Method for constructing stable cell strain capable of inducing gene silencing based on Piggybac system

Abstract

The invention relates to a method for constructing a stable cell strain capable of inducing gene silencing based on a Piggybac system. In particular, the present disclosure provides an engineered transposable element based on Piggybac comprising, from 5'to 3 ': a 5' ITR, a transposed region, a 3 'ITR. According to the present invention, the promoter, the Tet operon, the TetR, the shRNA design, the recombinant plasmid construction, the plasmid transfection and other aspects are optimized, such that the delivery, the integration and the validity verification of the Piggybac system-based induced gene silencing are achieved, the precise control and the reversible operation of the target nucleic acid expression level are achieved, and the quantitative data of the highly controllable silencing system is provided.

Description

Technical Field

[0001] This disclosure belongs to the fields of genetic engineering and cell engineering technology, specifically relating to a method for constructing stable cell lines capable of inducing gene silencing based on the Piggybac system. Background Technology

[0002] Stable cell lines with downregulated gene levels are frequently used in gene function research and drug development. Traditional gene knockout techniques have inherent limitations; for example, knockout can be lethal, preventing in-depth studies of gene function. In drug development, gene knockout can lead to changes in multiple signaling pathways, failing to provide convincing results for target validation.

[0003] To address these issues, researchers have developed systems for inducing gene silencing, which have been widely used in cell line and animal model construction due to their ability to provide varying degrees of gene reduction. Combined with RNA silencing technology, this system can achieve different levels of gene expression under the influence of inducers. Systems for inducing gene silencing include Cre-LoxP, Ecdysone, Lac repressor, and the Tet induction system. Among these, Tet-induced shRNA-mediated gene silencing is a powerful tool, but the system is relatively complex, requiring components such as the U6 promoter, Tet operon, Tet R gene, and shRNA. Although CRISPR technology can achieve fragment insertion, its insertion fragment length is limited and its efficiency is relatively low, often requiring multiple steps; therefore, it is currently mainly performed through viral transduction. Although viral transduction is highly efficient in various cell lines, it has the following drawbacks: 1. It may integrate multiple copies, affecting endogenous gene expression or causing genomic instability; 2. The viral vector has a limited carrying capacity, and larger gene fragments can reduce viral packaging titers; 3. The process is cumbersome, involving multiple processes such as viral packaging, requiring specific experimental conditions and taking a long time; 4. Gene integration is irreversible.

[0004] Given the aforementioned drawbacks, a more efficient, simple, and reversible method for inducible gene silencing is urgently needed. The emergence of transposons has provided a completely new perspective for constructing gene-editing cell lines. Transposons mainly include class I retroposomes and class II DNA transposons. Class II transposons directly cleave the DNA sequence containing the transposon using their own encoded transposases, achieving sequence transfer through a cut-and-paste process. The most common class II transposons include Sleeping Beauty transposons and Piggybac transposons. Sleeping Beauty transposons have strict host requirements and exhibit unstable transposition efficiency, limiting their widespread application. Piggybac transposons, on the other hand, are favored due to their specific integration site, precise cleavage, and widespread availability.

[0005] The Piggybac system comprises two vectors: a helper plasmid encoding a transposase and a transposon plasmid containing two inverted terminal repeats (ITRs) and the transposable region between them. The sequence to be integrated into the genome is cloned into this region. When the helper and transposon plasmids are co-transfected into target cells, the transposase produced by the helper plasmid recognizes the two ITR elements of the transposon and inserts the transposon region and the two ITR elements into the host genome. Insertion typically occurs at a host chromosomal locus containing a TTAA sequence, with TTAA repeats appearing flanking the transposon region. Because the helper plasmid enters the host cell transiently, it is gradually lost with cell division. With the loss of the helper plasmid, the transposon becomes permanently integrated into the host genome. When these host cells are transfected again with the helper plasmid, the integrated transposon is cleaved again, achieving scarless editing.

[0006] Compared to viral systems, the Piggybac system is safer, avoids immune tolerance issues, eliminates the need for viral packaging, production, and titer determination, simplifying the process. It also boasts a larger loading capacity, capable of accommodating large genes, including regulatory elements and multiple reading frames simultaneously. Furthermore, inserts can be removed by adding helper plasmids, achieving seamless editing and avoiding genotoxicity caused by persistent transposase presence. While Piggybac has been widely used for overexpression, its application in inducing gene silencing has not been reported. Summary of the Invention

[0007] I. Rotary element

[0008] To address the shortcomings of existing technologies, this disclosure provides a transposon element that does not require virus mediation. A transposon element is a non-free, functional DNA fragment in an organism that can self-replicate or self-cut copies and can continuously move within the genome of an organism in the same or different copies.

[0009] Specifically, this disclosure provides an engineered transposable element based on Piggybac, which includes from 5' to 3': a 5' reverse terminal repeat sequence (5'ITR), a transposable region, and a 3' reverse terminal repeat sequence (3'ITR).

[0010] PiggyBac transposons originate from lepidopteran insects and are classified as Group II transposons in eukaryotes. They are autonomous transposons with a short terminal inverted repeat (ITR) and an open coding frame (ORF). PiggyBac transposons primarily transpose via a cut-paste mechanism. The PiggyBac system exhibits high transposition efficiency and a wide host range.

[0011] In some embodiments, this disclosure provides a Piggybac-based engineered transpose element comprising, from 5' to 3': a 5'ITR, a transposeable region, and a 3'ITR, wherein the 5'ITR is shown in SEQ ID NO: 1; and the 3'ITR is shown in SEQ ID NO: 2.

[0012] In some implementations, the transposable region from 5' to 3' includes: a promoter controlling the shRNA, an shRNA specific to the target nucleic acid, a promoter controlling TetR, and a TetR coding sequence.

[0013] In other embodiments, the transposable region from 5' to 3' includes: a promoter controlling TetR, a TetR coding sequence, a promoter controlling shRNA, and shRNA specific to the target nucleic acid.

[0014] In some embodiments, the promoter controlling the shRNA is a U6 promoter. In some embodiments, the promoter controlling the shRNA is a U6 promoter with a Tet operon. In some specific embodiments, the promoter controlling the shRNA is a U6 promoter with two Tet operons, wherein the two Tet operons are inserted into the middle and end of the U6 promoter, respectively. In one specific embodiment, the promoter controlling the shRNA is shown in SEQ ID NO: 3 or 4.

[0015] Promoters are crucial regulatory elements that guide the expression pattern of coding sequences. Any suitable promoter can be used in this disclosure. In some embodiments, the promoter is an endogenous promoter. In some embodiments, the promoter is a heterologous promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in this disclosure. Promoters can be broadly classified into constitutive promoters or regulatory promoters, such as inducible promoters.

[0016] Constitutive promoters allow constitutive expression in host cells. Exemplary constitutive promoters considered herein include, but are not limited to, cytomegalovirus (CMV) promoters, human elongation factor-1α (hEF1α), ubiquitin C promoters (UbiC), glycerol phosphokinase promoters (PGK), and simian virus 40 early promoters (SV40). The efficiency of such constitutive promoters in driving transgene expression has been extensively compared in numerous studies. For example, the efficiency of CMV, hEF1α, UbiC, and PGK in driving chimeric antigen receptor expression in primary human T cells has been studied, concluding that the hEF1α promoter not only induces the highest levels of transgene expression but also maintains it ideally in CD4 and CD8 human T cells. In some embodiments, the promoter controlling TetR is an EF1α core promoter or a CMV promoter. EF1α core promoters and CMV promoters are well known in the art. In one specific embodiment, the promoter controlling TetR is an EF1α core promoter.

[0017] In some implementations, the TetR encoded sequence is as shown in SEQ ID NO: 5 or SEQ ID NO: 6.

[0018] In some implementations, the length of the target nucleic acid-specific shRNA (short hairpin RNA) is 47 bp to 57 bp, preferably 49 bp to 51 bp.

[0019] Target nucleic acid refers to the object targeted by the shRNA of this disclosure; target nucleic acid can be a gene, mRNA, etc. In this disclosure, target nucleic acid specifically refers to the gene to be silenced or its expression product. The nucleotide or amino acid information of the target nucleic acid is well known in the art, for example, but not limited to, it can be obtained from literature or databases. Those skilled in the art should understand that although a specific target nucleic acid sequence or its accession number is given, the target nucleic acid is not limited to the number in a specific database, and is also intended to cover any equivalent reference in any literature, book, or database in the prior art.

[0020] In some implementations, the shRNA comprises two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector consists of two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the polIII promoter. This is followed by 5 to 6 T molecules as transcription terminators.

[0021] Therefore, in some embodiments, the target nucleic acid-specific shRNA comprises two inverted repeat sequences and a stem-loop, the stem-loop being located between the two inverted repeat sequences. Based on common knowledge, those skilled in the art can design the two inverted repeat sequences in the shRNA according to the sequence of the target nucleic acid. One of the two inverted repeat sequences is a sense sequence, differing from the target nucleic acid by no more than 3, 2, or 1 nucleotides; the other is an antisense sequence, differing from the target nucleic acid by no more than 3, 2, or 1 mismatch. Furthermore, the length and composition of the loop sequence in the shRNA must be considered; the stem-loop sequence cannot be homologous to other sequences within the target gene. 1) Length of the loop sequence in shRNA: Generally, hairpin loops of 3-10 nt in length are suitable for designing shRNA. 2) Composition of the loop sequence in shRNA: Some studies suggest that loops with the composition uugauauccg and uucaagaga have higher repressive efficiency. When the sequence preceding the loop terminates with uu, the caccacc loop sequence is used. Two u's in the loop sequence are important for effective gene repression; however, there cannot be more than three consecutive u's in the loop sequence, as this may lead to premature termination of shRNA transcription. Therefore, although the Parp1 gene is used as an example in the embodiments, the engineered transposon elements of this disclosure are not limited to the sequence of the inverted repeat sequence in the target nucleic acid and shRNA.

[0022] In some specific implementations, the Piggybac-based engineered transposon element comprises, from 5' to 3': 5'ITR-U6 promoter-shRNA-EF1α core promoter specific to the target nucleic acid-TetR coding sequence-3'ITR.

[0023] In some specific implementations, the Piggybac-based engineered transposon element comprises, from 5' to 3': 5'ITR-EF1α core promoter-TetR coding sequence-U6 promoter-shRNA specific to the target nucleic acid-3'ITR.

[0024] II. Carrier

[0025] This disclosure also provides a Piggybac-based engineered transposable vector comprising the aforementioned Piggybac-based engineered transposable element. In some embodiments, the vector is a plasmid.

[0026] In some specific implementations, the Piggybac-based engineered transposon vectors also include elements selected from the following or combinations thereof: restriction enzyme cleavage sites, lactose operon, origin of replication, polyA, alternative tag, and T2A spacer sequence.

[0027] In some implementations, the Piggybac-based engineered transposon vectors contain one or more restriction enzyme sites, examples of which include, but are not limited to: AatII, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, AluI, AlwI, AlwNI, ApaI, ApaLI, ApeKI, ApoI, AscI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanII, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, Bc iVI, BclI, BfaI, BfuAI, BfuCI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, Bpu10I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, Bs eYI, BsgI, BsiEI, BsiHKAI, BsiWI, BslI, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp1286I, BspCNI, BspDI, BspEI, BspHI, BspMI, BspQI, BsrBI, BsrDI, BsrF C spCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DpnII, DraI, DraIII, DrdI, EaeI, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, Fat I, FauI, Fnu4HI, FokI, FseI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy166II, Hpy188I, Hpy188III, Hp y99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, KasI, KpnI, MboI, MboII, MfeI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspA1I, MspI, MwoI, NaeI, NarI,Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, NruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.B smAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaeR7I, PciI, PflFI, PflMI, PhoI, PleI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau3AI, Sau96I, SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, SmaI, SmlI, SnaBI, S peI, SphI, SspI, StuI, StyD4I, StyI, SwaI, T, TaqαI, TfiI, TliI, TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, XbaI, XcmI, XhoI, XmaI, XmnI or ZraI. ,

[0028] In some implementations, the Piggybac-based engineered transposon contains a replication origin, which is an active replication origin in E. coli. In some specific implementations, the replication origin is the Ori replication origin or the f1 Ori replication origin.

[0029] In some implementations, the Piggybac-based engineered transposable vector contains at least one prokaryotic antibiotic resistance tag and at least one eukaryotic antibiotic resistance tag.

[0030] In some specific implementation plans, the prokaryotic antibiotics are selected from: ampicillin, kanamycin, chloramphenicol, and neomycin.

[0031] In some specific implementation schemes, the eukaryotic antibiotics are selected from: puromycin and hygromycin.

[0032] In some implementations, the Piggybac-based engineered transposon vector also contains a reporter gene. A reporter gene is a gene encoding a detectable product, and therefore, detection of the reporter gene product can be used to assess the function of the target nucleic acid. The reporter gene can be fused to any suitable target nucleic acid to allow detection of whether the target nucleic acid is expressed or altered (e.g., cleaved by a transposase) under a given set of conditions. Non-limiting examples of reporter genes include: 3-galactosidase, 3-glucuronidase, glutathione S-transferase (GST), horseradish peroxidase (HRP), luciferase, chloramphenicol acetyltransferase (CAT), secretory alkaline phosphatase (SEAP), green fluorescent protein (GFP, e.g., eGFP), red fluorescent protein (RFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), catechol 2,3-oxygenase (xylE), and autofluorescein, including blue fluorescent protein (BFP).

[0033] In some embodiments, the terminator sequence comprises a nucleic acid sequence that marks the end of a gene or operon during transcription. This sequence mediates transcriptional termination by providing signals in the newly synthesized mRNA that trigger the release of mRNA from the transcription complex. These processes include direct interactions between the mRNA secondary structure and the complex and / or indirect activity of recruited termination factors. The release of the transcription complex frees up RNA polymerase and associated transcription mechanisms to initiate the transcription of new mRNA. Termination sequences include those known in the art. In some embodiments of this disclosure, the terminator sequence is a polyadenylation (poly-A) signal.

[0034] In some specific implementation schemes, the engineered transposable carrier based on Piggybac is Figure 2 The structure is shown.

[0035] III. System

[0036] This disclosure also provides a Piggybac-based gene silencing system comprising: any of the aforementioned Piggybac-based engineered transposon vectors; and a transposase or a nucleic acid encoding a transposase.

[0037] This disclosure provides a gene silencing system comprising an engineered transposon element that can be used to silence target nucleic acids. The transposon element and gene silencing system described herein can be used in a variety of applications, such as gene therapy and gene discovery research.

[0038] As described above, the gene silencing system of this disclosure can be delivered to host cells by physical, chemical, biological methods or combinations thereof. Various delivery vectors and methods are contemplated for use alone or in combination with this disclosure to achieve desired results for certain applications. Therefore, those skilled in the art will be able to best utilize various delivery methods and techniques to suit specific intended uses, such as gene therapy. Liposomes and other nanoparticles can accomplish this task. For example, two plasmids can be delivered to a patient: one providing expression of a transposase, and the other providing a transposon element (donor plasmid). These DNAs can be administered via injection in combination with liposomes. Upon entry into the cell, the transposase can bind to the transposon element in the donor plasmid, excise it, and then integrate it into the genome. Further details and exemplary transposon element delivery methods and techniques can be found, for example, Skipper, Kristian Alsbjerg et al., DNA transposon-based gene vehicles-scenes from an anevolutionary drive. Journal of Biomedical Science 20.1 (2013): 92.

[0039] In some embodiments, the transposase is a mammalian transposase.

[0040] Transposases catalyze the excision of transposons from donor polynucleotides (e.g., vectors) and subsequently integrate the transposons into target nucleic acids, such as the genome or extrachromosomal DNA of a target cell. In some embodiments, transposases bind to the terminal repeats of transposable elements.

[0041] In some embodiments, the transposase is shown in SEQ ID NO: 8.

[0042] IV. Reagent Kit

[0043] This disclosure provides a kit comprising any of the following: the aforementioned Piggybac-based engineered transposon element, the aforementioned Piggybac-based engineered transposon vector, and the aforementioned Piggybac-based gene silencing system.

[0044] In some implementations, the kit includes one or more containers. For example, the kit may provide one or more reaction or storage buffers. The buffer can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof.

[0045] In some embodiments, the kit includes culture medium, buffer, reagents, etc., to allow the use of the engineered transposon elements or gene silencing systems described herein. In some embodiments, the kit includes primers and reagents for use with the engineered transposon elements or gene silencing systems described herein.

[0046] In some implementations, the kit is packaged appropriately. Appropriate packaging includes, but is not limited to, vials, flexible packaging (e.g., resealable bags), etc. Additional components, such as instructions, may be optionally provided with the kit.

[0047] V. Uses and Methods

[0048] This disclosure provides a method for gene silencing, including the step of contacting a target nucleic acid in vitro with any of the following: the aforementioned Piggybac-based engineered transposon element, the aforementioned Piggybac-based engineered transposon vector, and the aforementioned Piggybac-based gene silencing system.

[0049] The term “inhibition” can be used interchangeably with “reduction,” “silence,” and “downregulation,” and includes any level of inhibition. Inhibition can be assessed by an absolute or relative reduction of the variable compared to a control level. This control level can be any type of control level used in the art, such as baseline levels before administration or levels from untreated subjects or samples. For example, the degree of inhibition of the target nucleic acid by the vector (or transposon element, or system, or shRNA) of this disclosure can be characterized by residual mRNA expression.

[0050] In some implementations, the method is performed in vitro.

[0051] In some implementations, the target nucleic acid is in a cell.

[0052] In some implementations, the target nucleic acid is genomic DNA.

[0053] In some embodiments, the gene silencing system inactivates target nucleic acids in cells. In some embodiments, the gene silencing system has induced transposition and reversible activity in cells.

[0054] In some embodiments, the target nucleic acid is located in a cell, which may be an animal cell, plant cell, algal cell, fungal cell, yeast cell, or bacterial cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0055] In some implementations, the mammalian cells are selected from immune cells, somatic cells, tumor cells, stem cells or iPSCs, or cell line models.

[0056] In some implementations, the cells are immune cells, such as T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), and macrophages.

[0057] In some embodiments, the cells are derived from epithelial tissue, muscle tissue, nerve tissue, or connective tissue, or any combination thereof. In some embodiments, the cells are derived from tissues selected from: liver, gastrointestinal tract, pancreas, kidney, lung, trachea, blood vessels, skeletal muscle, heart, skin, smooth muscle, connective tissue, cornea, genitourinary system, mammary gland, reproductive organs, endothelium, epithelium, fibroblasts, nerves, fat, bone, bone marrow, cartilage, pericytes, mesothelial cells, endocrine system, matrix, lymph, blood, endoderm, ectoderm, mesoderm, and combinations thereof. In some embodiments, the cells are derived from tissues selected from: connective tissue, muscle tissue, genitourinary tissue, gastrointestinal tissue, lung tissue, bone tissue, nerve tissue, and epithelial tissue, tissues derived from endoderm, tissues derived from mesoderm, tissues derived from ectoderm, or any combination thereof.

[0058] In some implementations, the cells are derived from tumors.

[0059] In some embodiments, the cells are diseased cells. Diseased cells may have altered metabolic, gene expression, and / or morphological characteristics. Diseased cells can be cancer cells, diabetic cells, and apoptotic cells. Diseased cells can be cells derived from a diseased subject.

[0060] This disclosure provides a method for producing stable cell lines with inducible gene silencing, comprising the steps of: 1) transforming the aforementioned Piggybac-based engineered transposon vector into cells to obtain transformed cells; 2) optionally, contacting the transformed cells with an inducer to silence target nucleic acids in the cells; preferably, the cells are mammalian cells.

[0061] Physical methods for introducing vectors into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, and electroporation. Methods for generating vector-containing cells are well known in the art. For example, see Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0062] In some implementations, the vector is introduced into the cell via electroporation / electroconversion. Attached Figure Description

[0063] Figure 1 : Schematic diagram of the Piggybac carrier structure in the prior art.

[0064] Figure 2 : A schematic diagram of the structure of the carrier constructed in this disclosure.

[0065] Figure 3A and Figure 3B Figure 1 shows the results of qPCR detection of Parp1 mRNA levels under different concentrations of inducers. Figure 3A SC-1; Figure 3B (SC-2).

[0066] Figure 4 Figure: Results of Parp1 protein expression levels under different concentrations of inducers detected by Western blot. Detailed Implementation

[0067] As used in this disclosure, the terms "transposon" and "transposon element" refer to a polynucleotide capable of being excised from a first nucleic acid (e.g., a vector) and integrated into a target site (e.g., a target nucleic acid in a cell or genomic or extrachromosomal DNA). A transposon comprises a nucleic acid sequence flanking a cis-acting nucleic acid sequence at the 5' end of the nucleic acid sequence and at least one cis-acting nucleic acid sequence at the 3' end of the nucleic acid sequence. The cis-acting nucleic acid sequence includes at least one ITR (TR, also known as an inverted ITR or terminal inverted repeat (TIR)) at each end of the transposon element to which transposase binding occurs.

[0068] The transposable elements described herein may or may not contain an open reading frame (ORF) encoding a transposase.

[0069] As used in this disclosure, the term "transposation" refers to the alteration of the position of a transposable element from the first nucleic acid and its integration into the target site.

[0070] As used in this disclosure, the term "terminal repeat" refers to the nucleic acid sequences located at both ends of a transposon element. A TR located at the 5' (upstream) is called a 5' ITR, and a TR located at the 3' (downstream) is called a 3' ITR. In class II transposon elements, the TRs are complementary to each other.

[0071] As used in this disclosure, the term "transposase" refers to a polypeptide that catalyzes the excision of a transposon from a first nucleic acid (e.g., a vector) and its integration into a target site (e.g., a target nucleic acid in a cell or genomic or extrachromosomal DNA).

[0072] In some implementations, the transposase binds to one or two ITRs.

[0073] The terms “nucleic acid,” “polynucleotide,” and “nucleic acid sequence” are used interchangeably to refer to polymeric forms of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues. “Oligonucleotide” refers to a short polynucleotide having no more than about 50 nucleotides.

[0074] A nucleotide is a compound composed of a purine base (or a pyrimidine base), a ribose sugar (or a deoxyribose sugar), and a phosphate group. In this disclosure, nucleotide should be understood broadly to include both ribonucleotides and deoxyribonucleotides. A person skilled in the art will clearly understand from the context whether a nucleotide refers to a ribonucleotide, a deoxyribonucleotide, or both. Adenine nucleotide is denoted as AMP, guanine nucleotide as GMP, cytosine nucleotide as CMP, uracil nucleotide as UMP, and thymine nucleotide as TMP. In sequences, nucleotides are often represented by lowercase letters a, g, c, u, and t, respectively.

[0075] As used in this disclosure, the term "operably linked" refers to a nucleic acid sequence that is functionally related to another nucleic acid sequence. For example, if a coding sequence is operably linked to a promoter sequence, this generally means that the promoter can promote transcription of the coding sequence. Because enhancers can function at intervals of thousands of bases from the promoter and intron sequences can have variable lengths, some nucleic acid sequences may be operably linked but not contiguous.

[0076] The term "complementary" or "anti-complementary" refers to the pairing of bases on one strand of a double-stranded nucleic acid in a complementary manner with bases on the other strand. In DNA, adenine (A) pairs with thymine (T); cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U); cytosine (C) pairs with guanine (G). The two strands are considered complementary when adenine on one strand pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine. The sequence of the complementary strand can be deduced from its sequence.

[0077] The term "perfect complementarity" means that there are no base mismatches between two nucleotide sequences.

[0078] The term "mismatch" refers to a situation in double-stranded nucleic acids where the bases at corresponding positions are not paired in a complementary manner.

[0079] As used in this disclosure, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is attached. Examples of vectors include, but are not limited to, bacteria, plasmids, bacteriophages, and granules, i.e., fragments capable of inserting into the host cell genome through homologous recombination.

[0080] As used in this disclosure, the term "plasmid" refers to a circular double-stranded DNA that can accept exogenous DNA fragments and replicate in prokaryotic or eukaryotic cells.

[0081] As used in this disclosure, a “fragment” of a sequence refers to a portion of a sequence. For example, a fragment of a nucleic acid sequence refers to a portion of a nucleic acid sequence, and a fragment of an amino acid sequence refers to a portion of an amino acid sequence.

[0082] As used in this disclosure, the term “engineering” refers to any operation that results in a detectable change in a polynucleotide or polypeptide, wherein such operation includes, but is not limited to, insertion, deletion, and substitution of a portion of a polynucleotide or amino acid sequence.

[0083] As used in this disclosure, the terms “transfected” or “transformed” or “transduced” refer to the process of transferring or introducing exogenous nucleic acids into a host cell. A “transfected” or “transformed” or “transduced” cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acids.

[0084] The term "in vivo" refers to the body of an organism in which cells are obtained.

[0085] "Ex vivo" or "in vitro" refers to the process of obtaining cells from an organism outside its body.

[0086] This article uses the term "about" to refer to a numerical value or parameter, including variations of that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0087] The term “about XY” used in this article has the same meaning as “about X to about Y”.

[0088] As used in this disclosure and the appended claims, the singular forms “a,” “an,” and “the” include the plural references unless the context clearly specifies otherwise.

[0089] The above-described solution will be further explained below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and do not limit the scope of this disclosure. The implementation conditions used in the embodiments can be illustrated by way of example; for any omissions, please refer to commonly used experimental manuals and the manufacturer's instructions for the reagents and instruments used. All chemical reagents are analytical grade, and all reagents and materials are commercially available.

[0090] Example 1. Construction of Piggybac-induced silencing vector

[0091] The following example uses the induction of Parp1 knockout as a target gene. Technicians understand that this approach also applies to other target genes. Following standard procedures, technicians can design specific shRNAs based on the target gene.

[0092] Commercially available Piggybac transposons include elements such as 3'ITR and 5'ITR homologous sequences, resistance (e.g., Ampicillin) selection markers, Tet-on 3G promoters, EGFP fluorescent markers, and the Tet-on 3G gene. Figure 1 ).

[0093] 1. Particle modification involves two steps:

[0094] 1) By seamless cloning, the Tet R gene was cloned into a vector, replacing the original Tet-on 3G gene, to obtain vector A;

[0095] 2) The gene fragment containing U6, the Tet operon, and shRNA was cloned into a vector, replacing the Tet-on promoter and the EGFP fragment, to obtain vector B. The modified plasmid map is shown below. Figure 2 As shown.

[0096] 2. The steps are as follows:

[0097] Step 1)

[0098] 1.1) Using primers P1-F and P1-R, a vector fragment F1 without the Tet-on 3G fragment was obtained by PCR;

[0099] 1.2) Synthesize the Tet R gene fragment, with 18bp homologous sequences at each end to the vector ends;

[0100] 1.3) Using the Gibson assembly seamless cloning method, the Tet R gene fragment was ligated into vector fragment F1 to obtain vector A;

[0101] Step 2)

[0102] 2.1) Using primers P2-F and P2-R, vector fragment F2 without the Tet-on3G promoter-EGFP (1398-2639) was obtained by PCR with vector A as a template;

[0103] 2.2) Synthesize gene fragment 2, which contains a U6 promoter fused with the Tet operon, shRNA (taking Parp1 as an example here), and terminator, with 18bp sequences homologous to the ends of the vector at both ends;

[0104] 2.3) Using the Gibson assembly seamless cloning method, fragment 2 was ligated to vector fragment F2 to obtain vector B.

[0105] Example 2. Production of stable cell lines for induced gene silencing (transposation in mammalian cells)

[0106] The engineered transposon elements, vectors, and systems disclosed herein are designed based on the framework of the commercially available Piggybac vector. Therefore, the target nucleic acids and cell types applicable to Piggybac are also applicable to the engineered transposon elements, vectors, and systems disclosed herein. Although this embodiment uses only the Parp1 gene and A549 cells as examples, those skilled in the art can extend the teachings of this disclosure to other mammalian cell types.

[0107] 1. Transform target cells A549 using a vector.

[0108] Following the Lonza electroporation kit instructions, vector B was transformed into A549 cells using Lonza 4D electroporation. A puro killing curve was plotted, and the minimum killing concentration was calculated. Two days after transfection, an appropriate concentration of puro was added to the culture medium for selection. Seven days after selection, cells were collected for monoclonalization.

[0109] 2. Monocloning

[0110] Dilute the cells obtained above to 0.5 cells per well using fresh culture medium and seed them into 96-well plates. Culture for 7-10 days. After single clones have grown, observe them under a microscope and digest them before transferring them to 48-well plates. Then, gradually transfer them to 24-well and 6-well plates for further culture.

[0111] 3. Monoclonal assay and functional identification

[0112] Single-clonal cells were collected, genomic DNA was extracted, and the key U6 and Tet R regions were amplified by PCR to check the integration success rate. Primers are shown in the table below. Sequencing results showed that the TetR and shRNA sequences were successfully inserted into the genome, consistent with expectations.

[0113] Two clones (labeled SC-1 and SC-2) were seeded in 6-well plates and treated with gradient concentrations of dox (polytetracycline hydrochloride) for 3-7 days. Cells were then collected for qPCR and Western blot identification.

[0114] The results showed that without the addition of dox, Parp1 expression was consistent with wild-type (WT), representing a very low background silencing value. After the addition of dox, both the mRNA and protein levels of the Parp1 gene decreased in a dose-dependent manner, with a decrease of approximately 70-90%, indicating the successful acquisition of a monoclonal cell line with induced Parp1 gene silencing.

[0115] After dox is withdrawn, the expression level of Parp1 is periodically monitored. It can be observed that the expression level of Parp1 returns to the normal level (WT), which represents the reversibility of this induction system.

[0116] Table 1. Sequence List

[0117]

[0118]

[0119] The method disclosed herein for constructing stable cell lines capable of inducing gene silencing based on the Piggybac system has the following advantages:

[0120] - Stable vectors have a large carrying capacity and can be cloned into a single vector through seamless cloning, which is efficient and fast. The resulting vectors are versatile and can be used for other gene silencing applications, requiring only one cloning step.

[0121] - No virus is required, and there is no need for virus packaging, titer determination, or other steps, saving time and costs.

[0122] The Piggybac system utilizes a highly efficient electroporation method, resulting in minimal cell damage and high transformation efficiency.

[0123] - The Piggybac fusion with the Tet induction system enables highly controllable and reversible gene silencing with low background. Without the addition of an inducer, the gene expression level is consistent with that of the wild type. With the addition of an inducer, gene expression decreases in a dose-dependent manner, and gene expression can return to normal levels after the inducer is removed.

[0124] The above content provides a further detailed explanation of this disclosure in conjunction with specific implementation plans, and it should not be assumed that the specific implementation of this disclosure is limited to these descriptions.

Claims

1. An engineered transpose element based on Piggybac, comprising, from 5' to 3': 5' inverted terminal repeat (5'ITR) Reseat area 3' inverted terminal repeat (3'ITR); in, The 5'ITR is shown in SEQ ID NO: 1; The 3'ITR is shown in SEQ ID NO: 2; The transposable region from 5' to 3' includes: a promoter controlling the shRNA, an shRNA specific to the target nucleic acid, a promoter controlling TetR, and a TetR coding sequence; or The transposable region from 5' to 3' includes: a promoter controlling TetR, a TetR coding sequence, a promoter controlling shRNA, and shRNA specific to the target nucleic acid.

2. The engineered transpose element based on Piggybac according to claim 1, wherein: The promoter controlling the shRNA is the U6 promoter; Preferably, the promoter controlling the shRNA is a U6 promoter with a Tet operon; more preferably, the promoter controlling the shRNA is a U6 promoter with two Tet operons, wherein the two Tet operons are inserted into the middle and 3' end of the U6 promoter, respectively.

3. The engineered transpose element based on Piggybac according to claim 1, wherein: Preferably, the promoter controlling the shRNA is shown in SEQ ID NO: 3 or 4; Preferably, the promoter controlling TetR is an EF1α core promoter or a CMV promoter, more preferably an EF1α core promoter; Preferably, the TetR encoding sequence is as shown in SEQ ID NO: 5 or SEQ ID NO: 6, and more preferably SEQ ID NO:

6.

4. The engineered transpose element based on Piggybac according to claim 1, wherein: The length of the target nucleic acid-specific shRNA is 47bp to 57bp, preferably 49bp to 51bp; The target nucleic acid-specific shRNA comprises two inverted repeat sequences and a stem loop, wherein the stem loop is located between the two inverted repeat sequences; One of the two inverted repeat sequences is a sense sequence that differs from the target nucleic acid by no more than 3, 2, or 1 nucleotides. The other of the two inverted repeat sequences is an antisense sequence, which has no more than 3, 2, or 1 mismatches with the target nucleic acid.

5. The engineered transpose element based on Piggybac according to any one of claims 1 to 4, wherein from 5' to 3' it comprises: 5'ITR-U6 promoter - shRNA specific to target nucleic acid - EF1α core promoter - TetR coding sequence - 3'ITR; or 5'ITR-EF1α core promoter-TetR coding sequence-U6 promoter-shRNA specific to target nucleic acid-3'ITR.

6. An engineered transpose carrier based on Piggybac, comprising the engineered transpose element based on Piggybac as described in any one of claims 1 to 5; Preferably, the carrier is a plasmid.

7. The engineered transposon vector based on Piggybac according to claim 6, further comprising elements selected from the following or combinations thereof: restriction enzyme sites, reporter genes, lactose operons, origin of replication, polyA, alternative tags, and T2A spacer sequences; in: Preferably, the origin of replication is an active origin of replication in Escherichia coli, more preferably the Ori origin of replication or the f1Ori origin of replication; Preferably, the selective label comprises at least one prokaryotic antibiotic resistance label and at least one eukaryotic antibiotic resistance label; More preferably, the prokaryotic antibiotic is selected from: ampicillin, kanamycin, chloramphenicol, neomycin; the eukaryotic antibiotic is selected from: puromycin, hygromycin; The reporter genes are selected from: 3-galactosidase, 3-glucuronidase, glutathione-S-transferase, horseradish peroxidase, luciferase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, and fluorescent protein.

8. A Piggybac-based gene silencing system, comprising: 1) The engineered transpose carrier based on Piggybac as described in claim 6 or 7; and 2) Transposases or nucleic acids encoding transposases; Preferably, the transposase is a mammalian transposase; More preferably, the transposase is shown in SEQ ID NO:

8.

9. A kit comprising any one of the following: The engineered transpose element based on Piggybac as described in any one of claims 1 to 5 The engineered transposon carrier based on Piggybac as described in claim 6 or 7 The Piggybac-based gene silencing system as described in claim 8.

10. A method for gene silencing, comprising the step of contacting a target nucleic acid with any of the following in vitro: The engineered transpose element based on Piggybac as described in any one of claims 1 to 5 The engineered transposon carrier based on Piggybac as described in claim 6 or 7 The Piggybac-based gene silencing system as described in claim 8.

11. A method for producing stable cell lines with inducible gene silencing, comprising the steps of: 1) Transform the Piggybac-based engineered transposon vector as described in claim 6 or 7 into cells to obtain transformed cells; 2) Optionally, the transformed cells are exposed to an inducer to silence the target nucleic acid in the cells; Preferably, the cell is a mammalian cell; Preferably, the conversion is an electrical conversion.

12. An engineered transposable carrier based on Piggybac, as shown in Figure 2.

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