A method for constructing a controllable immortalized cell line tool pig and its application

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a method for constructing a controllable immortalized cell line tool pig and its application. The construction method includes co-transfecting a transposon plasmid carrying an immortalization gene and a reversibly inducible expression system with a transposase expression plasmid to obtain monoclonal donor cells, followed by nuclear transfer to obtain transgenic pig clone embryos, and finally transplanting these embryos into sows for natural farrowing to obtain a controllable immortalized cell line tool pig. This invention also includes a method for preparing a controllable immortalized cell line by isolating tissue cells from the constructed controllable immortalized cell line tool pig and reversibly inducing immortalization, as well as the resulting controllable immortalized cell line. This invention breaks through the traditional approach of directly obtaining immortalized cell lines by using a live tool pig to pre-implant the immortalization system within a live pig, thereby achieving the systematic development of a pig immortalized cell line.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for constructing a controllable immortalized cell line tool pig and its application, which is used in the preparation of controllable immortalized cell lines. Background Technology

[0002] Pigs are important agricultural and economic animals, and also key large animal models in biomedical research, playing an irreplaceable role in disease models, drug screening, vaccine development, and organ transplantation research. However, compared to humans and mice, stable pig cell line resources are extremely scarce. As shown in Table 1, institutions such as the American Type Culture Collection (ATCC), the German Microbiology and Cell Culture Collection (DSMZ), the National Center for Type Culture Collection (NCACC) of the Chinese Academy of Sciences, and the China Center for Type Culture Collection (CCTCC) only preserve 15 immortalized pig cell lines, which severely restricts their basic research and industrial applications.

[0003] Table 1: Existing porcine cell lines and preservation institutions

[0004]

[0005] Currently, related research mainly relies on primary cells obtained directly from pigs. However, primary cells have many limitations: slow proliferation, limited passage times, and susceptibility to replicative senescence; the acquisition and isolation process is complex, dependent on live pigs, and costly; furthermore, there are large batch variations, resulting in poor experimental reproducibility, making it difficult to meet the needs of long-term, large-scale research. Therefore, developing safe, stable, and controllable immortalized pig cell lines is an urgent need in the fields of pig genetics and breeding and biomedical research.

[0006] The most commonly used method for cell immortalization is lentivirus-mediated gene delivery technology. This involves randomly integrating immortalization genes (such as SV40LT, hTERT, HPV-E6 / E7, etc.) into the genome of primary cells cultured in vitro using lentivirus vectors. Strong promoters continuously drive the expression of these genes, thereby enabling the cells to acquire unlimited proliferative capacity. The general technical approach is as follows:

[0007] 1) Vector construction: Selected immortalized genes (such as SV40LT, hTERT, HPV-E6 / E7, etc.) are cloned into lentiviral expression vectors, which usually contain strong promoters (such as CMV promoters) to drive the sustained, high-level expression of the target gene.

[0008] 2) Virus packaging: The constructed lentiviral vector and helper plasmid were co-transfected into 293T cells to package infective recombinant lentiviral particles.

[0009] 3) Cell infection: Collect viral supernatant and infect target primary cells;

[0010] 4) Screening and amplification: Cells that have successfully integrated the exogenous gene are screened using resistance markers on the vector (such as puromycin and neomycin), and then amplified and cultured to obtain immortalized cell lines;

[0011] 5) Application: The obtained immortalized cell lines will be used for subsequent experimental research.

[0012] For example, Chinese patent application CN 118497278A discloses a method for constructing an immortalized porcine peritoneal mesothelial cell line and its application, including the following steps: S10, providing a transfection system; S20, providing packaging cells, mixing the transfection system and the packaging cells, and packaging with lentivirus to obtain lentivirus particles; S30, providing isolated primary porcine peritoneal mesothelial cells, transducing the lentivirus particles with the porcine peritoneal mesothelial cells, and screening to obtain an immortalized porcine peritoneal mesothelial cell line.

[0013] For example, Chinese patent document CN 109706181A discloses a method for constructing an immortalized porcine hepatic stellate cell line, the immortalized porcine hepatic stellate cell line, and its application. The method isolates normal porcine hepatic stellate cells from porcine liver, transfects them with SV40LT overexpressing lentivirus, and obtains an immortalized porcine hepatic stellate cell line.

[0014] For example, Chinese patent application CN 113528453A discloses an immortalized porcine macrophage cell line, its construction method and application, which is obtained by co-transfecting the immortalized genes SV40LT and hTERT into primary porcine bone marrow-derived macrophages via lentiviral infection.

[0015] While these techniques using lentiviruses can obtain immortalized cells, they have the following significant drawbacks: 1) High risk of random integration: Lentiviral vectors integrate randomly into the host cell genome, potentially disrupting the function of important endogenous genes or activating the expression of proto-oncogenes, leading to malignant transformation and genomic instability; 2) Impaired cell function: Immortalized genes (such as SV40LT) are continuously and highly expressed under strong promoters, interfering with normal cell metabolism, differentiation, and signaling pathways, resulting in significant differences in cell function compared to the original cells, such as decreased differentiation capacity and abnormal metabolic state; 3) Genomic instability: Long-term suppression of cell cycle checkpoints such as p53 / Rb by viral oncogenes leads to the continuous accumulation of chromosomal abnormalities and mutations, affecting the long-term stability of cell lines and the reliability of experimental results; 4) Lack of controllable... 5) Limited efficiency of multi-gene delivery: Some cell types (such as primary porcine cells) cannot achieve stable immortalization with a single immortalization gene, while lentiviral vectors have limited carrying capacity (the size of the inserted fragment is no more than 4-5kb), and the infection efficiency of multi-gene co-delivery is extremely low, making it difficult to achieve the synergistic introduction of multiple genes; 6) Long cell construction cycle and complex process: This technical route is done once for each type of cell. For each type of tissue-derived cell, it is necessary to repeat the lengthy process of virus packaging, infection, and screening, which is inefficient and time-consuming, and seriously restricts the systematic development of porcine cell line resources.

[0016] In recent years, research teams have attempted to construct conditionally immortalized cell lines using the Tet-On system. The general technical procedure involves first introducing Tet-On-regulated immortalization genes into primary cells using vectors such as lentiviruses, obtaining stably transfected cell lines through drug screening, and then inducing immortalization gene expression by adding doxycycline (Dox) to obtain immortalized cells. The specific steps include: 1) constructing a Tet-On-regulated lentiviral vector; 2) infecting primary cells with lentivirus; 3) adding Dox to induce immortalization gene expression; and 4) obtaining immortalized cells.

[0017] For example, Chinese patent application CN 109022488A discloses a method for constructing a conditional cell immortalization lentiviral vector, and the pLVX-Tet3G-LargeT-T2A-mCherry-PuroR lentivirus packaged with this lentiviral vector and its application in establishing porcine ovarian granulosa cell lines. A reversible and inducible porcine ovarian granulosa cell line was established using the Tet-on-3G system. This cell line exhibits inducibility and reversibility. When Dox is added, cell proliferation can be stably maintained in vitro; when Dox is removed, ovarian granulosa cells can differentiate into functional terminal cells, secreting more estrogen and progesterone, but at this time, they lose their proliferative capacity; when Dox is added again, the cells will return to a proliferative state.

[0018] Compared to traditional continuous expression methods, this technical solution introduces controllability, but its core gene delivery method is still lentiviral vectors, thus failing to address the following issues: 1) The risk of random integration remains: Due to the continued use of lentiviral delivery, the risk of random integration persists, potentially leading to genomic instability and oncogenicity; 2) The construction process remains cumbersome: For each cell type, a series of steps such as lentiviral packaging, infection, and screening are still required, failing to fundamentally solve the problems of long construction cycles and low efficiency; 3) Poor versatility: This strategy still involves individual construction, failing to achieve the systematic and efficient acquisition of immortalized cell lines from various tissues from a universal resource library, and lacking the capability for large-scale cell resource development.

[0019] In summary, although existing technologies have addressed the controllability issue of porcine immortalized cell lines to some extent, problems remain unresolved, including genomic instability and potential safety risks arising from the random integration of immortalized genes in traditional methods, as well as the inefficiency and lengthy process of constructing individual cells for different tissues. These issues limit the application potential of porcine immortalized cell lines. Therefore, developing a systematic, efficient, and safe resource platform for obtaining multi-tissue porcine immortalized cell lines is of paramount importance. Summary of the Invention

[0020] To address the above-mentioned technical problems, this invention provides a technical solution for cell line tool pigs, namely, instead of directly generating immortalized cells, a controllable immortalization system is pre-placed in a living pig, making the living pig a resource library for cell line development, thereby providing a method for constructing controllable immortalized cell line tool pigs and their applications.

[0021] The present invention specifically provides the following technical solutions:

[0022] In a first aspect, the present invention provides a method for constructing a controllable immortalized cell line tool pig, comprising the following steps:

[0023] S1: Construct transposon plasmids that include immortalized genes and reversible inducible expression systems;

[0024] S2: Co-transfect porcine donor cells with the transposon plasmid and the transposase expression plasmid to obtain monoclonal donor cells;

[0025] S3: The monoclonal donor cell is nuclearly transferred to an enucleated oocyte to obtain a transgenic pig cloned embryo;

[0026] S4: The transgenic pig cloned embryo is transplanted into a recipient sow for natural birth, resulting in a controllable immortalized cell line tool pig.

[0027] Preferably, the immortalization genes mentioned in step S1 are selected from one or more of SV40LT, HPV-E6 / E7, Bmi-1, CDK4, EBV, and TERT. These genes can all achieve the goal of enabling cells to acquire continuous proliferation ability, and the choice of gene combination depends on the type of target cells and specific research needs.

[0028] Preferably, the inducer of the reversible inducible expression system described in step S1 is selected from one or more of doxycycline, ecdysone, rapamycin, and mifepristone. These inducers can all be applied to reversible, small-molecule induced gene expression regulation systems, enabling controllable expression with low background and high induction rate, and achieving precise on / off control of immortalized gene expression.

[0029] Preferably, the transposon in step S1 is selected from the PB transposon system, the SB transposon system, or the Tol2 transposon system. These transposon systems are all non-viral transposon systems with large fragment carrying capacity and stable integration capability, which can achieve efficient and stable integration of exogenous gene modules and avoid the risk of random integration by lentiviruses.

[0030] Preferably, the co-transfection in step S2 integrates the transposon plasmid into loci 43063259-43063260 on pig chromosome 4 and 127215595-127215596 on pig chromosome 13; or into pRosa26 or pH11 loci. These loci are safe sites on pig chromosomes, and the insertion of the foreign gene does not affect the host's normal growth, development, and reproduction, while allowing the foreign gene to be expressed stably for a long time.

[0031] Secondly, the present invention also provides a method for preparing a controllable immortalized cell line, comprising the following steps:

[0032] 1) Isolate tissue cells from the controllable immortalized cell line tool pig obtained by the above-described construction method of the present invention;

[0033] 2) Add an inducing agent to the culture medium of the tissue cells to induce reversible immortalization of the tissue cells and obtain a controllable immortalized cell line.

[0034] Thirdly, the present invention also provides the application of the above-described method in the preparation of controllable immortalized cell lines.

[0035] The present invention has the following beneficial effects:

[0036] This invention breaks with the traditional approach of directly obtaining immortalized cell lines. Instead of directly obtaining immortalized cell lines, it uses a live tool pig to pre-implant the immortalization system within the pig, thereby achieving the systematic development of immortalized cell lines. Specifically, this invention has the following beneficial effects:

[0037] First, this invention represents a paradigm shift from the traditional, piecemeal construction model to a system-wide development approach. The cell line tool pig constructed by this invention is a living cell resource bank. By cultivating only one tool pig, cells can be isolated from its various tissues at any time, and immortalized cell lines can be obtained through simple Dox induction. This shortens the lengthy cycle (several months to half a year) of traditional methods, where each type of cell is processed individually, to a universal model that allows for permanent use after a single construction, significantly improving the efficiency of porcine cell line resource development.

[0038] Secondly, this invention significantly improves the safety and genetic stability of immortalized cell lines. This invention uses the PB transposon system instead of lentivirus, avoiding the risk of random viral integration. Whole-genome sequencing identifies the precise integration site of the exogenous sequence, and karyotype analysis shows normal chromosome number and structure (2n=38, XY), ensuring a clear and stable genetic background for the cell line. Simultaneously, the reversible regulation through the Tet-On system prevents the continuous expression of immortalized genes, significantly reducing the potential risk of malignant transformation.

[0039] Third, this invention achieves reversible regulation of cell immortalization. By adding or removing Dox, the immortalized state of cells can be precisely controlled like a switch. When a large number of cells are needed, the switch is turned on to obtain unlimited proliferation capacity; when it is necessary to simulate normal in vivo function, the switch is turned off, and cell characteristics are restored. This reversibility, to a certain extent, solves the problem of impaired function in traditional immortalized cells.

[0040] Fourth, this invention overcomes the challenges of delivering large fragments or multiple genes. The PB transposon system used in this invention has a large carrying capacity, enabling the delivery of large gene fragments or multiple immortalized genes at once. This solves the problems of limited capacity of lentiviral vectors or poor immortalization effects of single genes, ensuring the success rate and stability of immortalization.

[0041] Fifth, this invention has strong versatility and can be adapted to construct cells from different tissue sources in pigs, making it applicable to a wide range of fields. Reversible immortalized cell lines have been successfully constructed in pig ear fibroblasts and adipose tissue SVF cells, fully demonstrating the excellent versatility of this technology, which can meet the needs of reversible immortalization modification of various somatic cells and has a wide range of applications. Attached Figure Description

[0042] Figure 1 The diagram shows the overall technical roadmap for constructing the cell line tool pig of the present invention and its immortalized cell line.

[0043] Figure 2A The image shows the pattern of the PB-Tet-On-SV40LT-P2A-EGFP-PuroR recombinant plasmid of the present invention.

[0044] Figure 2B The diagram shows a Sanger sequencing illustration of the PB-Tet-On-SV40LT-P2A-EGFP-PuroR recombinant plasmid of this invention.

[0045] Figure 3A The images show morphological photographs taken on days 6, 9, and 15 of the culture and screening process of the transgenic monoclonal cells obtained after successful transfection according to this invention.

[0046] Figure 3B The images show bright-field and GFP observations of five transgenic monoclonal cells (numbered PFF-PB-Tet-On-SV40LT-1 to 5) screened in this invention after Dox induction.

[0047] Figure 4A The present invention demonstrates the cloning experimental procedure for obtaining a cell line tool pig via somatic cell transplantation.

[0048] Figure 4B The images show 1-day-old and 180-day-old cloned piglets obtained by this invention.

[0049] Figure 5 The electrophoresis results of genotyping identification of the cloned piglets obtained in this invention are shown.

[0050] Figure 6 The PCR verification electrophoresis results of the gene integration sites of transgenic cloned piglets No. #0118 and No. #0121 of this invention are shown.

[0051] Figure 7 The results of Sanger sequencing of the gene integration site on chromosome 4 are shown.

[0052] Figure 8 The results of Sanger sequencing of gene integration sites on chromosome 13 are shown.

[0053] Figure 9 The results of karyotype analysis of transgenic cloned piglets No. #0118 and No. #0121 of this invention are shown.

[0054] Figure 10 The diagram illustrates the process of isolating, culturing, and immortalizing porcine ear fibroblasts from the constructed cell line of the present invention.

[0055] Figure 11 The diagram illustrates the process of isolating, culturing, and immortalizing porcine adipose SVF cells from the constructed cell line tool pig according to the present invention.

[0056] Figure 12 The images show morphological photographs of immortalized pig ear tissue cells obtained from the constructed cell line of the present invention at different passages.

[0057] Figure 13 The images show morphological photographs of immortalized porcine adipose SVF cells isolated and cultured from the constructed cell line tool pig according to the present invention at different passages.

[0058] Figure 14 The images show morphological photographs of immortalized pig ear tissue cells isolated and cultured from the constructed cell line of the present invention on days 1-6 after the removal of Dox induction.

[0059] Figure 15 The images show morphological photographs of immortalized porcine adipose SVF cells isolated and cultured from the constructed cell line of the present invention on days 1-6 after the removal of Dox induction. Detailed Implementation

[0060] The following detailed description provides further details through specific embodiments. However, it should be noted that the embodiments described below are merely for illustrating the invention more effectively and do not represent that the invention is limited to the given embodiments. Therefore, non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above description still fall within the scope of protection of this invention, and the scope of protection of the appended claims shall prevail.

[0061] The term "immortification" used in this invention refers to the process by which cultured cells, through physical, chemical, or biological methods, overcome the Hayflick limit and acquire the ability to continuously divide and proliferate indefinitely. Commonly used immortalization methods include telomerase activation, viral gene transfection, proto-oncogene activation, and tumor suppressor gene inhibition. Immortified cells are stable, homogeneous, and have consistent traits, making them ideal models for in vitro studies of cell proliferation, differentiation, apoptosis, and senescence, providing consistent and reproducible experimental results. They also allow for the immortalization of cells that are difficult to passage, proliferate slowly, and are prone to senescence, making them easier to culture and exhibiting stronger growth than primary cells, thus providing more cell resources and saving on cell separation cycles and costs. Immortified cells are also closely related to tumor cells and are important models for studying tumorigenesis mechanisms, contributing to a deeper understanding of the occurrence and development of cancer. Furthermore, they allow for the establishment of stable cell banks, forming genetically identical populations, facilitating related research by researchers at different times and locations.

[0062] Those skilled in the art will understand that the SV40LT used in this invention refers to the SimianVirus 40 Large T-antigen, a commonly used immortalization gene that enables cells to acquire unlimited proliferation capacity by inhibiting the activity of tumor suppressor proteins such as p53 and Rb.

[0063] Those skilled in the art will understand that HPV-E6 / E7, Bmi-1, CDK4, EBV, and TERT used in this invention are all commonly used immortalization genes in the field. HPV-E6 / E7 refers to the E6 (early 6th protein) and E7 (early 7th protein) proteins of human papillomavirus (especially type 16). E6 is responsible for degrading p53 tumor suppressor protein, and E7 is responsible for inhibiting Rb tumor suppressor protein, synergistically blocking the aging pathway. Bmi-1 refers to the B cell-specific Moronya murine leukemia virus insertion region 1 homolog, which achieves mild cell immortalization by inhibiting p16 tumor suppressor protein (p16INK4a). CDK4 refers to Cyclin-Dependent Kinase 4, which is the core engine kinase of the cell cycle and is key to promoting cell division and proliferation to achieve immortalization. EBV refers to Epstein-Barr Virus type 4, which achieves cell immortalization by regulating the inhibition of p16 tumor suppressor protein and promoting CDK4 activity. TERT refers to Telomerase Reverse Transcriptase. (transcriptase), hTERT stands for human telomerase reverse transcriptase, which is the catalytic subunit of telomerase. By maintaining telomere length, it enables cells to escape replicative senescence and achieve immortality.

[0064] Those skilled in the art will understand that the inducers used in this invention can be selected from doxycycline (Dox), ecdysone, rapamycin, mifepristone, etc., all of which are small molecule chemical reagents well known to those skilled in the art with reversible cell proliferation induction effects. Dox used in this invention is a tetracycline antibiotic, which acts as an inducer for the Tet-On system to turn the expression of the target gene on or off. The Tet-On system is a tetracycline-inducible expression system that reversibly activates the expression of downstream target genes in the presence of doxycycline (Dox); gene expression is turned off upon removal of doxycycline (Dox). The TRE3G promoter used in this invention is a modified promoter tightly regulated by the Tet-On system; it can only efficiently initiate the transcription of downstream genes after rtTA binds to Dox, thus achieving induction in conjunction with Dox.

[0065] Those skilled in the art will understand that the PB (PiggyBac Transposon System) used in this invention is a widely used tool in the fields of gene editing and gene transfer. It is a non-viral gene transfer tool that can efficiently and stably integrate exogenous DNA fragments into the host cell genome, offering advantages such as large carrying capacity, preferred integration sites, and good genetic stability. For example, PB transposons carrying specific genes (such as fluorescent protein genes, immortalization genes, etc.) can be introduced into cells, and through transposition, the genes are integrated into suitable locations in the genome, achieving long-term gene expression. In addition to the PB transposon system, this invention can also use the SB (Sleeping Beauty Transposon System) or Tol2 transposon system, both well-known to those skilled in the art. These are all non-viral DNA transposons, binary vectors, and can achieve stable integration of large fragments.

[0066] Those skilled in the art will understand that the somatic cell nuclear transfer technique used in this invention is a technique that involves transferring the nucleus of a donor cell into an enucleated oocyte, activating the cell, and then transferring the embryo to obtain a cloned pig with the same genetic background as the donor. The principles and operation of this technique are well known to those skilled in the art.

[0067] Those skilled in the art will understand that GFP (Green Fluorescent Protein) used in this invention refers to green fluorescent protein, a commonly used reporter gene for visually monitoring the expression of target genes; EGFP (Enhanced Green Fluorescent Protein) refers to enhanced green fluorescent protein, which is an artificially mutated and improved version of GFP.

[0068] Unless otherwise specified in this invention, any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose may be used. Even if the technologies, instruments, equipment, and materials used in this invention are specifically specified, it does not mean that this invention can only use these technologies, instruments, equipment, and materials, but merely represents the preferred solution of this invention. Those skilled in the art can still use any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose.

[0069] Example 1: Preparation of Cell Line Tool Pig

[0070] The technical roadmap for the overall construction process of the controllable immortalized cell line tool pig of this invention is as follows: Figure 1 As shown, the specific steps include:

[0071] (1) Construction of controllable immortalized gene expression vector

[0072] The coding sequences of the SV40LT immortalization gene and the reporter gene EGFP (Enhanced Green Fluorescent Protein) were amplified by polymerase chain reaction (PCR). Then, using homologous recombination technology, these genes were inserted into the PiggyBac (PB) transposon backbone plasmid containing the TRE3G promoter and the puromycin resistance gene (PuroR), constructing a recombinant plasmid named "PB-Tet-On-SV40LT-P2A-EGFP-PuroR".

[0073] The core components of this plasmid include: 1) the left and right arms of PB, used to mediate efficient / stable integration of exogenous modules; 2) Tet-On regulatory elements, including the trans-activator rtTA and the promoter TRE3G. In the absence of Dox, rtTA cannot bind to TRE3G, and transcription is shut down; in the presence of Dox, Dox binds to rtTA, and rtTA binds to TRE3G, initiating transcription of downstream genes; 3) the immortalization gene SV40LT, which efficiently and stably achieves cell immortalization; 4) the reporter gene EGFP, used for direct monitoring of the on / off state of gene expression; and 5) the puromycin resistance gene PuroR, used for subsequent screening of positive cell clones. The P2A portion is a commonly used linker element in this field for connecting two target genes.

[0074] The constructed plasmid map is as follows Figure 2A As shown; the sequence was verified to be correct by sequencing. Figure 2B A schematic diagram of the Sanger sequencing results for this plasmid is shown.

[0075] (2) Screening of monoclonal donor cells

[0076] Primary fetal fibroblasts (PFFs) were isolated and cultured from Rongchang pig fetuses at 30-35 days of gestation. The PB-Tet-On-SV40LT-P2A-EGFP-PuroR plasmid constructed in step (1) above was mixed with the PB transposase expression plasmid (System Biosciences, PB200A-1) at a molar ratio of 2.5:1 (total 10 μg). The mixed plasmid was co-transfected into the pig fetal fibroblasts using a NEPA 21 Type II electroporator (NEPA GENE) according to electroporation parameters #3 (perforation pulse: voltage 150V; pulse duration 5ms; pulse interval 50ms; number of pulses 2; decay rate 10%; pulse direction switching: none. Transfer pulse: voltage 20V; pulse duration 50ms; pulse interval 50ms; number of pulses 5; decay rate 40%; pulse direction switching: yes).

[0077] PB transposase recognizes the left and right arms of the transposon on the plasmid, efficiently and stably cleaving and integrating the entire expression module between the two arms into the host cell genome. The integration site is biased towards the TTAA sequence, reducing the risk of random integration. Forty-eight hours after transfection, 1 μg / mL of puromycin was added to the culture medium for drug selection, and cells were cultured for more than 10 days to kill untransfected cells. Finally, morphologically sound and stably growing positive monoclonal cell lines were obtained. Cell morphology photographs at 6, 9, and 15 days of culture are shown below. Figure 3A As shown.

[0078] Five monoclonal cell lines (numbered PFF-PB-Tet-On-SV40LT-1 to PFF-5, respectively) were used for functional validation. Induction was performed using 1 μg / mL Dox. Figure 3B As shown, all clones exhibited a clear GFP fluorescence signal, indicating that the Tet-On system functions normally in porcine cells and can precisely regulate the expression of exogenous genes through Dox.

[0079] (3) Somatic cell nuclear transfer

[0080] A single-clonal cell line with excellent growth and strong GFP fluorescence expression (numbered PFF-PB-Tet-On-SV40LT-4) was selected as the nuclear donor. Figure 4A The standard somatic cell nuclear transfer procedure shown describes the transfer of the nucleus of the monoclonal cell into an enucleated porcine oocyte to construct a cloned embryo. The constructed cloned embryos were then transferred into the oviducts of five recipient sows in natural estrus, successfully performing somatic cell nuclear transfer and obtaining cloned piglets. Figure 4B The cloned piglets at 1 day and 180 days of age are shown in Table 2 below.

[0081] Table 2: Status of somatic cell nuclear transfer and cloned piglets

[0082]

[0083] After the gestation period, a total of 6 cloned piglets were obtained (numbered #0118, #0119, #0120, #0121, #0122 and #0123 respectively). Two surviving cloned piglets, numbered #0118 and #0121, were used for subsequent experiments.

[0084] (4) Identification of transgenic pigs

[0085] Genotyping: Ear tissue was collected from piglets #0118 and #0121, and genomic DNA was extracted. Specific primers were designed targeting the TRE3G promoter, SV40LT gene, EGFP gene, TetOn3G gene, and PuroR gene sequences (as shown in Table 3 below, where primers with "F" represent forward primers and "R" represent reverse primers; β-actin represents β-actin and served as an internal control for electrophoresis results; these primer sequences correspond to SEQ ID NO: 1-12 in the sequence listing). Genotyping was performed using PCR. The electrophoresis lanes correspond to... Figure 5 The electrophoresis lanes shown indicate that the target band was amplified in all piglets, proving that the reversible immortalization system has been integrated into the genomes of piglets #0118 and #0121.

[0086] Table 3: Primer information for PCR identification of cloned pigs

[0087]

[0088] (5) Integration site analysis

[0089] The #0118 and #0121 cloned pigs underwent 30× whole-genome sequencing. The BWA alignment software (Burrows-Wheeler Aligner, version 0.7.17-r1188) was used to align to the pig genome reference sequence (Sscrofa 11.1, pig reference genome version 11.1) with default parameters. The SAM toolset (SAMtools, version 1.11) software was used to convert the SAM files (sequence alignment mapping files containing alignment information) generated by the BWA alignment software into BAM files (binary alignment mapping files). After sorting and removing duplicates, these BAM files were used to extract split reads for insertion site analysis.

[0090] The segmented reads of cloned pigs #0118 and #0121 are shown in Table 4 below. Analysis results show that the sequence of the plasmid "PB-Tet-On-SV40LT-P2A-EGFP-PuroR" was inserted and integrated into loci 43063259-43063260 on chromosome 4 and loci 127215595-127215596 on chromosome 13.

[0091] Table 4: Results of genome-wide analysis predicting exogenous sequence insertion sites

[0092]

[0093] Specific primers were further designed flanking the predicted integration site. The primer sequences are shown in Table 5, where primers with "F" in their names represent forward primers and those with "R" represent reverse primers; "chr4" represents chromosome 4, "chr13" represents chromosome 13, "Left" represents the left primer, and "Right" represents the right primer. These primer sequences correspond to SEQ ID NO: 13-20 in the sequence listing. PCR was performed using genomic DNA from #0118 and #0121 as templates to verify the foreign sequence integration sites predicted by whole-genome sequencing.

[0094] Table 5: Primer information for PCR identification of exogenous sequence integration sites

[0095]

[0096] like Figure 6 The electrophoresis results shown indicate that all PCR products were of the expected size. The PCR products were recovered and subjected to Sanger sequencing, as shown below. Figure 7 and Figure 8The sequencing results shown indicate that the exogenous sequence was integrated into the sequence between 43063259 and 43063260 on chromosome 4 and between 127215595 and 127215596 on chromosome 13.

[0097] (6) Karyotype analysis

[0098] Ear margin fibroblasts were isolated from transgenic pigs #0118 and #0121, and G-banded chromosome karyotype analysis was performed (Regen Biosciences, DM0001). Results are as follows: Figure 9 As shown, the cell chromosome number is 2n=38 (XY), consisting of 18 pairs of autosomes plus sex chromosomes. No abnormal chromosome number or obvious structural variations were detected, indicating that the cell line tool pig obtained in this invention has good genetic safety.

[0099] Example 2: Construction of a Controllable Immortalized Cell Line Based on a Cell Line Tool Pig

[0100] (1) Cell line tools: porcine cell isolation, culture and immortalization

[0101] Porcine ear fibroblasts (PEFs):

[0102] Cell line tool pig ear tissue was collected and washed with PBS containing penicillin-streptomycin to remove contaminants. Hair, epidermis, and subcutaneous fat were removed under aseptic conditions, and the dermis was cut into approximately 1 mm pieces. 3 After the tissue blocks were incubated in culture medium containing fetal bovine serum (FBS) for 5 minutes, the upper serum layer was aspirated, and the lower precipitate was inoculated into T25 culture flasks and incubated upside down in a 37°C, 5% CO2 incubator for approximately 8 hours to allow the tissue to adhere to the walls. Subsequently, DMEM containing 20% ​​FBS (Sigma, #F8318) and 4% penicillin-streptomycin was slowly added, and half of the culture medium was replaced every 2-3 days during the incubation period.

[0103] Figure 10 The images show the isolation and culture process of porcine ear fibroblasts. Spindle-shaped cells appear around the tissue blocks after 5-7 days. When the cell confluence reaches 80%-90%, the cells are digested and passaged. Ear tissue cells isolated from working pigs were induced to develop fibroblasts by adding 4 μg / mL Dox. After 10-14 days of induction, all cells showed obvious GFP fluorescence signals, indicating that the immortalization gene was successfully activated.

[0104] Stromal vascular fraction (SVF) cells in adipose tissue:

[0105] After collecting subcutaneous adipose tissue from the cell line tool pigs, it was washed with PBS containing penicillin-streptomycin. Under aseptic conditions, the adipose tissue was cut into 2-3 mm pieces.3 Small cells were digested with 0.1% collagenase I solution (Gibco, #17100017) in a 37°C water bath for 60 minutes. The digestion solution was filtered through a 70µm filter, and the cell pellet was collected by centrifugation. The cells were resuspended in high-glucose DMEM containing 20% ​​FBS and seeded into culture dishes. The cells were then cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were passaged. Adipate SVF cells isolated from porcine vegetative state cells (SVFs) were induced to develop SVFs by adding 4μg / mL Dox.

[0106] Figure 11 The photographs show the isolation and culture process of porcine adipose SVF cells. It can be seen that after 10-14 days of Dox induction, the cells all showed obvious GFP fluorescence signals, indicating that the immortalization gene was successfully activated.

[0107] (2) Cell line tool: Stability and ability of porcine cells to passage

[0108] Tool pig ear tissue cells and adipose SVF cells were further induced and passaged using 4 μg / mL Dox. Figure 12 and Figure 13 As shown, both cell lines can be stably passaged for more than 45 generations and maintain good proliferative activity, indicating that immortalized cell lines have been successfully obtained.

[0109] (3) Verification of the reversibility of immortalization

[0110] Dox was removed from immortalized porcine ear tissue cells and adipose SVF cells passaged to the 20th generation, and the cells were cultured again. Changes in GFP signal were observed under a fluorescence microscope on days 1, 2, 3, 4, 5, and 6 after Dox removal.

[0111] like Figure 14 and Figure 15 As shown, the GFP signal gradually weakened over time and almost completely disappeared by day 5; at the same time, the cell proliferation rate decreased significantly, indicating that the immortalization state was successfully reversed and the original state was restored.

[0112] In summary, this invention successfully constructed a tool pig cell line carrying a Tet-On-regulated immortalization gene module. In its construction strategy, this invention pre-integrates a reversible immortalization gene expression system into the genome of a live pig, making the pig itself a universal resource platform for developing immortalized cell lines. This represents a fundamental paradigm shift from "constructing cell lines one by one" to "systematic development of cell resource banks." Furthermore, this invention simultaneously introduces both the PB and Tet-On systems into the pig, achieving safe, stable, and large-fragment integration of exogenous gene modules, avoiding the risks of lentiviruses, and enabling precise and reversible on / off control of immortalization gene expression. Moreover, after isolating any tissue cells from the tool pig, immortalized cell lines can be induced simply by adding Dox in vitro, and the original state can be restored upon removing Dox. Multiple experiments have verified that this invention indeed yields a stable and reversibly regulated tool pig, and conveniently provides an inexhaustible resource of immortalized cell lines from various tissues and cells.

[0113] The above descriptions are merely embodiments of the present invention. Commonly known technical knowledge in the solutions is not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the filing date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical well-known technologies should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a controllable immortalized cell line tool pig, characterized in that... Includes the following steps: S1: Construct transposon plasmids that include immortalized genes and reversible inducible expression systems; S2: Co-transfect porcine donor cells with the transposon plasmid and the transposase expression plasmid to obtain monoclonal donor cells; S3: The monoclonal donor cell is nuclearly transferred to an enucleated oocyte to obtain a transgenic pig cloned embryo; S4: The transgenic pig cloned embryo is transplanted into a sow to give birth naturally, thereby obtaining a controllable immortalized cell line tool pig.

2. The construction method according to claim 1, characterized in that, The immortalization gene mentioned in step S1 is selected from SV40LT, HPV-E6 / E7, Bmi-1, CDK4, EBV, TERT One or more of them.

3. The construction method according to claim 1, characterized in that, The inducer of the reversible inducible expression system described in step S1 is selected from one or more of doxycycline, ecdysone, rapamycin, and mifepristone.

4. The construction method according to claim 1, characterized in that, The transposer mentioned in step S1 is selected from the PB transposer system, the SB transposer system, or the Tol2 transposer system.

5. The construction method according to claim 1, characterized in that, Step S2, co-transfection, integrates the transposon plasmid into loci 43063259-43063260 on chromosome 4 and 127215595-127215596 on chromosome 13 of pigs.

6. The construction method according to claim 1 or 5, characterized in that, Step S2, co-transfection, integrates the plasmid into the pRosa26 or pH11 site.

7. A method for preparing a controllable immortalized cell line, characterized in that, Includes the following steps: 1) Isolating tissue cells from the controllable immortalized cell line tool pig obtained by the construction method according to any one of claims 1-6; 2) Add an inducing agent to the culture medium of the tissue cells to induce reversible immortalization of the tissue cells and obtain a controllable immortalized cell line.

8. The application of the method according to any one of claims 1-7 in the preparation of controllable immortalized cell lines.

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