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WO2019228108A1 - Composition de réactif utilisée pour augmenter l'efficacité de transfection cellulaire - Google Patents

Composition de réactif utilisée pour augmenter l'efficacité de transfection cellulaire Download PDF

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Publication number
WO2019228108A1
WO2019228108A1 PCT/CN2019/084394 CN2019084394W WO2019228108A1 WO 2019228108 A1 WO2019228108 A1 WO 2019228108A1 CN 2019084394 W CN2019084394 W CN 2019084394W WO 2019228108 A1 WO2019228108 A1 WO 2019228108A1
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cells
transfection
actinomycin
solution
medium
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Chinese (zh)
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陈骐
傅雅娟
郑立群
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Fujian Normal University
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Fujian Normal University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a reagent composition for improving the efficiency of cell transfection.
  • Gene therapy is a new research field in contemporary medicine and biology. It introduces normal genes or therapeutic DNA sequences into target cells in a certain way to correct gene defects or play a therapeutic role, thereby achieving the purpose of treating diseases.
  • scientists have been studying effective ways to deliver plasmid DNA.
  • conventional exogenous expression systems are mainly divided into two types: one is the method of using viral vectors.
  • the three mainstream viral vectors include lentiviral vectors, retroviral viral vectors, and adenovirus (AAV) vectors. These methods are the mainstream choice for clinical trials such as gene therapy.
  • Viral vectors such as AAV vectors have the characteristics of high transfection rate and continuous and stable expression of foreign genes, but they need to be integrated into the host cell's chromosome to enable the continuous expression of foreign genes, so they have the risk of causing cancer and teratogenicity.
  • the disadvantage of poor safety has become the main reason for limiting the widespread use of viral vectors.
  • the other is the use of non-viral vectors.
  • This kind of non-viral vector is highly safe and easy to prepare. In terms of effects and development prospects, it is more advantageous than viral vectors in gene therapy.
  • the introduction efficiency and targeting of this type of vector are low.
  • Most host cells are highly resistant to foreign DNA, and the expression time of foreign genes is short after they are transferred into the host cells. The foreign genes are quickly shut down after transient expression. All have severely restricted the application and development of such carriers.
  • Non-viral vectors include liposomes, polymer vectors, nanogene transporters, and the like.
  • the general transfection methods are mostly liposome and electroporation methods.
  • the surface of the cationic liposome is positively charged, and the DNA molecule is encapsulated into a DNA-liposome complex by electrostatic interaction with the phosphate of the nucleic acid. It can be adsorbed on the negatively charged cell membrane and enter through the membrane fusion or endocytosis. Cells in vivo. Sometimes DNA is also transferred into cells through the direct penetration of small holes in the cell membrane.
  • the electroporation method or electroporation method uses a high-intensity electric field to instantly increase the permeability of the cell membrane, thereby sending foreign molecules, such as DNA, mRNA, proteins, and sugars, into the cytoplasm of the host cell.
  • the methods currently used for genetic modification of primary T cells are mainly performed by viral infection and electroporation. Viral methods often require higher virus titers when infecting primary cells and are generally less effective. For T cells, additional cytokines, such as IL-2 and IL-7, are needed for stimulation in order to have a certain viral infection efficiency.
  • the electroporation method has advantages in terms of ease of operation and experimental cycle compared to viral infection of primary cells. However, the popularity of this method is limited by many factors such as electrotransformation efficiency, cell survival rate, and differences in experimental equipment. In addition, primary cells are highly resistant to a variety of transfection methods, making the means for efficient gene editing of cells relatively scarce.
  • This article introduces a set of reagent compositions to inhibit the cellular immune pathways to improve the expression efficiency of the genes carried by the vector in the target cells, so as to be used in the clinical application of gene therapy.
  • the purpose of the present invention is to provide a reagent composition for improving the transfection efficiency of cells.
  • the reagent composition and a complex containing DNA and a transfection reagent or a lentivirus packaged with a target vector are separately added to the cell culture solution to improve the cell Transfection efficiency.
  • the reagent mixture is easy to operate, highly reproducible, and widely used. It has been successfully used in cell lines such as L929, BJ and mouse primary mouse fibroblasts, primary lung fibroblasts, T lymphocytes and other primary cells. .
  • the present invention adopts the following technical solutions:
  • a reagent composition for improving cell transfection efficiency comprises: a compound BX795, a compound (Rusolitinib) Ruxolitinib, a compound Tofacitinib, and a compound Actinomycin D ).
  • the reagent composition is composed of two or more substances of compound BX795 0.01-10 ⁇ M, compound Ruxolitinib 0.1-100 ⁇ M, compound Tofacitinib 0.1-100 ⁇ M, or compound Actinomycin D 0.1-100 nM.
  • the combination and preparation method of the reagent composition include the following:
  • Ruxolitinib solution 5 mg Ruxolitinib was added to 0.3260 ml of DMSO to obtain a 50 mM Ruxolitinib solution;
  • Tofacitinib solution 10 mg Tofacitinib Citrate was added to 0.3964 ml DMSO to obtain a 50 mM Tofacitinib Citrate solution;
  • the reagent concentration is as follows: BX795 is 0.01-10 ⁇ M, Ruxolitinib 0.11-100 ⁇ M, Tofacitinib is 0.1-100 ⁇ M, Actinomycin D 0.1-100 nM.
  • the method of the present invention is particularly suitable for the transfection of adherent cell lines, primary mouse fibroblasts, and primary mouse lung fibroblasts, and is suitable for a variety of cell lines that are difficult to transfect or have low transfection efficiency. .
  • the method of the present invention is applicable to various transfection methods, such as PEI transfection method, Lipofectamine lipid system transfection method, electrotransfection method, etc.
  • the method of the present invention has low biological toxicity, is stable in serum, has higher transfection efficiency, and is easy to operate as a gene transfection auxiliary reagent.
  • the reagent is formulated in the form of a mixture, and its transfection and expression effect is far superior to that of a single-component compound.
  • FIG. 1 GFP fluorescence effect of L929 cells transfected with each component.
  • Figure 2 The effect of mixed solution A on cell efficiency of L929 cells transfected by liposome transfection was detected by immunoblotting.
  • Fig. 3 Fluorescent effect diagram of the effect of a pair of mixed drugs on the transfection efficiency of L929 cells.
  • Figure 4 Fluorescence effect diagram of the effect of mixing three drugs on the transfection efficiency of L929 cells.
  • Figure 5 Fluorescence effect of mixed solution A on lung fibroblasts of mice transfected with liposome transfection and flow cytometric analysis.
  • Figure 6 Fluorescent effect of mixed solution A on lentiviral infection of human primary T cells.
  • BX795 solution 10 mg of BX795 (purchased from Selleck) was added to 1.6907 ml of DMSO to obtain a 10 mM BX795 mother liquor;
  • Ruxolitinib solution 5 mg of Ruxolitinib (purchased from APEXBIO) was added to 0.3260 ml of DMSO to obtain 50 mM Ruxolitinib solution;
  • Tofacitinib Citrate solution 10 mg Tofacitinib Citrate (purchased from APEXBIO) was added to 0.3964 ml of DMSO to obtain 50 mM Tofacitinib Citrate solution
  • the final concentration range of the compound BX795 on the cells may be 0.01-10 ⁇ M;
  • the final concentration of the compound Ruxolitinib on the cells may range from 0.1 to 100 ⁇ M;
  • the final concentration range of the compound Tofacitinib Citrate acting on the cells may be 0.1-100 ⁇ M;
  • the final concentration of the compound Actinomycin D on cells can range from 0.1 to 100 nM;
  • An endotoxin-removing plasmid extraction kit (purchased from Qiagen) was used to prepare an expression plasmid with the desired gene for transfection (the plasmid used in the present invention is an expression vector pEGFP-N1 containing a green fluorescent protein reporter gene, which is derived from Clontech Company), see the manufacturer's instructions for the method.
  • the plasmid was dissolved in 200 ⁇ l of ddH 2 O buffer, and its concentration was measured by a UV spectrophotometer.
  • Example 1 The effect of mixed solution A on cell transfection efficiency was verified in L929 cells
  • the test method is as follows:
  • liposome Lipofectamine 2000 was used to transfect L929 cells.
  • L929 cells were seeded at a suitable concentration in a 6-well plate and cultured in an incubator containing 5% CO 2 and 37 ° C for 12 hours. When the cells were completely adherent, the transfection could begin to reach 60-90%; before transfection; Change the medium for 1 hour. Replace the old medium with 1 ml of fresh DMEM complete medium containing 10% FBS. Add Dmix Complete A to the DMEM complete medium. The ratio of the mixture to the medium is 1: 1000.
  • the final concentration in the medium was 0.25 ⁇ M BX795, 5 ⁇ M Ruxolitinib, 5 ⁇ M Tofacitinib Citrate, 5 nM actinomycin D.
  • Prepare the transfection mixture In a 1.5 ml round-bottom sterile tube, mix 40 ⁇ l of Opti-MEM solution and 2 ⁇ l of Lipofectamin 2000 (Thermo Fisher), shake in a vortex shaker, and let stand for 5 minutes; add 2 ⁇ g pEGFP-N1 plasmid, gently shake and mix, and let stand for 20 minutes; add the DNA-Lipofecatamine mixture evenly to L929 cells, and gently shake while adding; after 12 hours, aspirate the medium and DNA Precipitate, wash twice with 1x PBS solution, and then replace with fresh medium; the medium needs to contain mixed solution A.
  • the test results are as follows: the untreated L929 cells are set as the first group, the single-component pretreated L929 cells are set as the second group of control groups, and the mixed solution A pretreated L929 cells are set as the experimental group.
  • the pEGFP-N1 plasmid was transfected into the control group and the experimental group, and the results are shown in FIG. 1.
  • the results in Figure 1 show that under bright field conditions, whether in the control group or the experimental group, the cells adhere to the wall. Although the cell proliferation of the experimental group is slightly weaker than that of the control group, its cell state is good and there are fewer dead cells; in dark field conditions The expression of GFP in the cells was observed below.
  • the fluorescence number of cells in the experimental group of the pretreated mixed solution A was higher than that of the two control groups, and the fluorescence intensity was strong. Staining efficiency and GFP expression efficiency, the results are far better than single-component pretreated or untreated cells.
  • the expression of GFP protein was then detected by immunoblot experiments ( Figure 2). The results showed that when the internal parameters were consistent (indicating that the number of cells was approximately the same), the expression level of GFP protein in the experimental group was much higher than that in the two control groups, which proved that Pretreatment of mixed solution A during infection can effectively promote the transfection and expression of GFP plasmid in L929 cells.
  • the test method is as follows:
  • the solvent DMSO was added to DMEM complete medium (1 ⁇ l DMSO / ml DMEM medium); the experimental group was L929 cells with two or two mixed drugs added to DMEM complete medium: BX795 + Ruxolitinib, BX795 + Tofacitinib Citrate, BX795 + Actinomycin D, Ruxolitinib + Tofacitinib Citrate, Ruxolitinib + Actinomycin D, Tofacitinib Citrate + Actinomycin D.
  • L929 cells were seeded in a 6-well plate and cultured in an incubator containing 5% CO 2 and 37 ° C for 12 hours. When the cells were completely adherent, and the cells reached 60-90%, transfection was started. Change the solution 1 hour before transfection.
  • test results are as follows: As shown in Figure 3, the transfection efficiency of GFP expressing L929 pre-treated with drug pairing is higher than that of the control group, which proves that drug pairing can effectively promote GFP plasmid in L929 cells during transfection. Transfection and expression.
  • the test method is as follows:
  • any three drug mixtures of BX795 (final concentration 0.25 ⁇ M), Ruxolitinib (final concentration 5 ⁇ M), Tofacitinib Citrate (final concentration 2.5 ⁇ M), and Actinomycin D (final concentration 5 nM) were divided into 4 in proportion.
  • L929 cells were seeded at a suitable concentration in a 6-well plate and cultured in an incubator containing 5% CO 2 and 37 ° C for 12 hours. When the cells were completely adherent, the transfection could begin to reach 60-90%; before transfection; Change the medium for 1 hour, and replace the old medium with 1 ml of fresh DMEM complete medium containing 10% FBS; add the above four groups of mixed solutions to the DMEM complete medium, the ratio of the mixed solution to the medium is 1: 1000; Prepare the transfection mixture: in a 1.5 ml round-bottom sterile tube, mix 40 ⁇ l of Opti-MEM solution and 2 ⁇ l of Lipofectamine 2000 (Thermo Fisher), shake in a vortex shaker, and let stand for 5 minutes; Add 2 ⁇ g of pEGFP-N1, gently shake and mix, and let stand for 20 minutes; add the DNA-Lipofectamine mixture evenly to L929
  • test results are as follows: pEGFP-N1 plasmid was transfected into the control group and the experimental group. The results are shown in Figure 4. 0.25 ⁇ M BX795, 5 ⁇ M Ruxolitinib, 2.5 ⁇ M Tofacitinib Citrate and 10 nM Actinomycin D are mixed with each other to pretreat L929 cells. The effect is similar to mixing two pairs. Compared with untreated L929 cells, the expression of GFP is greatly improved. The concentration can greatly exert the combined effect of drugs, and more effectively promote the transfection and expression of GFP plasmid in L929 cells.
  • the test method is as follows:
  • mice primary lung fibroblasts As an example, the effects of drug mixing on the transfection efficiency of primary adherent cells were examined. The experiment was divided into two groups. The control group was DMEM complete medium. DMSO / ml DMEM) mouse lung fibroblasts. The experimental group consisted of mouse lung fibroblasts with mixed solution A in DMEM complete medium. The final concentration of mixed solution A in the medium is 5 ⁇ M BX795, 5 ⁇ M Ruxolitinib, 5 ⁇ M Tofacitinib Citrate, 5 nM Actinomycin D.
  • lipofectin 2000 was used to transfect mouse lung fibroblasts.
  • the mouse lung fibroblast cells were seeded in a 6-well plate at an appropriate concentration, and cultured in an incubator containing 5% CO 2 and 37 ° C. for 12 hours.
  • the transfection began to reach 60-90%; Change the medium 1 hour before transfection, replace the old medium with 1 ml of fresh DMEM complete medium containing 10% FBS; add the above-mentioned mixed solution A to the DMEM complete medium, and the ratio of the mixed solution to the medium is 1: 1000 ;
  • the ratio of mixed solution A is 5 ⁇ M BX795, 5 ⁇ M Ruxolitinib, 2.5 ⁇ M Tofacitinib Citrate, and 10 nM Actinomycin D.
  • the pEGFP-N1 plasmid was transfected into the control group and the experimental group, and the results are shown in FIG. 5.
  • the results showed that when pEGFP-N1 plasmid was used to transfect mouse lung fibroblasts, the transfection efficiency was extremely low.
  • the number of successfully transfected cells in a field of view was single digits.
  • the cells of mixed solution A were pretreated and expressed GFP after transfection. The number of cells is greatly increased. The results indicate that mixed solution A can improve the transfection efficiency of primary adherent cells, such as mouse lung fibroblasts, and promote the expression of foreign genes in cells.
  • the test method is as follows:
  • lentivirus was used to infect human primary T cells.
  • the specific experimental methods are as follows:
  • PBMC Peripheral blood mononuclear cells
  • human peripheral blood was drawn, diluted 1: 1 with PBS, and the diluted mixed liquid was added to Ficoll with a density of 1.077, the volume ratio was 2: 1, centrifuged at 1000 rpm, and 18 ° C for 30 minutes.
  • the turbid layer at the interface was aspirated, washed with twice the volume of PBS, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded, and repeated-3 times.
  • the resulting cells were PBMC.
  • PBMC culture resuspend in 1640 medium containing 20% FBS 2.5 ⁇ g / ml CD3, 0.5 ⁇ g / ml CD28 1000 U / ml IL-2, 10 ng / ml IL-7 (1 can be added for every 5-10 ml blood) ml medium) was placed in a 6-well plate (or other adherent plate), and the cell suspension was collected after incubation at 37 ° C with 5% CO 2 for 2 hours. At this time, most of the macrophages had been removed. The suspension in the 6-well plate was transferred to an unattached culture flask and cultured for 4 days, and 1640 medium containing 10% FBS and 1000 U IL-2 was added depending on the cell density. During microscopy, the cells should show clumping.
  • lentivirus An endotoxin-removing plasmid extraction kit (purchased from Qiagen) was used to prepare an expression plasmid with the desired gene (the plasmid used in the present invention is an expression vector containing a green fluorescent protein reporter gene) pEGFP-N1, the plasmid required for virus assembly is psp2AX, VSVG); 293T cells were cultured in 10 cm plates using DMEM containing 10% FBS. When the cells grow to 70% -80%, lentiviral packaging can begin.
  • the total amount of plasmid was 20 ⁇ g, and it was dissolved in 250 ⁇ l Opti-MEM solution.
  • 60 ⁇ g PEI was dispensed into tubes and dissolved in Opti-MEM solution.
  • the configured solution was mixed and shaken vigorously for 15 seconds. After standing for 15 minutes, the solution was dropped evenly into the petri dish.
  • the solution was changed after 14 hours, and the supernatant was collected at intervals of 24 hours and 48 hours, which is the virus stock solution.
  • the collected virus stock was centrifuged at 2000 rpm for 5 minutes to remove larger cell debris.
  • the supernatant was filtered through a 0.45 ⁇ M filter membrane, and further concentrated by ultracentrifugation.
  • the obtained virus solution was stored in a refrigerator at -80 ° C.
  • T-cell infection preparation of plates required for infection: the day before the infection, the non-adherently treated 12-well plate was coated with Retronectin (TAKARA, 100T-A). Dilute Retronectin to 20 ⁇ g / ml with PBS, add 1 ml to each well, and overnight at 4 ° C. Before use, aspirate the liquid in the well, add PBS containing 2% BSA, and leave it at room temperature for 30 minutes. Aspirate the PBMC cultured in the flask, centrifuge and count. At the time of infection, 2 ⁇ 10 6 cells, 1640 medium containing 500 U / ml IL-2, 20% FBS, and virus solution were added to each well to a final volume of 2 ml. After culturing for about 5 days, supplement 1640 medium containing 500 U / ml IL-2 and 20% FBS according to the growth conditions. After 4 days, obvious fluorescence can be observed under a fluorescence microscope.
  • Retronectin TAKARA, 100T-A
  • human T cells can show partial fluorescence after being infected with lentivirus, indicating that the lentivirus successfully infected human T cells.
  • the human T cells pretreated with the mixed solution A were infected with lentivirus, and their fluorescence was relatively low.
  • the control group was significantly enhanced, indicating that mixed solution A had a significant enhancement effect on the T-cell lentivirus infection and the efficiency of foreign gene expression.

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Abstract

L'invention concerne une composition de réactif utilisée pour augmenter l'efficacité de transfection cellulaire, la composition étant composée de deux substances ou plus parmi BX795 à 0,01-10 μΜ, ruxolitinib à 0,1-100 μΜ, tofatib à 0,1-100 µΜ et actinomycine D à 0,1-100 nM. Un mélange de tels réactifs, un complexe contenant de l'ADN et un réactif de transfection ou un lentivirus emballé par un vecteur cible sont respectivement ajoutés à un milieu de culture cellulaire de façon à augmenter l'efficacité de transfection cellulaire.
PCT/CN2019/084394 2018-05-28 2019-04-25 Composition de réactif utilisée pour augmenter l'efficacité de transfection cellulaire Ceased WO2019228108A1 (fr)

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CN201810525471.2 2018-05-28

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WO2021141020A1 (fr) * 2020-01-06 2021-07-15 国立大学法人大阪大学 INTRODUCTION D'ACIDE NUCLÉIQUE À L'AIDE DE DEUX TYPES D'INHIBITEURS DE TBK1/IKKe
CN114686502A (zh) * 2022-04-28 2022-07-01 广州市花都区人民医院 一种在Raw264.7细胞中快速构建稳定高表达细胞株的方法
EP4071248A1 (fr) * 2021-04-07 2022-10-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Moyens et procédés pour améliorer le transfert de gènes ciblé par des récepteurs
CN115873787A (zh) * 2022-12-28 2023-03-31 云舟生物科技(广州)股份有限公司 一种高效转染细胞株及其制备方法和应用
CN119530302A (zh) * 2025-01-22 2025-02-28 青岛华赛伯曼医学细胞生物有限公司 提高til细胞的慢病毒转导效率及转导稳定性的方法及试剂盒

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CN108715865B (zh) * 2018-05-28 2021-09-21 福建师范大学 用于提高细胞转染效率的试剂组合物
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CN117070570A (zh) * 2023-08-28 2023-11-17 南通大学 提高脂质体转染试剂的转染效率的方法

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WO2014199166A1 (fr) * 2013-06-12 2014-12-18 University Court Of The University Of St Andrews Procédé d'augmentation de la vitesse de croissance virale et/ou du titre viral dans des cellules
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CN108715865A (zh) * 2018-05-28 2018-10-30 福建师范大学 用于提高细胞转染效率的试剂组合物

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021141020A1 (fr) * 2020-01-06 2021-07-15 国立大学法人大阪大学 INTRODUCTION D'ACIDE NUCLÉIQUE À L'AIDE DE DEUX TYPES D'INHIBITEURS DE TBK1/IKKe
EP4071248A1 (fr) * 2021-04-07 2022-10-12 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Moyens et procédés pour améliorer le transfert de gènes ciblé par des récepteurs
WO2022214588A1 (fr) * 2021-04-07 2022-10-13 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Moyens et procédés pour améliorer le transfert de gènes ciblant un récepteur
CN114686502A (zh) * 2022-04-28 2022-07-01 广州市花都区人民医院 一种在Raw264.7细胞中快速构建稳定高表达细胞株的方法
CN115873787A (zh) * 2022-12-28 2023-03-31 云舟生物科技(广州)股份有限公司 一种高效转染细胞株及其制备方法和应用
CN119530302A (zh) * 2025-01-22 2025-02-28 青岛华赛伯曼医学细胞生物有限公司 提高til细胞的慢病毒转导效率及转导稳定性的方法及试剂盒

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