Method for sorting cells by using membrane protein

Abstract

Provided is a polynucleotide comprising an expression cassette of a membrane protein and one or more additional expression cassettes for expressing one or more additional genes. The expression cassette of the membrane protein is connected to the one or more additional expression cassettes. The membrane protein comprises a transmembrane domain and an extracellular domain. The extracellular domain comprises a recognition sequence. The recognition sequence can be specifically recognized by an antibody of the recognition sequence. Further provided are a vector comprising the polynucleotide and a cell, and a method for transferring an exogenous gene into the cell by using the polynucleotide or the vector and screening the cell.

Description

Cell sorting methods using membrane proteins

[0001] Priority information

[0002] This application claims priority and benefit to patent application 202410542476.1, filed with the China National Intellectual Property Administration on April 30, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of transgenics, and more particularly to polynucleotides, vectors, vector combinations, cells, kits, and methods for transferring one or more exogenous genes into cells and for screening cells containing or integrating said one or more exogenous genes into their genome. Background Technology

[0004] With the rapid development of biotechnology, stable transformed cell lines are playing an increasingly important role in both basic and applied research. The techniques for constructing stable transformed cell lines involve the stable integration of one or more exogenous genes into the host cell genome, and the use of genes such as resistance genes to screen for target cell populations. Common multi-gene integration methods are as follows:

[0005] 1) By designing vectors containing multiple exogenous genes, multiple genes can be integrated simultaneously using transferase or viral vector-mediated methods, followed by selective screening using specific combinations of resistance genes. This method is limited by the limited types of available resistance genes and can only meet the needs of relatively simple cell line construction. Furthermore, resistance gene screening requires multiple steps, which is time-consuming and costly.

[0006] 2) Construct a composite expression vector containing multiple independent expression cassettes, each driven by an independent expression element. Integrate this composite vector into the host cell genome using a transferase or viral vector-mediated approach. Combined with single-gene screening, multi-gene integrated cell lines can be obtained. The limitations of this method are the complexity of plasmid construction, the potential for reduced transfer efficiency due to excessively large vector sizes, and the inability to flexibly adjust the integration levels of different genes.

[0007] While screening for antibiotic resistance genes is a common strategy for constructing stable cell lines, it also has certain limitations. Long-term antibiotic use may induce drug-resistant mutations in cells, affecting cell line stability and expression efficiency; some antibiotics may negatively impact cell growth and metabolism, thereby affecting the expression and function of the target protein. Furthermore, for cell lines intended for clinical application, detailed descriptions of the construction process are required, demonstrating that the final product does not contain any antibiotic residues or resistance genes harmful to human health.

[0008] The development of stable cell lines is crucial for the production processes of therapeutic proteins (including antibodies), viral vectors, and more. The constructed production cell lines not only need to efficiently express the target gene but also maintain high stability and proper product bioactivity.

[0009] Traditional methods for constructing and screening stable transformed cell lines using resistance genes and antibiotic drugs have several drawbacks. First, the screening process is time-consuming, typically requiring a week or more to complete the screening of a single resistance gene, and usually cannot use different antibiotic drugs simultaneously. Excessive screening time may lead to gene mutations or other adverse changes in cells. Second, the range of antibiotics suitable for mammalian cell screening is limited, thus failing to meet the need for simultaneously screening a larger number of exogenous genes. Furthermore, different cell types exhibit varying sensitivities to antibiotics. Different drug concentrations need to be adjusted for different cell types, resulting in poor applicability.

[0010] Fluorescent proteins (GFPs) play a crucial role in protein localization and cellular imaging analysis as commonly used molecular tools. However, recent studies have revealed that GFPs may generate reactive oxygen species such as hydrogen peroxide during maturation and folding, potentially inducing oxidative stress in cells and thus affecting normal cellular physiological functions. For example, enhanced green fluorescent protein (eGFP) releases a large amount of H₂O₂ during maturation. This byproduct can alter gene expression, affecting multiple biological signaling pathways and interfering with the normal functioning of related biological processes (Ganini D et al., Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells. Redox Biol. 2017; 12:462-468). Furthermore, GFPs also have limitations in optical performance. Most GFPs have wide excitation and emission spectral bandwidths, inevitably leading to spectral overlap, which limits the possibility of using multiple different colored GFPs simultaneously in the same system. Because spectral overlap causes interference between fluorescence signals, increases background noise, and affects analytical resolution and sensitivity, the application of multicolor GFPs is restricted.

[0011] Given the shortcomings of existing methods, there is an urgent need for a more efficient technology for the construction and screening of multi-gene integrated cell lines. Summary of the Invention

[0012] To address the problems encountered in the construction of multi-gene integrated cells as described above, this disclosure provides the following.

[0013] In one aspect, this disclosure provides a polynucleotide (hereinafter referred to as "the polynucleotide of this disclosure") comprising an expression cassette for a membrane protein and one or more additional expression cassettes for expressing one or more additional genes, the expression cassette for the membrane protein being linked to the one or more additional expression cassettes, the membrane protein comprising a transmembrane domain and an extracellular domain, the extracellular domain comprising a recognition sequence capable of being specifically recognized by an antibody of the recognition sequence.

[0014] In one embodiment of the polynucleotide disclosed herein, the expression cassette of the membrane protein includes a nucleic acid sequence encoding the membrane protein and a nucleic acid sequence encoding a signal peptide for guiding the membrane protein to the cell membrane.

[0015] In one embodiment of the polynucleotide disclosed herein, the recognition sequence is a protein tag sequence.

[0016] In one embodiment of the polynucleotide disclosed herein, the polynucleotide includes an expression cassette region comprising an expression cassette of the membrane protein and one or more additional expression cassettes, at least one of the sides of the expression cassette region having an integration sequence that enables the expression cassette region to be integrated into the genome of the cell when the polynucleotide is transduced into the cell.

[0017] In one embodiment of the polynucleotide disclosed herein, the expression cassette of the membrane protein has cleavage sequences on both sides, which enable the expression cassette of the membrane protein to be cleaved from the expression cassette region.

[0018] In one aspect, this disclosure provides a vector (hereinafter referred to as "the vector of this disclosure") comprising the polynucleotide of this disclosure as described above.

[0019] In one aspect, this disclosure provides a cell (hereinafter referred to as "the cell of this disclosure") comprising the polynucleotide of this disclosure as described above or the carrier of this disclosure as described above.

[0020] In one aspect, this disclosure provides a method for transferring a foreign gene into a cell (hereinafter referred to as "the method of this disclosure"), the method comprising transferring a polynucleotide of this disclosure as described above or a vector of this disclosure as described above into the cell.

[0021] In one embodiment of the method disclosed herein, the expression cassette region is integrated into the genome of the cell.

[0022] In one embodiment of the method disclosed herein, the method further includes incubating the cells with an antibody of the recognition sequence after transfecting the polynucleotide of the present disclosure as described above or the vector of the present disclosure as described above into the cells.

[0023] In one embodiment of the method disclosed herein, the antibody recognizing the sequence is labeled with a positive sorting marker that can be detected by flow cytometry.

[0024] In one embodiment of the method disclosed herein, the positive sorting marker is a fluorescein-based marker.

[0025] In one embodiment of the method disclosed herein, the method further includes sorting the cells positive for the positive sorting marker by flow cytometry.

[0026] In one embodiment of the method disclosed herein, the method further includes, after transfecting the polynucleotide of the present disclosure as described above or the vector of the present disclosure as described above into the cell, cutting off the expression cassette of the membrane protein from the expression cassette region.

[0027] In one embodiment of the method disclosed herein, the method further includes, after cleaving the expression cassette of the membrane protein from the expression cassette region, incubating the cells with an antibody capable of specifically binding to the extracellular domain of the membrane protein, wherein the antibody capable of specifically binding to the extracellular domain of the membrane protein is labeled with a negative sorting marker detectable by flow cytometry. In one embodiment, the negative sorting marker is a fluorescein-based marker.

[0028] In one embodiment of the method disclosed herein, the method further includes sorting cells negative for the negative sorting marker by flow cytometry.

[0029] In one aspect, this disclosure provides cells obtained by the methods of this disclosure as described above.

[0030] In one aspect, this disclosure provides the use of the polynucleotides of this disclosure as described above, the vectors of this disclosure as described above, or the cells obtained by the methods of this disclosure as described above in the preparation of stable cell lines with single or multiple exogenous genes.

[0031] In one aspect, this disclosure provides a carrier combination (hereinafter referred to as "the carrier combination of this disclosure") comprising a plurality of carriers of this disclosure as described above, each expressing a distinct identification sequence.

[0032] In one aspect, this disclosure provides a kit (hereinafter referred to as "the kit of this disclosure") comprising: the vector combination of this disclosure as described above; and a plurality of positive sorting antibodies corresponding to the different recognition sequences, each labeled with a different marker detectable by flow cytometry. In one embodiment, the marker is a fluorophore marker.

[0033] In one embodiment of the kit disclosed herein, the kit further comprises one or more negative sorting antibodies labeled with one or more distinct markers detectable by flow cytometry. In one embodiment, the marker is a fluorescein-based marker. In one embodiment, the extracellular domains of the membrane proteins expressed by the vectors in the vector assembly have the same or similar sequences, except for the recognition sequence, such that the extracellular domains can be recognized by the same negative sorting antibody.

[0034] This disclosure aims to overcome the limitations of existing methods in terms of multi-gene integration efficiency, screening flexibility, and cost control, thereby enabling the rapid and economical construction of multi-gene integrated cell lines with ideal expression characteristics. By introducing non-antibiotic markers, the risk of cell mutations caused by long-term antibiotic use is avoided, ensuring the safety of the final product. Simultaneously, this technology can reflect the integration and expression levels of different exogenous genes, achieving flexible and efficient multi-gene screening, significantly shortening the cell line construction cycle, and reducing development costs. Compared with resistance gene screening methods and fluorescent protein screening methods, it can significantly improve the complexity and diversity of cell line construction. After cell line construction is completed, membrane protein sequences can also be removed, thereby ensuring the stability of the final cells. Attached Figure Description

[0035] Figure 1 shows a schematic diagram of the structure of a polynucleotide according to one embodiment of the present disclosure.

[0036] Figure 2 shows the results of flow cytometry detection according to one embodiment of the present disclosure.

[0037] Figure 3 shows the results of flow cytometry detection according to another embodiment of this disclosure.

[0038] Figure 4 shows the results of flow cytometry detection according to another embodiment of this disclosure.

[0039] Figure 5 shows the results of gel electrophoresis detection according to one embodiment of the present disclosure.

[0040] Figure 6 shows the results of virus titer detection according to one embodiment of this disclosure.

[0041] Detailed description of the invention

[0042] The following embodiments are used to illustrate the technical solution of the present invention, and should not be considered as limiting the scope and spirit of the present invention.

[0043] In one aspect, the polynucleotide of this disclosure comprises an expression cassette for a membrane protein and one or more additional expression cassettes for expressing one or more additional genes, the expression cassette for the membrane protein being linked to the one or more additional expression cassettes, the membrane protein comprising a transmembrane domain and an extracellular domain, the extracellular domain comprising a recognition sequence capable of being specifically recognized by an antibody of the recognition sequence. In one example, the expression cassette for the membrane protein comprises a nucleic acid sequence encoding the membrane protein, the nucleic acid sequence encoding the membrane protein comprising a nucleic acid sequence encoding the transmembrane domain and a nucleic acid sequence encoding the extracellular domain, the nucleic acid sequence encoding the extracellular domain comprising a nucleic acid sequence encoding the recognition sequence. In one example, the one or more additional genes may be one or more exogenous genes to be transferred into a cell (and may be further integrated into the cellular genome). In one instance, the transmembrane domain and the extracellular domain originate from the same membrane protein, such as a naturally occurring membrane protein. In one instance, the extracellular domain of a naturally occurring membrane protein may be selected, and a segment thereof may be designated as a recognition sequence. In another instance, a foreign sequence (e.g., a nucleic acid sequence encoding a protein tag) may be inserted into or fused with the nucleic acid sequence encoding the extracellular domain of a naturally occurring membrane protein to obtain a chimeric or fused membrane protein. In one instance, the transmembrane domain and the extracellular domain originate from different membrane proteins. There are no particular restrictions on which membrane protein the transmembrane domain and / or the extracellular domain may originate from, as long as, after expression, the membrane protein has a transmembrane domain anchored to the cell membrane and an extracellular domain exposed outside the cell containing a recognition sequence that can be specifically recognized by an antibody. In one instance, the membrane protein from which the transmembrane domain and / or the extracellular domain originates may be a synthetic membrane protein or a naturally occurring membrane protein, such as one derived from eukaryotes, mammals, humans or mice, such as human or mouse CD4, CD8, CD20, EMMPRIN / CD147, TMEM176B proteins, etc.

[0044] When used herein, the term "expression cassette" refers to a polynucleotide construct containing all the elements required for expression. An expression cassette typically contains the target gene sequence and an operatively linked promoter and terminator sequence. When used herein, the term "operatively linked" means that the segments are arranged to be operative during transcription from the promoter to the stop codon.

[0045] In one instance, the specific sequence or type of recognition sequence that can be used in this disclosure is not particularly limited, as long as it has an antibody capable of specifically recognizing and binding to it. In one instance, the recognition sequence is a protein tag sequence. In this disclosure, the term "protein tag" (or "tagged protein") has its common meaning in the art, referring to a subdomain or peptide sequence in a fusion protein that is linked to the target protein, providing a useful and convenient tool for improving the solubility of recombinant proteins, simplifying protein purification, and allowing easy tracking during protein expression and purification. The protein tags or tagged proteins that can be used in this disclosure are not particularly limited, and can be various protein tags or tagged proteins commonly used in the art, including but not limited to His tag, GST tag, MBP tag, Flag tag, HA tag, Myc tag, SNAP tag, Halo tag, SUMO tag, GFP tag, Strep tag, StrepII tag, Trx tag, Avi tag, S tag, VSVG tag, V5 tag, and T7 tag. In one example, the HA tag may have the amino acid sequence shown in SEQ ID NO:24, the StrepII tag may have the amino acid sequence shown in SEQ ID NO:25, the Flag tag may have the amino acid sequence shown in SEQ ID NO:26, and the V5 tag may have the amino acid sequence shown in SEQ ID NO:27.

[0046] In one example, the nucleic acid sequence encoding the extracellular domain of the membrane protein of this disclosure may include one or more copies (e.g., 1, 2, 3, 4, 5, 6 or more) of a recognition sequence encoding a nucleic acid sequence. In another example, the nucleic acid sequence encoding the extracellular domain of the membrane protein of this disclosure may include one or more copies (e.g., 1, 2, 3, 4, 5, 6 or more) of a protein tag sequence encoding a nucleic acid sequence. As those skilled in the art will understand, the location of the recognition sequence within the extracellular domain is not particularly limited, as long as it can be specifically bound by its antibody when exposed to the extracellular environment.

[0047] In one example, the expression cassette of the membrane protein further includes a nucleic acid sequence encoding a signal peptide. There are no particular limitations on the signal peptide that can be used in this disclosure, as long as it enables the membrane protein to be directed to the cell membrane.

[0048] In one example, the polynucleotide includes an expression cassette region comprising an expression cassette of the membrane protein and one or more additional expression cassettes. In one example, at least one of the flanking sides of the expression cassette region has an integration sequence that enables the expression cassette region to be integrated into the cell's genome when the polynucleotide is transduced into the cell. Methods for integrating a target nucleic acid fragment into the cell's genome are known in the art, including but not limited to transposon system-mediated integration methods, viral vector-mediated integration methods, gene editing system-mediated integration methods, site-specific recombinase-mediated integration methods, and linearized template-mediated integration methods. In one example, the expression cassette region is integrated into the cell's genome using a transposon system, and the expression cassette region has transposession recognition sequences flanking it as integration sequences. The transposon systems that can be used are not particularly limited and may include DNA transposon systems and retrotransposon systems commonly used in the art, including but not limited to: the Tol1 transposon system, the Tol2 transposon system, the Frog Prince transposon system, the Minos transposon system, the Hsmar1 transposon system, the Helraiser transposon system, the ZB transposon system, the Intruder transposon system, the SPINON transposon system, the TcBuster transposon system, the Passport transposon system, the Yabusame-1 transposon system, the Uribo2 transposon system, the PiggyBac transposon system, the Sleeping Beauty transposon system, the LINE1 retrotransposon system, the MaLR retrotransposon system, and various variants or derivatives of the above transposon systems. In one example, a viral vector, such as a retrovirus, like a lentiviral vector, is used to integrate the expression cassette region into the genome of a cell, and the expression cassette region is flanked by, for example, long terminal repeat (LTR) sequences of the viral vector as integration sequences. The viral vectors that can be used are not particularly limited, as long as they can integrate the target nucleic acid fragment into the cell genome. In one example, a gene editing system, such as a CRISPR-Cas system, a TALEN system, or a ZFN system, is used to integrate the expression cassette region into the cell genome, and the expression cassette region has homologous sequences flanking, for example, the genomic integration site (using homology-mediated repair (HDR) mechanisms) as integration sequences. The gene editing system that can be used is not particularly limited, as long as it can integrate the target nucleic acid fragment into the cell genome.In one example, a site-specific recombinase system, such as the Cre site-specific recombinase system, the Flp site-specific recombinase system, the phiC31 site-specific recombinase system, the Dre site-specific recombinase system, or the Bxb1 site-specific recombinase system, is used to integrate the expression cassette region into the cell's genome, and at least one of the flanking sites of the expression cassette region has a recognition sequence of the site-specific recombinase as an integration sequence. The site-specific recombinase system that can be used is not particularly limited, as long as it can integrate the target nucleic acid fragment into the cell's genome. In one example, gene delivery technologies, such as electroporation, calcium phosphate transfection, liposome transfection, or other non-viral vectors and viral vector delivery methods, are used to deliver the polynucleotides of this disclosure into cells and integrate the expression cassette region into the cell's genome, and the expression cassette region has homologous sequences flanking the genomic integration site (utilizing homology-mediated repair (HDR) mechanisms) as integration sequences.

[0049] In one example, after the expression cassette region of the polynucleotide disclosed herein is transferred into a cell and integrated into the cell's genome, the expression cassette of the membrane protein is cleaved from the expression cassette region, thereby causing the cell to cease expressing the membrane protein. Methods for cleaving the target nucleic acid fragment from its original location in the vector or genome are known in the art, including but not limited to transposon system-mediated cleavage methods, site-specific recombinase-mediated cleavage methods, and gene editing system-mediated cleavage methods. In one example, a gene editing system, such as a CRISPR-Cas system, a TALEN system, or a ZFN system, is used to cleave the expression cassette of the membrane protein from the expression cassette region. The gene editing system that can be used is not particularly limited, as long as it is capable of cleaving the target nucleic acid fragment. In one example, the expression cassette of the membrane protein has cleaving sequences on both sides, which enable the expression cassette of the membrane protein located between the cleaving sequences to be cleaved from the expression cassette region. In one example, a gene editing system, such as a CRISPR-Cas system, a TALEN system, or a ZFN system, is used to cut the expression cassette of the membrane protein from the expression cassette region, and the expression cassette of the membrane protein has specific sequences on both sides that can be recognized by editing elements (e.g., a complex of sgRNA and Cas9) as cut sequences. In another example, a transposon system is used to cut the expression cassette of the membrane protein from the expression cassette region, and the expression cassette of the membrane protein has transposase recognition sequences on both sides as cut sequences. The transposon system that can be used is not particularly limited and can be as described above. In one example, where a transposon system is used for both integrating the expression cassette region into the genome of a cell and cutting the expression cassette of the membrane protein from the expression cassette region, the transposon system used to cut the expression cassette of the membrane protein from the expression cassette region is different from the transposon system used to integrate the expression cassette region into the genome of the cell to avoid cutting one or more additional expression cassettes (containing the target exogenous nucleic acid fragment to be integrated) in the expression cassette region. In one example, a site-specific recombinase system is used to cleave the expression cassette of the membrane protein from the expression cassette region, and the expression cassette of the membrane protein has site-specific recombinase recognition sequences on both sides as cleavage sequences. The site-specific recombinase system that can be used is not particularly limited and may include site-specific recombinase systems commonly used in the art, such as the Cre site-specific recombinase system, the Flp site-specific recombinase system, the bacteriophage phiC31 site-specific recombinase system, the Dre site-specific recombinase system, and the bacteriophage Bxb1 site-specific recombinase system.

[0050] In one aspect, this disclosure provides vectors comprising the polynucleotides of this disclosure. The types of vectors that can be used in this disclosure are not particularly limited and can be vectors commonly used in the art for delivering target nucleic acids. In one example, the vector is a non-viral vector. In one example, the vector is a plasmid vector. In one example, the vector is a lipid nanoparticle (LNP) or liposome. In one example, the vector is a viral vector, such as an adeno-associated virus vector, a retroviral vector, or a lentiviral vector.

[0051] In one aspect, this disclosure provides cells comprising the polynucleotides or vectors of this disclosure. The source of the cells is not particularly limited. In one example, the cells are derived from eukaryotic cells. In one example, the cells are derived from mammalian cells. In one example, the cells are derived from human cells.

[0052] In one aspect, this disclosure provides a method for transferring one or more exogenous genes into a cell, the method comprising transferring one or more of the disclosed polynucleotides or one or more of the disclosed vectors into the cell. In one example, the method is used to transfer one exogenous gene into a cell. In one example, the method is used to transfer multiple exogenous genes into a cell. In one example, the method transiently transfers one or more exogenous genes into a cell. In one example, the method stably integrates one or more exogenous genes into the genome of a cell. In one example, the method of this disclosure transfers one exogenous gene into a cell, and the exogenous gene is located in one of the disclosed polynucleotides or one of the disclosed vectors. In one example, the method of this disclosure transfers multiple exogenous genes into a cell, and the multiple exogenous genes are respectively located in multiple of the disclosed polynucleotides or multiple of the disclosed vectors.

[0053] In one example, the method of this disclosure further includes integrating the expression cassette region into the genome of a cell. For aspects of integrating the expression cassette region into the genome of a cell, please refer to the description above.

[0054] In one example, the method of this disclosure further includes incubating the cells with an antibody of the recognition sequence after transfecting the polynucleotide or vector of this disclosure into the cells. In one example, the antibody of the recognition sequence is used in a subsequent positive sorting step (and is therefore referred to as a positive sorting antibody). In one example, the antibody of the recognition sequence is labeled with a marker that can be detected by flow cytometry. In one example, the marker is used in a subsequent positive sorting step (and is therefore referred to as a positive sorting marker). In one example, the positive sorting marker is a fluorescein marker. Fluorescein markers that can be used as positive sorting markers of this disclosure are not particularly limited, as long as they can be used to label antibodies and can be detected and sorted by flow cytometry, including but not limited to, for example, FITC (fluorescein isothiocyanate), PE (phycoerythrin), PerCP (polydoxyphylla chlorophyll protein), APC (allophycocyanin), PE-Cy7, Pacific Blue, Alexa Fluor. TM A series of luciferins, such as Alexa Fluor TM 305, Alexa Fluor TM 405, Alexa Fluor TM 488, Alexa Fluor TM 532, Alexa Fluor TM 700, etc., Brilliant Violet TM Series of fluoresceins, such as Brilliant Violet TM 421, Brilliant Violet TM 480, Brilliant Violet TM 605, Brilliant Violet TM 650, etc., DyLight TM Series of fluoresceins, such as DyLight TM 488, DyLight TM 550, DyLight TM 650, etc.

[0055] In one instance, the method of this disclosure further includes sorting cells positive for the positive sorting marker by flow cytometry. The terms "flow cytometry" or "flow cytometer" have their common meaning as understood by those skilled in the art. Methods for detecting and sorting cells carrying specific markers using flow cytometry for positive / negative purposes are well known to those skilled in the art.

[0056] In one example, the method of this disclosure involves transferring multiple exogenous genes into cells, each located within a multiple polynucleotide or vector disclosed herein. The multiple polynucleotides or vectors express multiple distinct recognition sequences. After transferring the multiple polynucleotides or vectors into cells, the cells are incubated with multiple antibodies corresponding to the multiple distinct recognition sequences. The antibodies are labeled with multiple distinct markers (e.g., fluorescein markers) detectable by flow cytometry. When sorting cells positive for the markers by flow cytometry, the target cells are cells co-positive for the multiple markers.

[0057] In one example, the method of this disclosure further includes, after transfecting the polynucleotide or vector of this disclosure into a cell, cleaving the expression cassette of the membrane protein from the expression cassette region. For aspects regarding cleaving the expression cassette of the membrane protein from the expression cassette region, please refer to the above description.

[0058] In one example, the method further includes, after cleaving the expression cassette of the membrane protein from the expression cassette region, incubating the cells with an antibody capable of specifically binding to the extracellular domain of the membrane protein. In one example, the antibody capable of specifically binding to the extracellular domain of the membrane protein is used in a subsequent negative sorting step (and is therefore referred to as a negative sorting antibody). In one example, the negative sorting antibody is the same as the positive sorting antibody. In one example, the negative sorting antibody is different from the positive sorting antibody; for example, the positive sorting antibody is capable of specifically binding to a recognition sequence, and the negative sorting antibody is capable of specifically binding to other parts of the extracellular domain besides the recognition sequence. In one example, the negative sorting antibody specifically binds to a recognition sequence in the extracellular domain. In one example, the negative sorting antibody specifically binds to other parts of the extracellular domain besides the recognition sequence. In one example, the negative sorting antibody is labeled with a marker detectable by flow cytometry. In one example, the marker is used in a subsequent negative sorting step (and is therefore referred to as a negative sorting marker). In one example, the negative sorting marker is a fluorophore. Fluorescent markers that can be used as negative sorting markers of this disclosure are not particularly limited, as long as they can be used to label antibodies and can be detected and sorted by flow cytometry. As will be understood by those skilled in the art, markers that can be used as positive sorting markers of this disclosure can also be used as negative sorting markers of this disclosure. In one example, a negative sorting marker is used, and the antibody labeled by the negative sorting marker can specifically bind to all of the one or more extracellular domains used. In one example, multiple negative sorting markers are used, which are distinguishable by flow cytometry, and the multiple negative sorting markers are used to label different antibodies, which are respectively specifically bound to the multiple extracellular domains used. As those skilled in the art will understand, after the expression cassette of the membrane protein is cleaved from the expression cassette region, the cell will no longer express the membrane protein (recognition sequence), and the expressed membrane protein (recognition sequence) will be degraded or lost after passage, resulting in the cell surface no longer being able to bind labeled antibodies (the bound markers will be quenched, degraded, or lost). Therefore, after further culture, the use of positive sorting antibodies or positive sorting markers in cells treated with the cleavage of the membrane protein expression cassette does not substantially affect the use of the same negative sorting antibodies or negative sorting markers as the positive sorting antibodies or positive sorting markers.

[0059] In one example, the method further includes sorting cells negative for the negative sorting marker by flow cytometry. Methods for detecting and negatively sorting cells carrying specific markers by flow cytometry are well known to those skilled in the art.

[0060] In one aspect, this disclosure provides cells obtained by the methods of this disclosure as described above, said cells containing or having one or more exogenous target genes integrated into their genome and not containing any resistance selection genes, and preferably not containing any exogenous gene sequences for selection (including expression cassettes of membrane proteins in polynucleotides of this disclosure).

[0061] In one aspect, this disclosure provides the use of the polynucleotides of this disclosure, the vectors of this disclosure, or cells obtained by the methods of this disclosure in the preparation of stable cell lines with single or multiple exogenous genes. When used herein, the term "stable cell line with single or multiple exogenous genes" refers to a cell line in which one or more exogenous gene sequences have been inserted or integrated into its genome.

[0062] In one aspect, this disclosure provides a vector combination comprising multiple vectors of this disclosure, each vector expressing a distinct recognition sequence. In one example, the vector combination of this disclosure is used to prepare cell lines stably integrated with multiple exogenous genes, the coding sequence of each (or several) of the exogenous genes being located in an expression cassette region (the one or more additional expression cassettes) of one of the vectors in the vector combination. After transfection of cells using the vector combination of this disclosure, the presence or absence of one or more recognition sequences on the cell surface can determine whether the corresponding one or more exogenous genes have been transfected into the cells or integrated into the cell's genome.

[0063] In one aspect, this disclosure provides a kit comprising: the vector combination of this disclosure; and a plurality of positive sorting antibodies corresponding to the distinct recognition sequences thereof, the plurality of positive sorting antibodies being labeled with distinct markers detectable by flow cytometry, such as fluorescein markers. A description of the positive sorting antibodies and the markers (positive sorting markers) can be found in the above description.

[0064] In one example, the kit further comprises one or more negative sorting antibodies labeled with one or more distinct markers, such as fluorescein markers, detectable by flow cytometry. In another example, the extracellular domains of membrane proteins expressed by the vectors in the vector assembly have the same or similar sequences as the recognition sequence, such that the extracellular domains can be recognized by the same negative sorting antibody. In this case, a single negative sorting antibody can be used to recognize cells that are co-negative with multiple recognition sequences. Further description of the negative sorting antibodies and the markers (negative sorting markers) can be found above.

[0065] Example 1: Plasmid Construction Method

[0066] The molecular cloning techniques used in the following examples, such as DNA fragment amplification, restriction endonuclease digestion of DNA fragments, gel recovery of DNA fragments, ligation of two DNA fragments, transformation of the ligation product into competent cells, plasmid extraction, preparation, and identification, are all well-known and mature techniques in the art. The following reagents are involved in the following examples: high-fidelity DNA polymerase (Akerui); OK Clon DNA ligation kit (Akerui, AG11803); restriction endonuclease (NEB); DNA fragment gel recovery kit (OMEGA, D2500-02); plasmid miniprep kit (OMEGA, D6943-02); chemicompetent cells (XL-10gold). Plasmids 06.01.2752 (SEQ ID NO:1), 06.01.2804 (SEQ ID NO:2), and 06.01.2793 (SEQ ID NO:3) were synthesized by GenScript. Plasmid 06.01.2752 carries the transposase recognition sequence (5' and 3' inverted repeat sequences) of the Uribo2 transposon system. Plasmid 06.01.2804 carries the Uribo2 transposase coding sequence. Plasmid 06.01.2793 carries the coding sequence of a human codon-optimized phiC31 site-specific recombinase. Plasmid sequencing was performed by Qingke Biotechnology Co., Ltd.

[0067] Table 1 shows the primers used for plasmid construction. Table 2 shows the plasmid number, name, and abbreviation. Table 3 shows a description of the function of the elements appearing in the plasmid. The element sequence information used in the plasmids involved in the following embodiments is an example of implementing this disclosure. Those skilled in the art will expect that replacing the element sequences on the plasmids used in the following embodiments with other element sequences with similar biological functions can achieve the same or similar effects as this disclosure.

[0068] Figure 1 shows a schematic diagram of the structure of the polynucleotide of this disclosure as an example. When the coding sequence of the tag protein is inserted at "Insertion Position 1" shown in Figure 1, the corresponding sequence or variant is named "D1", and when the coding sequence of the tag protein is inserted at "Insertion Position 2" shown in Figure 1, the corresponding sequence or variant is named "D2". PF and PR shown in Figure 1 are the corresponding positions of PCR primers used to verify the cleavage efficiency of the membrane protein expression cassette. The "SpeI restriction site" shown in Figure 1 is the restriction site used for inserting the target exogenous gene using the OK Clon DNA Ligation Kit.

[0069] The specific plasmid construction method is as follows:

[0070] 1. Construction of plasmid 06.01.2755: Two fragments amplified by fusion PCR were ligated using plasmid mCd8a pcDNA3.1-3xFlag-C (Ubibio, 53452) as a template and primers 231120-C-mCD8-F1 (SEQ ID NO:28) and 231120-FP-SPGS-R (SEQ ID NO:30), and 231120-FP-SPGS-F1 (SEQ ID NO:31) and 231120-C-mCD8(TMD)-R (SEQ ID NO:29). The ligation product was double-digested with restriction endonucleases ClaI and KpnI-HF, and then ligated with plasmid 06.01.2752, which had also been double-digested with ClaI and KpnI-HF. This yielded plasmid 06.01.2755 carrying G4S at insertion site 1 as shown in Figure 1. The plasmid 06.01.2755 encodes the nucleic acid sequence (SEQ ID NO:4, hereinafter referred to as mCD8α-D1) of the linker sequence mCD8α (truncation of the intracellular region).

[0071] 2. Construction of plasmid 06.01.2756: Using plasmid mCd8a pcDNA3.1-3xFlag-C (Ubisoft Bio, 53452) as a template, plasmids 231120-C-mCD8-F1 (SEQ ID NO:28), 231120-FP-CD8H-2XGS-TMD-R (SEQ ID NO:32), 231120-mCD8H-2XGS-TMD-F (SEQ ID NO:33), and 231120-C-mCD8(TMD)-R (SEQ ID NO:33) were constructed. Two fragments were ligated using primers NO:29 for PCR amplification. The ligation product was double-digested with restriction endonucleases ClaI and KpnI-HF, and then ligated with plasmid 06.01.2752, which was also double-digested with ClaI and KpnI-HF. This yielded plasmid 06.01.2756, which carries the coding nucleic acid sequence (SEQ ID NO:6, hereinafter referred to as mCD8α-D2) of mCD8α (truncation of the intracellular region) with the G4S linker sequence inserted at insertion position 2 as shown in Figure 1.

[0072] 3. Construction of plasmid 06.01.2758: Using plasmid 06.01.2755 as a template, PCR amplification was performed using 231120-C-mCD8-F1 (SEQ ID NO:28) and 231120-C-SP-1XTag#1-R1 (SEQ ID NO:38) as primers. The amplified fragment was double-digested with restriction endonucleases ClaI and BamHI-HF, and ligated with plasmid 06.01.2752, which was also double-digested with ClaI and BamHI-HF, to obtain plasmid 06.01.2758 carrying the coding nucleic acid sequence (SEQ ID NO:8, hereinafter referred to as HA-D1) of mCD8α (truncation of the intracellular region) with the HA tag coding nucleic acid sequence inserted at insertion position 1 as shown in Figure 1.

[0073] 4. Construction of plasmid 06.01.2760: Using plasmid 06.01.2756 as a template, PCR amplification was performed using 231120-C-1xTag#1-Hinge-F1 (SEQ ID NO:34) and 231120-C-mCD8(TMD)-R (SEQ ID NO:29) as primers. The amplified fragment was double-digested with restriction endonucleases KpnI-HF and BamHI-HF, and ligated with plasmid 06.01.2756, which was also double-digested with KpnI-HF and BamHI-HF, to obtain plasmid 06.01.2760 carrying the coding nucleic acid sequence of mCD8α (truncation of the intracellular region) with the HA tag coding nucleic acid sequence inserted at insertion position 2 as shown in Figure 1 (SEQ ID NO:10, hereinafter referred to as HA-D2).

[0074] 5. Construction of plasmid 06.01.2763: Using plasmid 06.01.2755 as a template, PCR amplification was performed using 231120-C-mCD8-F1 (SEQ ID NO:28) and 231204-C-SP-1xTag#2-R (SEQ ID NO:39) as primers. The amplified fragment was double-digested with restriction endonucleases ClaI and BamHI-HF, and ligated with plasmid 06.01.2755, which was also double-digested with ClaI and BamHI-HF, to obtain plasmid 06.01.2763 carrying the coding nucleic acid sequence of mCD8α (truncation of the intracellular region) with the Flag tag coding nucleic acid sequence inserted at insertion position 1 as shown in Figure 1 (SEQ ID NO:12, hereinafter referred to as Flag-D1).

[0075] 6. Construction of plasmid 06.01.2765: Using plasmid 06.01.2756 as a template, PCR amplification was performed using 231121-C-1xTag#2-Hinge-F (SEQ ID NO:35) and 231120-C-mCD8(TMD)-R (SEQ ID NO:29) as primers. The amplified fragment was double-digested with restriction endonucleases KpnI-HF and BamHI-HF, and ligated with plasmid 06.01.2756, which was also double-digested with KpnI-HF and BamHI-HF, to obtain plasmid 06.01.2765 carrying the coding nucleic acid sequence of mCD8α (truncation of the intracellular region) with the Flag tag coding nucleic acid sequence inserted at insertion position 2 as shown in Figure 1 (SEQ ID NO:14, hereinafter referred to as Flag-D2).

[0076] 7. Construction of plasmid 06.01.2773: Using plasmid 06.01.2755 as a template, PCR amplification was performed using 231120-C-mCD8-F1 (SEQ ID NO:28) and 231122-C-SP-1xTag#4-R (SEQ ID NO:40) as primers. The amplified fragment was double-digested with restriction endonucleases ClaI and BamHI-HF, and ligated with plasmid 06.01.2755, which was also double-digested with ClaI and BamHI-HF, to obtain plasmid 06.01.2773 carrying the coding nucleic acid sequence (SEQ ID NO:16, hereinafter referred to as StrepII-D1) of mCD8α (truncation of the intracellular region) with the StrepII tag coding nucleic acid sequence inserted at insertion position 1 as shown in Figure 1.

[0077] 8. Construction of plasmid 06.01.2775: Using plasmid 06.01.2775 as a template, PCR amplification was performed using 231122-C-1xTag#4-Hinge-F (SEQ ID NO:36) and 231120-C-mCD8(TMD)-R (SEQ ID NO:29) as primers. The amplified fragment was double-digested with restriction endonucleases KpnI-HF and BamHI-HF, and ligated with plasmid 06.01.2776, which was also double-digested with KpnI-HF and BamHI-HF, to obtain plasmid 06.01.2775 carrying the coding nucleic acid sequence of mCD8α (truncation of the intracellular region) with the StrepII tag coding nucleic acid sequence inserted at insertion position 2 as shown in Figure 1 (SEQ ID NO:18, hereinafter referred to as StrepII-D2).

[0078] 9. Construction of plasmid 06.01.2778: Using plasmid 06.01.2755 as a template, PCR amplification was performed using 231120-C-mCD8-F1 (SEQ ID NO:28) and 231122-C-SP-1xTga#5-R (SEQ ID NO:41) as primers. The amplified fragment was double-digested with restriction endonucleases ClaI and BamHI-HF, and ligated with plasmid 06.01.2755, which was also double-digested with ClaI and BamHI-HF, to obtain plasmid 06.01.2778 carrying the coding nucleic acid sequence (SEQ ID NO:20, hereinafter referred to as V5-D1) of mCD8α (truncation of the intracellular region) with the V5 tag coding nucleic acid sequence inserted at insertion position 1 as shown in Figure 1.

[0079] 10. Construction of plasmid 06.01.2780: Using plasmid 06.01.2756 as a template, PCR amplification was performed using 231122-C-1xTag#5-Hinge-F (SEQ ID NO:37) and 231120-C-mCD8(TMD)-R (SEQ ID NO:29) as primers. The amplified fragment was double-digested with restriction endonucleases KpnI-HF and BamHI-HF, and ligated with plasmid 06.01.2756, which was also double-digested with KpnI-HF and BamHI-HF, to obtain plasmid 06.01.2780 carrying the coding nucleic acid sequence (SEQ ID NO:22, hereinafter referred to as V5-D2) of mCD8α (truncation of the intracellular region) with the V5 tag coding nucleic acid sequence inserted at insertion position 2 as shown in Figure 1.

[0080] 11. Construction of plasmid 06.01.2781: Using plasmid 19BF074 (see Chinese Patent Application 202010366440.4, which is incorporated herein by reference) as a template, PCR amplification was performed using primers 231204-C-okclone-rtTA-Tag#1-F (SEQ ID NO:42) and 231204-C-okclone-rtTA-Tag#1-R (SEQ ID NO:43). The amplified fragment was ligated into plasmid 06.01.2758, which was digested with restriction endonuclease SpeI-HF, using the OK Clon DNA ligation kit, thereby obtaining plasmid rtTA, which carries the Tet-ON system regulatory protein. adv The plasmid 06.01.2781 contains the sequence of HA-D1 linked to the sequence of the Cumate system regulatory protein CymR (hereinafter referred to as rtTA / CymR-HA-D1).

[0081] 12. Construction of plasmid 06.01.2782: Using plasmid 18BF074 (see Chinese patent application 202010366440.4) as a template, and 231204-C-okclone-gag / pol-Tag#1-F (SEQ ID NO:44) and 231204-C-okclone-gag / pol-Tag#1-F (SEQ ID NO:45) as primers, PCR amplification was performed. The amplified fragment was ligated into plasmid 06.01.2763 digested with restriction endonuclease SpeI-HF using the OK Clon DNA ligation kit, thereby obtaining plasmid 06.01.2782 carrying the sequence of Flag-D1 linked to the lentiviral packaging gene gag / pol (hereinafter referred to as gag / pol-Flag-D1).

[0082] 13. Construction of plasmid 06.01.2783: Using plasmid 18BF068 (see Chinese patent application 202010366440.4) as a template, and 231204-C-okclone-VSVG / Rev-Tag#4 / 5-F (SEQ ID NO:46) and 231204-C-okclone-VSVG / Rev-Tag#4 / 5-R (SEQ ID NO:47) as primers, PCR amplification was performed. The amplified fragment was ligated into plasmid 06.01.2773 digested with restriction endonuclease SpeI-HF using the OK Clon DNA ligation kit, thereby obtaining plasmid 06.01.2783 carrying the sequence of StrepII-D1 linked to the envelope glycoprotein VSV-G coding sequence (hereinafter referred to as VSV-G-StrepII-D1).

[0083] 14. Construction of plasmid 06.01.2784: Using plasmid 18BF071 (see Chinese patent application 202010366440.4) as a template, and PCR amplification was performed using primers 231204-C-okclone-VSVG / Rev-Tag#4 / 5-F (SEQ ID NO:46) and 231204-C-okclone-VSVG / Rev-Tag#4 / 5-R (SEQ ID NO:47). The amplified fragment was ligated into plasmid 06.01.2778, which was digested with restriction endonuclease SpeI-HF, using the OK Clon DNA ligation kit, thereby obtaining plasmid 06.01.2784 carrying the sequence of V5-D1 linked to the lentiviral packaging gene Rev (hereinafter referred to as Rev-V5-D1).

[0084] Table 1. Primer List

[0085] Table 2. Plasmid ID, Composition Description and Abbreviation

[0086] Table 3. Description of plasmid functional elements

[0087] Table 4. Sequence Description

[0088] Example 2: Single-label instantaneous rotation measurement

[0089] 293T cells (ATCC, CRL-3216) were seeded at a rate of 8E+05 cells per well in 6-well plates (Corning, 3516). The culture medium was 2 ml of DMEM complete medium. After 24 hours of culture, transfection was performed using the PEI method. 200 μl of transfection reagent containing 5 μg of total plasmid was added to each well. The plasmid loading amounts for each group are shown in Table 5 below (the remainder was filled with plasmid 06.01.2752 used as an empty vector). The total plasmid to PEI mass ratio was 1:4. Three hours after transfection, the culture medium in the 6-well plates was replaced with an equal volume of DMEM complete medium, and the cells were cultured for another 24 hours. After trypsin digestion, each cell was injected with 100 μl of Alexa Fluor diluted 1:300. TM 700 (AF700) fluorescently labeled anti-HA Tag antibody (Cell Signaling, 17561S), Brilliant Violet TMCell samples were incubated for 30 minutes with fluorescein-labeled anti-Flag Tag antibody (Biolegend, 637322), FITC-labeled anti-StrepII Tag antibody (Genscript, A01736), or APC-labeled anti-V5 Tag antibody (Abcam, ab72560). After incubation, cells were washed with PBS containing 1% FBS, and fluorescence signals were detected using a flow cytometer (NovoCyte, 3130).

[0090] The results are shown in Figure 2. The AF700-anti HA indicator shows that groups HA-D1 and HA-D2 are using Alexa Fluor. TM The results of incubation with 700 fluorescein-labeled anti-HA tag antibody and flow cytometry analysis of corresponding channel signals were obtained. BV421-anti-Flag shows the results of incubation with BV421 fluorescein-labeled anti-Flag tag antibody and flow cytometry analysis of corresponding channel signals in the Flag-D1 and Flag-D2 groups. FITC-anti-StrepII shows the results of incubation with FITC fluorescein-labeled anti-StrepII tag antibody and flow cytometry analysis of corresponding channel signals in the StrepII-D1 and StrepII-D2 groups. APC-anti-V5 shows the results of incubation with APC fluorescein-labeled anti-V5 tag antibody and flow cytometry analysis of corresponding channel signals in the V5-D1 and V5-D2 groups.

[0091] As shown in Figure 2, the percentage of positive cells after transient transfection was 29.20% in the HA-D1 group, 26.03% in the HA-D2 group, 26.48% in the Flag-D1 group, 38.10% in the Flag-D2 group, 31.20% in the StrepII-D1 group, 28.73% in the StrepII-D2 group, 34.14% in the V5-D1 group, and 31.93% in the V5-D2 group. Based on these results, it can be determined that membrane proteins with different recognition sequences (protein tags) can be recognized by their corresponding antibodies.

[0092] Table 5. Transfection Information Table

[0093] Example 3: Construction and screening of multi-tag stable cell lines

[0094] In this embodiment, multiple exogenous genes were integrated into the cell genome using the transposon system (Uribo2) according to the method disclosed herein, and the constructed cells were screened. Specifically, 293T cells (ATCC, CRL-3216) were seeded at a rate of 8E+05 cells per well in a 6-well plate (Corning, 3516) with 2 ml of DMEM complete medium. After culturing for 24 hours, transfection was performed according to the PEI method. At transfection, 200 μl of transfection reagent containing 5 μg of total plasmid was added to each well. The plasmid composition was as follows: plasmid 06.01.2781 (rtTA / CymR-HA-D1), plasmid 06.01.2782 (gag / pol-Flag-D1), plasmid 06.01.2783 (VSV-G-StrepII-D1), plasmid 06.01.2784 (Rev-V5-D1), and plasmid 06.01.2804 (Uribo2 transposase) were in a mass ratio of 1:1:1:1:0.6. The total plasmid content to PEI mass ratio was 1:4. Three hours after transfection, the medium in the 6-well plate was replaced with an equal volume of DMEM complete medium, and the cells were cultured for another 24 hours. The cells were then digested with trypsin and then mixed with 100 μl of Alexa Fluor diluted 1:300. TM Cells were co-incubated for 30 minutes with 700 fluorescein-labeled anti-HA tag antibody (Cell Signaling, 17561S), BV421 fluorescein-labeled anti-Flag tag antibody (Biolegend, 637322), FITC fluorescein-labeled anti-StrepII tag antibody (Genscript, A01736), and APC fluorescein-labeled anti-V5 tag antibody (Abcam, ab72560). After incubation, cells were washed with PBS containing 1% FBS, and the percentage of 4-tag positive cells before sorting was detected using a Beckman CytoFLEX SRT flow cytometer ("Before Sorting" in Figure 3). After detection, 4-tag positive cells were sorted using a Beckman CytoFLEX SRT flow cytometer. After culturing in 6-well plates for 5 days, cells were digested with trypsin and then mixed with 100 μl of Alexa Fluor diluted 1:300. TMCells were co-incubated for 30 minutes with 700 fluorescein-labeled anti-HA tag antibody (Cell Signaling, 17561S), BV421 fluorescein-labeled anti-Flag tag antibody (Biolegend, 637322), FITC fluorescein-labeled anti-StrepII tag antibody (Genscript, A01736), and APC fluorescein-labeled anti-V5 tag antibody (Abcam, ab72560). After incubation, the cells were washed with PBS containing 1% FBS, and then the 4-tag positive cells were re-sorted using a Beckman CytoFLEX SRT flow cytometer. These cells were named 293T-HA-Flag-StrepII-V5 cells (“After sorting” in Figure 3).

[0095] As shown in Figure 3, the graph corresponding to "before sorting" shows the proportion of positive cells 24 hours after co-transfection with the four plasmids: the proportion of double positivity for HA and V5 tags was 19.00%, and the proportion of positivity for all four tags was 18.96% (19.00% * 99.83%). The graph corresponding to "after sorting" shows the proportion of positive cells after two sorting operations using flow cytometry: the proportion of double positivity for HA and V5 tags was 91.93%, and the proportion of positivity for all four tags was 90.43% (91.93% * 98.37%).

[0096] Further, the expression level of the exogenous gene (lentiviral packaging gene) in the obtained 4-tag positive cells was determined (by measuring viral transduction titer). Specifically, 293T-HA-Flag-StrepII-V5 cells were seeded at a rate of 8E+05 cells / well in 6-well plates (Corning, 3516) using 2 ml of DMEM complete medium. After 24 hours of culture, the cells were transfected using the PEI method with the transgenic plasmid pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene, #12252, 2 μg). Three hours after transfection, the medium was changed to DMEM complete medium containing 1 μg / ml doxycycline (DOX), 200 μg / ml 4-isopropylbenzoic acid (Cumate), and 5 mmol / L sodium butyrate. After 48 hours of further culture, the supernatant was collected, diluted at different ratios, and then transduced into 293T cells. The EGFP expression ratio of 293T cells at different dilution points was detected using an Eisen flow cytometer (NovoCyte 3130) to calculate the transduction titer of the prepared lentivirus. The results showed (“293T-HA-Flag-StrepII-V5” in Figure 6) that the transduction titer of the lentivirus prepared from 293T-HA-Flag-StrepII-V5 cells was 2.7 E6TU(EGFP) / ml.

[0097] Based on the above results, it can be determined that the cell construction and sorting method based on membrane proteins in this disclosure can be used to quickly screen cell lines that stably integrate and express multiple exogenous genes.

[0098] Example 4: Deletion of expression cassettes for membrane proteins

[0099] In this embodiment, the expression cassette of a membrane protein with a protein tag coding sequence was removed from the genome of a cell (293T-HA-Flag-StrepII-V5) using a phiC31 site-specific recombinase system (as mentioned above, the expression cassette of the membrane protein has recognition sites attP / attB of the phiC31 recombinase on both sides), and the effect of the above removal was detected by using a fluorescently labeled anti-mCD8α antibody (which can detect four chimeric membrane proteins at once).

[0100] Specifically, 293T-HA-Flag-StrepII-V5 cells were seeded at a rate of 8E+05 cells per well in 6-well plates (Corning, 3516) using 2 ml of DMEM complete medium. After 24 hours of culture, the cells were transfected with plasmid 06.01.2793 (5 μg) using the PEI method. Three hours after transfection, the medium in the 6-well plates was replaced with an equal volume of DMEM complete medium to obtain 293T-HA-Flag-StrepII-V5 (phiC31 cleaved) cells. After culturing for another 5 days, the cells were trypsinized and then incubated for 30 minutes with a 1:300 dilution of FITC-labeled anti-mCD8α antibody (BD, 553030). After incubation, the cells were washed with PBS solution containing 1% FBS, and then FITC-negative cells were sorted using a Beckman CytoFLEX SRT flow cytometer. These cells were designated as 293T-HA-Flag-StrepII-V5 (phiC31 shearing + negative selection) cells.

[0101] The results are shown in Figure 4. In Figure 4, "293T control" shows the flow cytometry results after incubating the original 293T cells with FITC-labeled anti-mCD8α antibody; "HA-Flag-StrepII-V5" shows the flow cytometry results after incubating 293T-HA-Flag-StrepII-V5 cells with FITC-labeled anti-mCD8α antibody; "HA-Flag-StrepII-V5 (phiC31 cleavage)" shows the flow cytometry results after incubating 293T-HA-Flag-StrepII-V5 (phiC31 cleavage) cells with FITC-labeled anti-mCD8α antibody; and "HA-Flag-StrepII-V5 (phiC31 cleavage + negative selection cells)" shows the flow cytometry results after incubating 293T-HA-Flag-StrepII-V5 (phiC31 cleavage + negative selection) cells with FITC-labeled anti-mCD8α antibody. As shown in Figure 4, the mCD8α positivity rate of 293T-HA-Flag-StrepII-V5 cells (S4) was 99.04%, the mCD8α positivity rate of 293T-HA-Flag-StrepII-V5 (phiC31 cleavage) cells (S3) was 70.98%, and the mCD8α positivity rate of 293T-HA-Flag-StrepII-V5 (phiC31 cleavage + negative selection) cells (S2) could be reduced to 7.89%.

[0102] Simultaneously, genomic DNA was extracted from each cell using a DNA extraction kit (Aikerui, AG21009) as a PCR template. PF (5'-AATACTATCTTGCAGATAGC-3') and PR (5'-AAGGCCTGTTTAAACACCGG-3') were used as primers, and Phusion was employed. TM Amplification was performed using High-Fidelity DNA Polymerase (Thermo, F530S). The amplification products were analyzed by gel electrophoresis. The results are shown in Figure 5. Theoretically, if the expression cassette of the membrane protein is not cleaved, the amplification will yield a 2240 bp product, while if the expression cassette of the membrane protein is cleaved, the amplification will yield a 299 bp product.

[0103] In Figure 5, NTC shows the gel electrophoresis results without a template control; S1 shows the gel electrophoresis results of the original 293T cells as a control; S2 shows the gel electrophoresis results of 293T-HA-Flag-StrepII-V5 cells (phiC31 cleavage + negative selection) (theoretically, only a 299bp band should be seen); S3 shows the gel electrophoresis results of 293T-HA-Flag-StrepII-V5 cells (phiC31 cleavage) (theoretically, both a 2240bp and a 299bp band should be seen due to the lack of sorting); S4 shows the gel electrophoresis results of 293T-HA-Flag-StrepII-V5 cells (theoretically, only a 2240bp band should be seen); P shows the gel electrophoresis results of amplification using plasmid 06.01.2760 as a template as a control (theoretically, only a 2240bp band should be seen). The results shown in Figure 5 are consistent with expectations. Based on the above results, it can be determined that after phiC31 shearing and flow cytometry negative sorting, the cell genome basically no longer contains expression cassettes of membrane proteins.

[0104] Furthermore, the effect of cleaving the expression cassette of the membrane protein from the cell genome on the expression level of the uncleaved target exogenous gene (lentiviral packaging gene) was examined (by measuring viral transduction titer). Specifically, 293T-HA-Flag-StrepII-V5 cells (phiC31 cleavage + negative selection) were seeded at 8E+05 cells / well in 6-well plates (Corning, 3516) in 2 ml of DMEM complete medium. After 24 hours of culture, the cells were transfected with the transgenic plasmid pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene, #12252, 2 μg) according to the PEI method. Three hours after transfection, the medium was changed to DMEM complete medium containing 1 μg / ml doxycycline (DOX), 200 μg / ml 4-isopropylbenzoic acid (Cumate), and 5 mmol / L sodium butyrate. After 48 hours of further culture, the supernatant was collected, diluted at different ratios, and then transduced into 293T cells. The EGFP expression ratio of 293T cells at different dilution points was detected using an Eisen flow cytometer (NovoCyte 3130) to calculate the transduction titer of the prepared lentivirus. The results showed (in Figure 6, “293T-HA-Flag-StrepII-V5 (phiC31 cleavage + negative selection)”) that the transduction titer of the lentivirus prepared from 293T-HA-Flag-StrepII-V5 (phiC31 cleavage + negative selection) cells was 2.5E6 TU(EGFP) / ml, which was not significantly lower than that of 293T-HA-Flag-StrepII-V5 cells that had not undergone cleavage and negative selection (2.7E6 TU(EGFP) / ml, as described in Example 3).

[0105] Based on the above results, it can be determined that, after shearing and negative sorting, the membrane proteins used for screening can be removed from more than 90% of the cells, and the removal does not affect the expression and function of the target exogenous genes that need to be integrated into the cell genome.

[0106] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this disclosure. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0107] Although embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present disclosure. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present disclosure.

Claims

1. A polynucleotide, wherein, The polynucleotide includes an expression cassette for a membrane protein and one or more additional expression cassettes for expressing one or more additional genes, the expression cassette for the membrane protein being linked to the one or more additional expression cassettes, the membrane protein including a transmembrane domain and an extracellular domain, the extracellular domain including a recognition sequence that can be specifically recognized by an antibody of the recognition sequence.

2. The polynucleotide of claim 1, wherein, The expression cassette for the membrane protein contains a nucleic acid sequence encoding the membrane protein and a nucleic acid sequence encoding a signal peptide, the signal peptide being used to guide the membrane protein to the cell membrane.

3. The polynucleotide of claim 1, wherein, The identification sequence is a protein tag sequence.

4. The polynucleotide of claim 1, wherein, The polynucleotide includes an expression cassette region containing the expression cassette of the membrane protein and one or more additional expression cassettes, and at least one of the sides of the expression cassette region has an integration sequence that enables the expression cassette region to be integrated into the genome of the cell when the polynucleotide is transferred into the cell.

5. The polynucleotide of claim 1, wherein, The expression cassette of the membrane protein has cleavage sequences on both sides, which enable the expression cassette of the membrane protein to be cleaved from the expression cassette region.

6. The vector of claim 5, wherein, The vector comprises a polynucleotide according to any one of claims 1-5.

7. A cell, wherein, The cell contains a polynucleotide according to any one of claims 1-5 or a vector according to claim 6.

8. A method of transferring a foreign gene into a cell, wherein, The method includes transferring a polynucleotide according to any one of claims 1-5 or a vector according to claim 6 into the cell.

9. The method of claim 8, wherein, The expression cassette region is integrated into the genome of the cell.

10. The method of claim 8, wherein, The method further includes incubating the cells with the antibody after transferring the polynucleotide according to any one of claims 1-5 or the vector according to claim 6 into the cells.

11. The method of claim 10, wherein, The antibody is labeled with a positive sorting marker that can be detected by flow cytometry, preferably a fluorescein-based marker.

12. The method of claim 11, wherein, The method also includes sorting cells that are positive for the positive sorting marker by flow cytometry.

13. The method of claim 8, wherein, The method further includes, after transferring the polynucleotide according to any one of claims 1-5 or the vector according to claim 6 into the cell, cutting off the expression cassette of the membrane protein from the expression cassette region.

14. The method of claim 13, wherein, The method further includes, after cutting the expression cassette of the membrane protein from the expression cassette region, incubating the cells with an antibody that specifically binds to the extracellular domain of the membrane protein, wherein the antibody that specifically binds to the extracellular domain of the membrane protein is labeled with a negative sorting marker that can be detected by flow cytometry, wherein the negative sorting marker is preferably a fluorescein-based marker.

15. The method of claim 14, wherein, The method also includes sorting cells that are negative for the negative sorting marker by flow cytometry.

16. A cell, wherein, The cells are obtained by the method according to any one of claims 8-15.

17. Use of the polynucleotide according to any one of claims 1-5, the vector according to claim 6, or the cell according to claim 7 in the preparation of a stable cell line with a single exogenous gene or multiple exogenous genes.

18. A combination of vectors wherein, The carrier combination includes multiple The carrier according to claim 6 expresses different recognition sequences.

19. A kit, wherein, The kit contains: The carrier assembly according to claim 18; and A variety of positive sorting antibodies, corresponding to the different recognition sequences, are labeled with different markers that can be detected by flow cytometry, such as fluorescein markers.

20. The kit of claim 19, wherein, The kit also includes one or more negative sorting antibodies labeled with one or more distinct markers, such as fluorescein markers, that can be detected by flow cytometry; preferably, the extracellular domains of the membrane proteins expressed by the vectors in the vector assembly have the same or similar sequences, except for the recognition sequence, so that the extracellular domains can be recognized by the same negative sorting antibody.

Images