condition of TK organoids.

[0054] Expanding the successfully gene-edited organoids: When cells reach 80% contact ratio, passage them at a 1:3 ratio. Digest in a TrypLE 37℃ water bath for 5 minutes, then pipette 10 times; repeat once. Centrifuge, discard the supernatant, mix well, and passage at a 1:3 ratio. All cell culture flasks used for passage must be coated with Matrigel. The number of cells expanded depends on specific experimental requirements.

[0055] S05. RNA-seg technology was used to perform phenotypic analysis on TK organoids, including analysis of cell proliferation, differentiation, migration and signal transduction pathways, to assess the impact of K-ras mutations on organoid function.

[0056] The surface morphology of organoids was observed using an optical microscope, and the number and diameter changes of organoids were counted. The internal structure of organoids was observed by labeling them with GFP (green fluorescent protein), revealing a hollow, cavitary structure. Figure 4 4% paraformaldehyde was added to each well overnight to fix the organoids. They were dehydrated and embedded in paraffin according to standard procedures. After sectioning (5 μm), they were stained with hematoxylin and eosin using hematoxylin and eosin. IHC / IF (antibodies: TTF-1, CK7, Ki-67) and hematoxylin were used for nuclear or cytoplasmic staining. Phenotypic changes in lung organoids from WT, Vector (Trp53), and TK (Trp53 / Kras-lsl-G12D) mice were compared on day 5. Hematoxylin and eosin staining (HE staining) Figure 5 It was found that all groups of organoids grew stably; the alveoli of WT and Vector were vacuolated and uniform in size; and the alveoli of TK organoids were larger in diameter and more numerous, and showed actual structures. Figure 6 , 7 8). IHC staining and IF were used to confirm the pathological status of TK organoids. CK7 nonspecific positivity and TTF-7 negativity indicated that TK organoids had not yet differentiated into lung cancer. Scanning electron microscopy revealed that Ki-67+ cells had a higher proliferative capacity in TK organoids than in normal and Vector lung organoids. Figure 9 , 10 ).

[0057] S06. Luciferase-labeled organoids were orthotopically transplanted into mouse lung parenchyma via percutaneous intrapulmonary injection. The luciferase-labeled TK organoids were digested in TrypLE at 37°C, then mechanically dissociated, centrifuged, and resuspended in 30% matrix gel to prepare an organoid suspension. Mice were anesthetized with isoflurane gas, and the hair on the left chest was shaved and disinfected. A 0.5 cm lateral ventral incision was made to expose the intercostal muscles. Then, 300,000 cells (75 μL each) were injected into the left lobe of the lung over 10 seconds, followed immediately by suturing the incision. Figure 11 ).

[0058] S07. After the above operations are completed ( Figure 12Postoperatively, the mice's weight was monitored regularly. Figure 17 ), changes in lung tissue volume ( Figure 14 Observations showed that the nodules nearly disappeared within the first four weeks after vaccination, exhibited slow growth after four weeks, and then showed a rapid growth trend after approximately six weeks. Therefore, the size of the lung nodules was observed using MicroCT technology at weeks 4, 6, and 10. Figure 13 In this study, lung nodules in mice were captured and their morphology was observed. Representative volumetric views and GFP images of lung tissue in the TK model at weeks 4, 6, and 10 showed that the tumor volume in the TK lung nodule carcinoma transformation model group gradually increased. Figure 15 ,18), fluorescence signal enhancement ( Figure 16 ).

[0059] S08. At weeks 4, 6, and 10, mouse lung tissue was collected for HE and IHC staining (method as in 9) to assess the biological characteristics of the tumor. The results showed that lung nodule tissue did not show positive expression of lung cancer tumor markers TTF-1, Ki-67, and CK7 at 4 weeks; however, over time, the expression of lung cancer tumor markers TTF-1, Ki-67, and CK7 gradually increased, indicating an increase in tissue malignancy. Figure 19 ).

[0060] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless defined as herein.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for constructing an animal model of pulmonary tuberculosis transformation, characterized in that, One-quarter of the lung tissue from 5-week-old mice with the Trp53 gene missing was used for lung epithelial organoid culture. After extracting mouse lung tissue and constructing an expression vector containing the K-ras target gene, plasmids were extracted and packaged into retroviruses using calcium phosphate transfection. The viral particles were used to infect the organoids. The successfully edited organoids were passaged and amplified at a ratio of 1:

3. The successfully edited organoids in the logarithmic growth phase were identified by HE and IHC staining and then an organoid suspension was prepared. 75 μL of the suspension containing 300,000 organoids was transplanted orally into the left lobe of the mouse lung within 10 seconds, and the incision was immediately sutured. The weight, behavior and health status of the mice were monitored regularly after surgery. The presence of tumor cell fluorescence was observed weekly using the IVISSpectrum imaging system and the size of lung nodules was observed using MicroCT technology. Lung tissues of the transplanted mice were collected at weeks 4, 6 and 10 for HE and IHC / IF staining of lung adenocarcinoma markers Ki-67, TTF-1 and CK-7 to evaluate the biological characteristics of the tumor. The in situ transplantation into the left lobe of the mouse lung specifically involves injecting the organoid suspension into the left lobe of the mouse lung. The CDS region sequence of the K-ras target gene is shown in SEQ ID No. 1; The primer sequences used to construct the expression vector are: the forward primer sequence is 5′-ATGACTGAGTATAAACTTGTGGTGG-3′, and the reverse primer sequence is 5′-TCACATAACTGTACACCTTGTCCTT-3′; The lung nodules nearly disappeared within 4 weeks immediately after vaccination, then began to grow slowly after 4 weeks, and then began to grow rapidly after about 6 weeks.

2. The construction method according to claim 1, characterized in that, The organoid infection process also includes verifying the gene editing efficiency of the organoids. The cell genome of the infected organoids is extracted, and q-PCR is used to verify the gene editing efficiency of the organoids to ensure that the K-ras gene editing of the organoids is successful.

3. The construction method according to claim 1, characterized in that, All cell culture flasks used for passage and amplification must be coated with matrix gel.

4. The application of the pulmonary nodule carcinoma transformation animal model constructed by the construction method according to any one of claims 1-3 in the exploration of the malignant evolution of pulmonary nodules and the pathogenesis mechanism of lung cancer.

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