FLP-IN-DF-1 cell line as well as construction method and application thereof

Abstract

The invention relates to the related technical field of molecular biology and cell engineering, in particular to an FLP-IN-DF-1 cell line and a construction method and application thereof, and the construction method comprises the following steps: step 1, constructing CRISPR / Cas9 site-specific cleavage plasmid containing a chicken HIPP11 safety site sequence; step 2, constructing a donor plasmid Donor-FRT containing a homologous arm of a safety site of the HIPP11; 3, jointly transfecting plasmids in the step 1 and the step 2 into a chicken DF-1 cell line to obtain an FLP-IN-DF-1 cell line for expressing red fluorescence; and step 4, under the action of Flp recombinase, transfecting an exogenous gene donor plasmid containing an FRT site to the FLP-IN-DF-1 cell line obtained in the step 3, and driving the expression of a target gene through an optimized strong promoter to realize site-specific transfer and efficient expression of the exogenous gene in the chicken DF-1 cell line. Different from an existing Flp-In system depending on a random integration strategy, the construction method disclosed by the invention emphasizes fixed-point integration and promoter optimization, and improves the homogeneity, stability and screening efficiency of exogenous gene expression.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and cell engineering, and in particular to the FLP-IN-DF-1 cell line, its construction method, and its applications. Background Technology

[0002] In the fields of molecular biology and cell engineering research, constructing stable cell lines expressing exogenous genes is a crucial foundation for applications such as protein function verification, vaccine development, and industrial production of exogenous proteins. Currently, the construction of stable cell lines mainly relies on the following methods:

[0003] Random integration. When using plasmid transfection, lentiviral, or retroviral vector-mediated gene delivery methods, exogenous genes are typically integrated into the host genome randomly. These methods result in uncontrollable integration sites, significant positional effects, uneven expression levels of the exogenous gene, poor stability, and large differences between cell lines.

[0004] Transposon-mediated integration. Transposon systems such as PiggyBac can improve integration efficiency, but their integration events still occur randomly at multiple sites in the genome, and multi-copy integration may lead to genomic structural instability, thereby affecting the genetic stability of cell lines and subsequent applications.

[0005] The Flp-In system. This system mediates the specific integration of exogenous genes into FRT sites via Flp recombinase, which can improve expression consistency to some extent. However, FRT sites usually need to be pre-introduced into the host genome via random integration, and their final insertion position is uncontrollable. Therefore, the expression stability of exogenous genes is still affected by the integration site. In addition, existing Flp-In platforms are mainly suitable for mammalian cells and have insufficient adaptability to avian cells such as DF-1. There is still a lack of directly applicable site-specific integration systems.

[0006] In summary, current technologies for DF-1 cells cannot yet achieve a site-specific integration system with controllable integration sites, stable expression, and suitability for large-scale construction. Therefore, there is an urgent need to develop a novel site-specific integration method adapted to DF-1 cells, and to ensure screening efficiency and stable expression of exogenous genes by optimizing promoter and selection marker design, so as to obtain cell lines with uniform expression and genetic stability. Summary of the Invention

[0007] The purpose of this invention is to provide the FLP-IN-DF-1 cell line, its construction method, and its application, in order to solve the problem mentioned in the background art of the inability to achieve a stable expression platform for exogenous genes with controllable integration sites, stable expression, and high screening efficiency in avian cells (such as DF-1 cells).

[0008] To achieve the above objectives, the present invention provides the following technical solution: FLP-IN-DF-1 cell line, wherein the cell line uses CRISPR / Cas9 technology to knock in the FRT sequence at the HIPP11 safe site of chicken embryonic fibroblast DF-1, and uses Flp recombinase to achieve site-specific integration of exogenous genes at the FRT site, thereby constructing a stable and controllable gene expression platform.

[0009] Preferably, the exogenous gene is a target gene, and the DNA fragment of the target gene is less than 10kb.

[0010] Preferably, the cell line maintains stable exogenous gene expression through puromycin selection and maintains stable integration of the target gene during continuous passage.

[0011] Preferably, the cell line achieves stable integration of a single FRT sequence at the HIPP11 safe site.

[0012] Preferably, the site-directed integration of the FRT sequence at the HIPP11 safe site of the cell line with the exogenous gene is mediated by Flp recombinase and does not depend on random integration mechanisms.

[0013] Preferably, the cell line uses CRISPR / Cas9 technology to precisely insert the FRT sequence into the DF-1 cell genome, and uses Flp recombinase to achieve efficient integration of the exogenous gene at this site.

[0014] Preferably, the donor plasmid of the resistance gene in the cell line uses the CMV promoter to drive the expression of the resistance gene, and the target gene is expressed through the CBh promoter.

[0015] The method for constructing the FLP-IN-DF-1 cell line includes the following steps:

[0016] 1. CRISPR / Cas9 site-specific cleavage: sgRNAs targeting the HIPP11 safe site were designed, and a double-stranded cleavage was generated at this site using the CRISPR / Cas9 system, providing an insertion window for precise integration of the FRT sequence;

[0017] 2. Donor plasmid construction and FRT integration: The FRT sequence and its upstream and downstream homologous arms are constructed into the donor plasmid, and the FRT sequence is precisely inserted into the HIPP11 site through homologous recombination;

[0018] 3. Promoter optimization and screening strategy: The CMV promoter is used to drive the puromycin resistance gene, which increases the expression of the resistance gene and enhances the screening pressure, thereby efficiently screening for cells that have correctly integrated FRT;

[0019] 4. Monoclonal screening and expansion culture: FLP-IN-DF-1 monoclonal cells that stably integrate FRT were obtained through resistance screening and monoclonal culture, and then expanded and identified.

[0020] The FLP-IN-DF-1 cell line is used for:

[0021] 1. Stable expression of exogenous genes, suitable for constructing a highly consistent and reproducible stable expression system in avian cells;

[0022] 2. Gene function research, used to verify the effects of regulatory elements, gene mutants, and protein function;

[0023] 3. Exogenous protein expression and production, achieving controllable expression and avoiding expression fluctuations caused by position effects;

[0024] 4. Establishment of a site-directed integration expression system based on Flp recombination to support subsequent modular expression of multiple genes or stable transgene construction.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. Achieve true site-specific integration: Exogenous genes are always integrated into the same safe genomic site, overcoming the problems of expression differences, position effects and expression instability caused by random integration.

[0027] 2. Promoter optimization improves expression stability: The CMV strong promoter drives the selection marker, improving the efficiency of resistance selection; the CBh promoter drives the expression of the target gene, avoiding interference with the CMV promoter and improving the stability and consistency of gene expression after integration.

[0028] 3. Applicable to avian cell systems: It solves the long-standing problem of the lack of a usable platform for the Flp-In system in avian cells (especially DF-1 cells), and provides an efficient gene expression tool for fields such as virology, immunology, and vaccine research.

[0029] 4. High construction efficiency and good reproducibility: By combining the CRISPR / Cas9+FRT system, single-copy, reproducible engineered cell lines can be stably obtained. Attached Figure Description

[0030] Figure 1 The image shows the PCR amplification results of various functional fragments of the donor plasmid. Lane 1 is the amplified band of the plasmid backbone sequence, lane 2 is the amplified band of the FRT site sequence, lane 3 is the amplified band of the upstream homologous arm of the chicken HIPP11 site, and lane 4 is the amplified band of the downstream homologous arm of the chicken HIPP11 site.

[0031] Figure 2 This is a schematic diagram of the basic structural elements of a donor plasmid.

[0032] Figure 3 A schematic diagram illustrating the co-transfection of DF-1 cells with a CRISPR / Cas9 site-specific cleavage plasmid and a donor plasmid.

[0033] Figure 4 These are monoclonal cell clusters obtained after screening for monoclonal drugs.

[0034] Figure 5 The PCR results and sequencing verification diagram for amplifying the FRT sequence in the FLP-IN-DF-1 cell genome are shown above. The sequence alignment results of the PCR amplification products are shown above, and the sequencing peak diagram is shown below. Sequencing of the recovered products confirmed that the FRT site was correctly inserted into the chicken HIPP11 safe site, which was used to confirm that the FRT site was successfully integrated into the host cell genome.

[0035] Figure 6 The image shows the expression of red fluorescent protein in the stable FLP-IN-DF-1 cell line during the expansion culture process, including a bright field image, a Cherry fluorescence image, and an overlay of the two. It can be seen that the cell population still maintains a stable and uniform red fluorescence signal after long-term passage culture, with a red fluorescence positivity rate of 100%, confirming the stability of cell line expression.

[0036] Figure 7 Image of the precipitate after centrifugation of FLP-IN-DF-1 cells.

[0037] Figure 8 The results show the copy numbers of the ACTB gene (left) and FRT site (right) in FLP-IN-DF-1 cells, detected by digital titer PCR (ddPCR).

[0038] Figure 9 This is a schematic diagram of the operation process for constructing a stable FLP-IN-DF-1 cell line in an embodiment of the present invention.

[0039] Figure 10 This is a schematic diagram of fluorescence images showing the expression of the target gene in cells after FLP recombination. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The principle of this invention is based on the specific recombination and integration mechanism of the FRT sequence in the Flp-In system. First, a single FRT site is introduced into the genomic HIPP11 safe site using CRISPR / Cas9, making the integration location of the exogenous gene controllable, uniform, and stable. Then, Flp recombinase catalyzes FRT-FRT specific recombination, precisely inserting the donor vector carrying the target gene into this site. Simultaneously, by optimizing the promoter combination (CMV for selection markers and CBh for target gene expression), sufficient expression driving force is ensured before and after integration, improving selection efficiency and supporting long-term stable expression of the target gene.

[0042] Example 1: Construction of donor plasmid

[0043] The FRT sequence was amplified from the pFRT-lacZeo plasmid and inserted between the CMV promoter and the Cherry sequence (a red fluorescent protein). The knock-in fragment was amplified using pre-designed primers, and the amplification product was verified by 1% agarose gel electrophoresis. Figure 1 As shown, a specific amplification band of approximately 1.1 kb can be obtained.

[0044] Subsequently, the backbone sequence, upstream and downstream homologous arms of HIPP11, and the knock-in sequence were mixed at a mass ratio of 100 ng: 50 ng: 50 ng: 80 ng and added to a homologous recombinase system for ligation. The ligation product was transformed into competent DH5α cells, cultured overnight at 37°C, and single clones were picked for sequencing verification. The sequencing results are as follows: Figure 2 As shown, the FRT sequence was completely inserted, and the donor plasmid was successfully constructed. The positive cloning rate of this step was approximately 65–85% (n=20).

[0045] Example 2 Construction and screening of FLP-IN-DF-1 cell line

[0046] 2.1 Resuscitation and Plating of DF-1 Cells

[0047] After resuscitation, DF-1 cells were cultured at 39°C and 5% CO2. After 3–4 stable passages, the cells were seeded into 60 mm culture dishes, and transfection was performed when the cell density reached approximately 70%–80%.

[0048] 2.2 Co-transfection of CRISPR / Cas9 cutting plasmid and donor plasmid

[0049] The cleavage plasmid (PX458-sgRNA) and the donor plasmid were co-transfected into DF-1 cells using the Lip3000 transfection reagent. The specific transfection steps are as follows:

[0050] (1) Add 250 μl of Opti-MEM medium to a 1.5 mL sterile centrifuge tube, add 6 μg of the cutting plasmid PX458-sgRNA and 6 μg of the donor plasmid and mix well, then add 22 μl of P3000 reagent and mix well.

[0051] (2) Add 250 μl of Opti-MEM medium and 24 μl of ILP3000 medium to a 1.5 mL sterile centrifuge tube and mix well;

[0052] (3) Mix the reagents in the two centrifuge tubes, incubate at room temperature for 15 min, and then evenly drop the mixture into a 60 mm dish for transfection;

[0053] (4) Replace with fresh culture medium 12 hours after transfection;

[0054] (5) Observe the cells under a fluorescence microscope after 24 hours. Green fluorescence (GFP) indicates successful expression of the cleaved plasmid, and red fluorescence (Cherry) indicates expression of the donor plasmid. When the two overlap, they appear yellow. Figure 3 As shown, in typical experiments, the GFP positivity rate after transfection is about 30-40%, and the Cherry positivity rate is about 40-50% (fluorescence analysis).

[0055] 2.3 Drug screening yields integrated positive clones

[0056] Puromycin screening was performed 36 hours after transfection, following the steps below:

[0057] (1) 36 hours after transfection, the cells were digested and passaged. The cells in each 60 mm dish were mixed by pipetting and passaged into two 10 cm dishes to reduce the cell density.

[0058] (2) About 12-24 hours after passage, after observing that the cells have successfully adhered to the wall and have normal morphology and no vacuoles inside, the original culture medium is discarded and replaced with a complete culture medium containing 2 μg / mL puromycin for screening.

[0059] (3) Change the culture medium containing 2 μg / mL puromycin once a day for a total of one week;

[0060] (4) The culture medium containing 1.5 μg / mL puromycin was changed every two days for a total of three weeks. At this time, the cells that had not stably integrated the knock-in sequence into the genome had basically died. The cells that had stably integrated the sequence could proliferate normally under drug screening, thus forming monoclonal cell clusters. The monoclonal clusters expressed stable red fluorescence, which could be seen under a microscope as follows. Figure 4 .

[0061] Typical drug screening data: Primary screening survival rate: 1–5%; Monoclonal formation rate: approximately 20–40 clones / 10cm dish.

[0062] Example 3 Identification of the FLP-IN-DF-1 cell line

[0063] Subsequently, monoclonal strains were collected using a monoclonal loop for expansion culture. The location of the monoclonal strains was observed under a microscope, and black markers were used to mark the bottom of the dish. A 6mm sterile glass monoclonal loop was dipped into Dow Corning vacuum silica gel and placed over the marked area. Trypsin was added to the loop for digestion, and the digested strains were then transferred to 12-well plates for expansion culture.

[0064] Gene knock-in effect was identified by gene sequencing. Different FLP-IN-DF-1 cells were collected, and total DNA was extracted from each cell line using a DNA extraction kit. PCR amplification was performed using HIPP11 primers. The primers were:

[0065] HIPP11-F:5'-GTGAGCTTGTATTGCAGCGTTC-3'

[0066] HIPP11-R:5'-GTCTGTAGCAACTGCTCTGGAA-3'

[0067] Mix 10 pmol of forward and reverse primers, 100 ng of total cellular DNA, and 10 μl of 2×KODONEMIX, then add enzyme-free water to a final volume of 20 μl. Perform agarose gel electrophoresis on the PCR products. Recover the PCR products from the identified amplified samples and perform sequencing for identification. Figure 5 Sequencing of the recovered products confirmed that the FRT site was correctly inserted. Typical integration efficiency: approximately 25% of the screened clones were correctly integrated clones (n=50).

[0068] FLP-IN-DF-1 cells were passaged and cultured for 10 generations in medium containing 0.5 μg / mL puromycin. Microscopic observation revealed stable red fluorescence signals, such as... Figure 6 The positive rate of red fluorescence was 100%. Cells expressing high levels of Cherry exhibited a distinct red hue, such as... Figure 7 .

[0069] To identify a single insertion at the FRT site: 5 ng of DNA was extracted from FLP-IN-DF-1 cells. A PCR system was prepared according to the SniperddPCR manual. Primers were used to identify the copy number of the ACTB gene and the copy number of the insertion site FRT. ACTB is a known single-copy gene of DF-1, and was therefore used to correct the FRT copy number. If the FRT site has only one insertion in the genome, the ACTB gene copy number should be twice the FRT copy number at the insertion site, as shown in the results. Figure 8 ACTB: Approximately 2 copies / cell

[0070] FRT: Approximately 1 copy / cell, indicating that the donor is integrated into the genome singly. Detection primers are as follows:

[0071] ACTB-F:5'-GTGCGTGACATCAAGGAGAAGC-3'

[0072] ACTB-R:5'-AGGAGTCAGACTTACCCAAGAAAGAT-3'

[0073] FRT-F:5'-GCCTCCTCCGAGCGGATGTA-3'

[0074] FRT-R:5'-GTCTTGACCTCAGCGTCGTAGTG-3'

[0075] Example 4: Application of the FLP-IN-DF-1 cell line

[0076] 4.1 Construction of PCDNA5-FRT vector based on CBh promoter

[0077] In FLP-IN-DF-1 cells, the knock-in sequence uses the CMV promoter to express the target protein. To avoid promoter interference caused by the same strong promoter in stable cells, the CBh promoter was used instead of the CMV promoter to drive the expression of the target gene in the PCDNA5 plasmid.

[0078] Primers for amplifying the backbone sequence:

[0079] P5-F:5'-CATAGAAGACACCGGGACCGATC-3'

[0080] P5-R:5'-AACGCGTATATCTGGCCCGTACA-3'

[0081] Primers for amplifying the CBh promoter:

[0082] CBH-F:5'-TGTACGGGCCAGATATACGCGTTcgttac…-3'

[0083] CBH-R:5'-GATCGGTCCCGGTGTCTTCTATGCCaacct…-3'

[0084] The backbone sequence and insert sequence were mixed at a mass ratio of 100 ng:50 ng and ligated with homologous recombinase. The ligation product was directly added to competent DH5α cells, plated, and single clones were picked and sequenced to obtain the PCDNA5-FRT plasmid. An arbitrary target protein sequence was inserted into the multiple restriction enzyme sites (MCS) of the PCDNA5-FRT plasmid. In this experiment, the EGFP sequence was used as an example to construct the PCDNA5-FRT-EGFP plasmid.

[0085] 4.2 Flp recombinase-mediated integration and screening of target genes

[0086] The pOG44 plasmid expresses Flp recombinase, which allows the PCDNA5 plasmid to be recombinated and inserted into the FRT site. The principle is as follows: Figure 9 .

[0087] The co-transfection procedure was similar to that described above. After 24 hours, selection was performed using 200 μg / mL hygromycin B for 7 days. FLP-IN-DF-1-EGFP cells stably expressing green fluorescence after selection were as follows: Figure 10 As shown. EGFP positivity rate: 80–95%.

[0088] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The FLP-IN-DF-1 cell line, characterized by: The cell line was constructed by knocking the FRT sequence into the HIPP11 safe site of chicken embryonic fibroblast DF-1 cells using CRISPR / Cas9 technology, and by using Flp recombinase to achieve site-specific integration of exogenous genes at the FRT site, thus building a stable and controllable gene expression platform.

2. The FLP-IN-DF-1 cell line according to claim 1, characterized in that: The exogenous gene is a target gene, and the DNA fragment of the target gene is less than 10kb.

3. The FLP-IN-DF-1 cell line according to claim 1, characterized in that: The cell line maintains stable exogenous gene expression through puromycin selection and maintains stable integration of the target gene during continuous passage.

4. The FLP-IN-DF-1 cell line according to claim 1, characterized in that: The cell line achieved stable integration of a single FRT sequence at the HIPP11 safe site.

5. The FLP-IN-DF-1 cell line according to claim 1 or 2, characterized in that: The cell line's site-directed integration of the FRT sequence at the HIPP11 safe site with the exogenous gene is mediated by Flp recombinase and does not depend on random integration mechanisms.

6. The FLP-IN-DF-1 cell line according to claim 1, characterized in that, The cell line uses CRISPR / Cas9 technology to precisely insert the FRT sequence into the DF-1 cell genome, and uses Flp recombinase to achieve efficient integration of the exogenous gene at this site.

7. The FLP-IN-DF-1 cell line according to claim 1, characterized in that, In the cell line, the donor plasmid of the resistance gene is expressed using the CMV promoter, and the target gene is expressed using the CBh promoter.

8. A method for constructing the FLP-IN-DF-1 cell line according to any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Design sgRNA targeting the HIPP11 safe site and use the CRISPR / Cas9 system to generate a double-stranded nick at this site to provide an insertion window for the precise integration of the FRT sequence; (2) The FRT sequence and its upstream and downstream homologous arms were constructed into the donor plasmid, and the FRT sequence was precisely inserted into the HIPP11 site through homologous recombination; (3) The CMV promoter is used to drive the puromycin resistance gene, which increases the expression of the resistance gene and enhances the screening pressure, thereby efficiently screening for cells that have been correctly integrated with FRT; (4) FLP-IN-DF-1 monoclonal cells with stable integration of FRT were obtained through resistance screening and monoclonal culture.

9. The application of the FLP-IN-DF-1 cell line according to any one of claims 1 to 7 in gene function research.

10. The use of the FLP-IN-DF-1 cell line according to any one of claims 1 to 7 in the production of exogenous proteins.

Images