CN108715865A - Reagent composition for improving cell transfecting efficiency - Google Patents
Reagent composition for improving cell transfecting efficiency Download PDFInfo
- Publication number
- CN108715865A CN108715865A CN201810525471.2A CN201810525471A CN108715865A CN 108715865 A CN108715865 A CN 108715865A CN 201810525471 A CN201810525471 A CN 201810525471A CN 108715865 A CN108715865 A CN 108715865A
- Authority
- CN
- China
- Prior art keywords
- cell
- reagent
- compound
- transfection
- actinomycin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to the reagent compositions for improving cell transfecting efficiency, belong to biotechnology, the reagent composition includes 2 kinds or two or more material composition in 0.01-10 μM of compound BX795,0.1-100 μM of compound R uxolitinib, 0.1-100 μM of compound Tofacitinib, 0.1-100 nM of compound Actinomycin D.The slow virus of these reagent mixed liquors and the compound containing DNA and transfection reagent or the purposeful carrier of packaging is separately added into cell culture fluid, to improve the transfection efficiency of cell.The reagent mixture is easy to operate, repeatable strong, and use scope is big, has successfully applied in the primary cells such as the cell lines such as L929, BJ and mouse primary lung fibroblast, T lymphocytes.
Description
Technical field
The invention belongs to biotechnologies, and in particular to the reagent composition for improving cell transfecting efficiency.
Background technology
Gene therapy is a new research field of contemporary medical science and biology, by certain way by normal gene
Or medicative DNA sequence dna imports target cell to correct the defect of gene or play therapeutic effect, to reach treatment disease
The purpose of disease.In order to reach ideal therapeutic effect, scientist is studying always the effective way of transmission Plasmid DNA.Often at present
The heterogenous expression system of rule is broadly divided into two classes：One kind is by the way of viral vectors, and three kinds of viral vectors of mainstream include
Slow virus（lentiviral）Carrier, reverse transcription（retroviral）Viral vectors and adenovirus（AAV）Carrier, these methods
It is the mainstream selection of the clinical tests such as gene therapy.Viral vectors such as AAV carriers have high transfection efficiency, continually and steadily express external source
The characteristics of gene, but it need to be integrated into the chromosome of host cell can enable foreign gene continuous expression, therefore have
There is carcinogenic, teratogenesis danger.The main reason for this poor defect of safety has become limiting virus carrier extensive use.It is another
Class is by the way of non-virus carrier.This kind of non-virus carrier is safe, easily prepared, in gene therapy, from effect
It is seen in development prospect, has more superiority compared with viral vectors.But the importing efficiency and targeting of such carrier are relatively low, larger portion
Point host cell is strong to exogenous DNA repellence, and foreign gene be transferred to host cell after expression time it is short, it is outer after transient expression
Source gene quick closedown, these all seriously constrain the application and development of such carrier.Non-virus carrier includes liposome, high score
Subcarrier, Nanoparticulate Carriers for Gene Delivery etc..
For existing ordinary cells strain or primary cell, general transfection method mostly uses liposome method and electroporation
Method.Cationic-liposome surface is positively charged, and DNA molecular is rolled into DNA- fat by electrostatic interaction with the phosphate radical of nucleic acid
Liposome complex can be adsorbed on the negatively charged cell membrane in surface, enter cell body by the fusion or endocytosis of film
It is interior.Sometimes DNA is passed into cell also by the direct osmosis of aperture on cell membrane.Electroporation or electric robin are
By the electric field action of high intensity, membrane passage is instantaneously improved, thus by exogenous molecules, such as DNA, mRNA, albumen, sugar
Class etc. is sent into the endochylema of host cell.But for many cell strains or primary cell, exogenous DNA passes through non-virus carrier side
Method fails to complete transcription and translation into destination protein after entering cell, and expression efficiency is relatively low, often up to less than requirement of experiment.And lead to
It is also not high to cross the efficiency that electric shocking method and viral method are transfected, and experimental implementation cumbersome.How exogenous DNA is improved in cell
Interior is expressed as a technical barrier.In terms of clinical treatment, the disease of hemopoietic system genetic dysfunction is treated, is such as joined
Close immune deficiency（SCID）Or the novel means for the treatment of cancer, such as Chimeric antigen receptor T cell immunotherapy（CAR-T）Have
Rely in the means of a high-efficiency transfection T cell.The method that primary T cells carry out genetic modification is used to now mainly to feel by virus
Dye and the mode of electroporation carry out.When viral method infects primary cell, generally require higher virus titer and usually
Effect is not satisfactory.For T cell, it is also necessary to which additional cell factor can just have if IL-2 and IL-7 are stimulated
Certain viral efficiency of infection.And the method for electroporation compares in operation ease and on experimental period viral method infector generation carefully
Born of the same parents have more advantage.However, the universal of the method is limited by the factors such as electric transfer efficient, cell survival rate, laboratory apparatus difference.Add
Primary cell for a variety of transfection methods all have stronger repellence so that about cell carry out high efficiency gene editor hand
Section is more deficient.
A group reagent composition is described herein by the inhibition to cellular immunity access, to improve the base that carrier is delivered
Because of the expression efficiency in target cell, for the clinical application of gene therapy.
Invention content
The purpose of the present invention is to provide the reagent composition for improving cell transfecting efficiency, reagent composition with contain
The slow virus of the compound or the purposeful carrier of packaging of DNA and transfection reagent is separately added into cell culture fluid, to improve cell
Transfection efficiency.The reagent mixture is easy to operate, repeatable strong, and use scope is big, and it is thin successfully to have applied to L929, BJ etc.
In the primary cells such as born of the same parents system and mouse primary l cell, primary lung fibroblast, T lymphocytes.
To achieve the above object, the present invention adopts the following technical scheme that：
Reagent composition for improving cell transfecting efficiency, the reagent composition include：Compound BX795, compound（Shandong
Suo Li replaces Buddhist nun）Ruxolitinib, compound support method replace cloth（Tofacitinib）, compound actinomycin D（Actinomycin
D）.
The wherein described reagent composition is by compound BX795 0.01-10 μM, compound R uxolitinib 0.1-100 μ
M, 2 kinds or 2 kinds in compound Tofacitinib 0.1-100 μM or compound Actinomycin D 0.1-100 nM
The above material composition.
The combination of reagent composition and preparation method, including it is as follows：
1）Prepare BX795 solution：10 mg BX795 are added in 1.6907 ml DMSO, 10 mM BX795 mother liquors are obtained；
2）Prepare Ruxolitinib solution：5 mg Ruxolitinib are added in 0.3260 ml DMSO, 50 mM are obtained
Ruxolitinib solution；
3）Prepare Tofacitinib solution：10 mg Tofacitinib Citrate are added in 0.3964 ml DMSO, are obtained
To 50 mM Tofacitinib Citrate solution；
4）Prepare Actinomycin solution Ds：1 mg Actinomycin D are added in 79.65 μ l DMSO, 10 mM are obtained
Actinomycin solution Ds；
5）Arbitrary 2 kinds of compound combinations in BX795, Ruxolitinib, Tofacitinib or Actinomycin D are matched
The reagent mixed liquor of specific final concentration is made.
6）By arbitrary 3 kinds of compound groups in BX795, Ruxolitinib, Tofacitinib or Actinomycin D
Close the reagent mixed liquor for being configured to specific final concentration.
7）By 4 kinds of compound combinations in BX795, Ruxolitinib, Tofacitinib and Actinomycin D, match
The reagent mixed liquor of specific final concentration, i.e. mixed liquor A is made.
8）The reagent concentration is as follows：BX795 is 0.01-10 μM, Ruxolitinib is 0.11-100 μM,
Tofacitinib is 0.1-100 μM, Actinomycin D 0.1-100 nM.
The advantage of the invention is that：
1. the method for the present invention is particularly suitable for each attached cell system, primary mouse embryo fibroblasts, Primary mouse lung fiber finer
The transfection of born of the same parents is suitable for a variety of difficult cell lines transfected or transfection efficiency is low.
2. the method for the present invention is suitable for a variety of rotaring transfecting modes, such as PEI infection protocols, Lipofectamine lipoids systems
Infection protocol, electric robin etc.
3. the method bio-toxicity of the present invention is small, stablize in serum, transfection efficiency higher and gene transfection easy to operate
Auxiliary reagent.
4. the present invention uses form of mixtures reagent preparation, improves transfection and expression effect is far superior to single component
Compound.
Description of the drawings
Fig. 1 adds L929 cell transfecting GFP fluorescent effect figures after each component.
Fig. 2 Western blots detect the cell efficiency influence that mixed liquor A transfects lipofection in L929 cells.
The fluorescent effect figure of Fig. 3 influences of the hybrid medicine to the transfection efficiency of L929 cells two-by-two.
The fluorescent effect figure of influence of the tri- kinds of drug mixing of Fig. 4 to the transfection efficiency of L929 cells.
Fluorescent effect figure and flow cytometry of Fig. 5 mixed liquor As to lipofection transfected lung fibroblast
Figure.
Fig. 6 mixed liquor As infect slow virus method in the fluorescent effect figure of people's primary T cells.
Specific implementation mode
The step of preparing the mixed liquor is as follows：
I. the preparation of reagent
1. the configuration of mother liquor
1）Prepare BX795 solution：By 10 mg BX795（It is purchased from Selleck companies）It is added in 1.6907 ml DMSO, obtains 10
MM BX795 mother liquors；
2）Prepare Ruxolitinib solution：By 5 mg Ruxolitinib（It is purchased from APEXBIO companies）0.3260 ml is added
In DMSO, 50 mM Ruxolitinib solution are obtained；
3）Prepare Tofacitinib Citrate solution：By 10 mg Tofacitinib Citrate（It is purchased from APEXBIO public affairs
Department）It is added in 0.3964 ml DMSO, obtains 50 mM Tofacitinib Citrate solution
4）Prepare Actinomycin solution Ds：By 1 mg Actinomycin D（It is purchased from Selleck companies）79.65 μ are added
In l DMSO, 10 mM Actinomycin solution Ds are obtained
2. the preparation of reagent mixed liquor
1）By above-mentioned BX795, Ruxolitinib, Tofacitinib Citrate and Actinomycin in 1 ml DMSO
Solution D, combination of two are configured to arbitrary 2 kinds of compound compositions of specific final concentration；
2）By above-mentioned BX795, Ruxolitinib, Tofacitinib Citrate and Actinomycin in 1ml DMSO
Solution D, three or three formulated in combination at specific final concentration arbitrary 3 kinds of compound compositions；
3）By above-mentioned BX795, Ruxolitinib, Tofacitinib Citrate and Actinomycin in 1ml DMSO
Solution D, formulated in combination is at 4 kinds of compound compositions of specific final concentration, as mixed liquor A；
The final concentration range that the compound BX795 acts on cell can be 0.01-10 μM；
The final concentration range that the compound R uxolitinib acts on cell can be 0.1-100 μM；
The final concentration range that the compound Tofacitinib Citrate act on cell can be 0.1-100 μM；
The final concentration range that the compound Actinomycin D act on cell can be 0.1-100 nM；
3. transfection is prepared with plasmid
With the endotoxic big extraction reagent kit of plasmid of removal（Purchased from Qiagen companies）Prepare the table that target gene is connected with needed for transfection
Up to plasmid（Plasmid used in the present invention is the expression vector pEGFP-N1 containing Green fluorescent protein fusion vector, is derived from
Clontech companies）, method is shown in producer's explanation.With the ddH of 200 μ l2O buffer solution plasmids, ultraviolet specrophotometer survey it
Concentration.
Below by embodiment, the present invention is further described
Embodiment 1 verifies influence of the mixed liquor A to cell transfecting efficiency in L929 cells
Test method is as follows：
This experiment selects 2000 infection protocols of liposome Lipofectamine to transfect L929 cells.It is by suitable concentration that L929 is thin
Born of the same parents are inoculated in 6 orifice plates, are containing 5% CO2It is cultivated 12 hours in 37 DEG C of incubators, it is long to 60-90% when cell is completely adherent
It can start to transfect；Transfection changes liquid in first 1 hour, and old culture is changed with the fresh DMEM complete mediums containing 10% FBS of 1 ml
Base；It is added mixed liquor A in DMEM complete mediums, the ratio of mixed liquor and culture medium is 1:1000；Mixed liquor A is in the medium
Final concentration of 0.25 μM of BX795,5 μM of Ruxolitinib, 5 μM of Tofacitinib Citrate, 5 nM
actinomycin D.Prepare transfection mixture：In the round bottom sterile tube of 1.5 ml, by the Opti-MEM solution and 2 of 40 μ l
The Lipofectamin 2000 of μ l（Thermo Fisher companies）Mixing, be put in whirlpool concussion instrument shake it is even, stand 5 minutes；Add
The pEGFP-N1 plasmids for entering 2 μ g gently shake mixing, stand 20 minutes；DNA-Lipofecatamine mixed liquors are uniformly added
Enter in L929 cells, side edged gently shakes mixing；After 12 hours, sucks culture medium and precipitated with DNA, the PBS solution with 1 times is clear
Wash 2 times, then replaced with fresh medium；It is needed containing mixed liquor A in culture medium.It is tested using fluorescence microscope after 48 hours
Group and control group（Only with solvent not medicine treatment）The luciferase expression situation of cell utilizes flow cytomery L929 cells
GFP expression efficiencies, and expression quantity of the GFP albumen in cell is detected by the method for immunoblotting.
Test result is as follows：Untreated L929 cells are set as first group, it is that one-component, which pre-processes L929 cells,
Two groups of control groups, the pretreated L929 cells of mixed liquor A are as experimental group.By pEGFP-N1 plasmid transfections control group and experiment
Group, the results are shown in Figure 1.Fig. 1 is the result shows that under the conditions of light field, either control group or experimental group, the adherent jail of cell,
Although the cell Proliferation of experimental group is slightly weaker than control group, its cell state is good, and dead cell is few；Cell is observed under dark fieid conditions
The experimental group cell fluorescence number of the expression of GFP, Pretreatment Mixed Liauid A is higher than two control groups, and fluorescence intensity is stronger,
Illustrate in transfection Pretreatment Mixed Liauid A can increase substantially L929 cells transfection efficiency and GFP expression efficiencies, result it is remote
Pretreated better than one-component or untreated cell.The expression of GFP albumen is detected by immunoblot experiment later
(Fig. 2), the results showed that the expression of the GFP albumen that under internal reference unanimous circumstances (shows that cell quantity is roughly the same) in experimental group
Amount is far above two groups of control groups, it was demonstrated that in transfection, Pretreatment Mixed Liauid A can effectively facilitate GFP plasmids and turn in L929 cells
Dye and expression.
Embodiment 2 verifies influence of the hybrid medicine to transfection efficiency two-by-two in L929 cells
Test method is as follows：
This experiment configures BX795 in proportion（0.25 μM of final concentration）,Ruxolitinib（5 μM of final concentration）,Tofacitinib
Citrate（2.5 μM of final concentration）,Actinomycin D（5 nM of final concentration）Mixed liquor two-by-two, totally 6 groups, control group is
Solvent DMSO is added in DMEM complete mediums（1 μ l DMSO/ml DMEM culture mediums）；Experimental group is in DMEM complete mediums
The L929 cells of hybrid medicine two-by-two are added：BX795+Ruxolitinib, BX795+Tofacitinib Citrate, BX795+
Actinomycin D, Ruxolitinib+ Tofacitinib Citrate, Ruxolitinib+Actinomycin D,
Tofacitinib Citrate+Actinomycin D 。
This experiment still selects 2000 infection protocols of liposome Lipofectamine to transfect L929 cells.L929 cells are connect
Kind is containing 5% CO in 6 orifice plates2It is cultivated 12 hours in 37 DEG C of incubators, when cell is completely adherent, length to 60-90% is opened
Begin to transfect；Transfection changes liquid in first 1 hour, and old culture medium is changed with the fresh DMEM complete mediums containing 10% FBS of 1 ml；DMEM
It is separately added into above-mentioned 7 groups of mixed liquors in complete medium, the ratio of mixed liquor and culture medium is 1:1000；Prepare transfection mixing
Object：In the round bottom sterile tube of 1.5 ml, by the Lipofectamine 2000 of the Opti-MEM solution of 40 μ l and 2 μ l
（Thermo Fisher companies）Mixing, be put in whirlpool concussion instrument shake it is even, stand 5 minutes；The pN1-GFP plasmids of 2 μ g are added, gently
It is light to shake mixing, stand 20 minutes；DNA-Lipofectamine mixed liquors are uniformly added into L929 cells, side edged is gently
Shake mixing；After 12 hours, sucks culture medium and precipitated with DNA, cleaned 2 times with 1 times of PBS solution, then change fresh cultured
Base；It is needed in culture medium containing each group mixed liquor.The glimmering of fluorescence microscope experimental group and cellular control unit is used after 48 hours
Light expression.
Test result is as follows：As shown in figure 3, after being mixed two-by-two with drug the transfection expression GFP of pretreated L929 effect
Rate be more than control group, it was demonstrated that transfection when drug mix two-by-two after can effectively facilitate transfection of the GFP plasmids in L929 cells with
Expression.
Embodiment 3 verifies influence of three kinds of drugs mixing to transfection efficiency in L929 cells
Test method is as follows：
This experiment is in proportion by BX795（0.25 μM of final concentration）,Ruxolitinib（5 μM of final concentration）,Tofacitinib
Citrate（2.5 μM of final concentration）, Actinomycin D（5 nM of final concentration）Arbitrary three kinds of drug mixtures, be divided into 4
Group, system are respectively：1 control group is that solvent DMSO (1 μ l DMSO/ml DMEM are added in DMEM complete mediums）；2-4 is real
It is that the L929 cells of three kinds of drugs mixing are added in DMEM complete mediums to test group：BX795+Ruxolitinib+
Tofacitinib Citrate, BX795+Ruxolitinib+Actinomycin D, BX795+ Tofacitinib
Citrate+Actinomycin D, Ruxolitinib+ Tofacitinib Citrate+Actinomycin D.
This experiment still selects 2000 infection protocols of liposome Lipofectamine to transfect L929 cells.By suitable concentration
By L929 cell inoculations in 6 orifice plates, containing 5% CO2It is cultivated 12 hours in 37 DEG C of incubators, it is long when cell is completely adherent
It can start to transfect to 60-90%；Transfection changes liquid in first 1 hour, is changed with the fresh DMEM complete mediums containing 10% FBS of 1 ml
Old culture medium；It is separately added into above-mentioned 4 groups of mixed liquors in DMEM complete mediums, the ratio of mixed liquor and culture medium is 1:
1000；Prepare transfection mixture：In the round bottom sterile tube of 1.5 ml, by the Opti-MEM solution of 40 μ l and 2 μ l
Lipofectamine 2000（Thermo Fisher companies）Mixing, be put in whirlpool concussion instrument shake it is even, stand 5 minutes；It is added 2
The pEGFP-N1 of μ g gently shakes mixing, stands 20 minutes；It is thin that DNA-Lipofectamine mixed liquors are uniformly added into L929
In born of the same parents, side edged gently shakes mixing；It after 12 hours, sucks culture medium and DNA and precipitates, clean 2 times with 1 times of PBS solution, so
Replaced with fresh medium afterwards；It is needed in culture medium containing each group mixed liquor.After 48 hours using fluorescence microscope experimental group and
The luciferase expression situation of cellular control unit.
Test result is as follows：By pEGFP-N1 plasmid transfections control group and experimental group, the results are shown in Figure 4,0.25 μM
BX795,5 μM of Ruxolitinib, 2.5 μM of Tofacitinib Citrate, 10 tri- kinds of medicines of nM Actinomycin D
Object mutually mix pretreatment L929 cells it is similar with the effect mixed two-by-two, compared with untreated L929 cells, greatly carry
The expression of high GFP, it was demonstrated that the joint that can be greatly played drug after drug mutually mixes in transfection using suitable concentration is made
With more efficiently facilitating transfection and expression of the GFP plasmids in L929 cells.
Embodiment 4 verifies influence of the mixed liquor A to cell transfecting efficiency in the primary lung fibroblast of mouse
Test method is as follows：
Primary Stage Data is improved largely to the transfection efficiency of L929 cells after showing drug mixing, but for the effect of primary cell
Fruit does not understand also.By taking mouse primary lung fibroblast as an example, to the shadow of primary adherent cell transfecting efficiency after detection drug mixing
It rings.This experiment is divided into two groups, and control group is that solvent DMSO (1 μ l DMSO/ml DMEM are added in DMEM complete mediums）It is small
Mouse lung fibroblast, experimental group are that the mouse lung fibroblast of mixed liquor A is added in DMEM complete mediums.Mixed liquor A is being trained
Support final concentration of 5 μM of BX795,5 μM of Ruxolitinib, 5 μM of Tofacitinib Citrate, 5 nM in base
Actinomycin D。
2000 infection protocol transfected lung fibroblasts of liposome Lipofectamine are selected to this experiment.By suitable
Mouse lung fibroblast cell inoculation in 6 orifice plates, is being contained 5% CO by concentration2It is cultivated 12 hours in 37 DEG C of incubators, when thin
Born of the same parents are completely adherent, long to start to transfect to 60-90%；Transfection changes liquid in first 1 hour, complete with the fresh DMEM containing 10%FBS of 1 ml
Full culture medium changes old culture medium；It is added above-mentioned mixed liquor A in DMEM complete mediums, the ratio of mixed liquor and culture medium is 1:
1000；The proportioning of mixed liquor A is 5 μM of BX795,5 μM of Ruxolitinib, 2.5 μM of Tofacitinib Citrate, 10
nM Actinomycin D.Prepare transfection mixture：In the round bottom sterile tube of 1.5 ml, by the Opti-MEM solution of 40 μ l
With the Lipofectamine 2000 of 2 μ l（Thermo Fisher companies）Mixing, be put in whirlpool concussion instrument shake it is even, stand 5 points
Clock；The pEGFP-N1 of 2 μ g is added, gently shakes mixing, stands 20 minutes；DNA-Lipofectamine mixed liquors are uniformly added
Enter in mouse lung fibroblast, side edged gently shakes mixing；After 12 hours, sucks culture medium and precipitated with DNA, with 1 times of PBS
Solution cleans 2 times, then replaced with fresh medium；It is needed in culture medium containing mixed liquor A as described above.Using glimmering after 48 hours
The luciferase expression situation of light microscope observation experiment group and cellular control unit utilizes flow cytomery mouse lung fibroblast
Cell GFP expression efficiencies.
Test result is as follows：
By pEGFP-N1 plasmid transfections control group and experimental group, the results are shown in Figure 5.The result shows that pEGFP-N1 plasmid transfections are small
When mouse lung fibroblast, transfection efficiency is extremely low, and it is units that successful number of cells is transfected under a visual field, and pre-processes mixing
The cell of liquid A, the cell quantity that GFP is expressed after transfection increase significantly.The result illustrates mixed liquor A to primary adherent cell, such as
Mouse lung fibroblast can improve its transfection efficiency, promote the expression of foreign gene in the cell.
Embodiment 5 verifies influence of the mixed liquor A to cell transfecting efficiency in primary T cells
Test method is as follows：
This experiment selects slow virus method to infect people's primary T cells.Specific experimental method is as follows：
1）The isolation and culture of T cell
Peripheral blood mononuclear cells (PBMC) refers to the cell with single core in peripheral blood, including lymphocyte, monocyte
Deng.In order to carry out PBMC separation, human peripheral is extracted, 1：1 dilutes with PBS, and it is 1.077 that density, which is added, in the mixing liquid after dilution
Ficoll, volume ratio 2：1,1000 rpm centrifuge, 18 DEG C 30 minutes.The turbidity screen for drawing interface, is washed with 2 times of volume PBS
It washs, 2000 rpm are centrifuged 5 minutes, and abandon supernatant, are repeated -3 times, the cell of gained is PBMC.PBMC is cultivated：With containing 20%
FBS 2.5 μ g/ml CD3,0.5 μ g/ml CD28 1000 U/ml IL-2,10 ng/ml IL-7 1640 culture medium be resuspended
（1 ml culture mediums can be added in every 5-10 ml blood）It is placed in 6 orifice plates（Or on the tablet of other adherent processing）, 37 DEG C of 5% CO2
Cell suspension is collected after incubating 2 hours, has been removed most macrophage at this time.Suspension in 6 orifice plates is transferred to not adherent
It handles in culture bottle, cultivates 4 days, 1640 culture mediums of visual cell's density addition containing 10% FBS, 1000 U IL-2.Period mirror
Clustering phenomena should be presented in inspection, cell.
2）T cell slow-virus infection step
1. the preparation of slow virus：With the endotoxic big extraction reagent kit of plasmid of removal（Purchased from Qiagen companies）It prepares needed for transfection
It is connected with the expression plasmid of target gene（Plasmid used in the present invention is the expression vector containing Green fluorescent protein fusion vector
PEGFP-N1, plasmid needed for virus assembly are psp2AX, VSVG）；293T cells are cultivated in 10cm tablets, used medium is
DMEM containing 10% FBS.Slow virus packaging can be started by waiting for that cell is grown to 70%-80%.The pUC pUC of selection is three plasmid systems
System.With pNL:psp2AX:VSVG=4:3:2 ratio is packed, and it is 20 μ g that total amount, which is added, in plasmid, is dissolved in 250 μ l
Opti-MEM solution.It is in charge of 60 μ g PEI of configuration, is dissolved in Opti-MEM solution.The solution configured is mixed and is acutely shaken
It swings 15 seconds, after standing 15 minutes, uniformly instills in culture dish.Liquid is changed after 14 hours, is spaced 24 hours and 48 hours and is collected later
Supernatant, as virus stock solution used.2000 rpm of collected virus stock solution used centrifuges 5 minutes to remove larger cell fragment.Supernatant
Liquid is further concentrated with 0.45 μM of membrane filtration, ultracentrifugation, and the virus liquid of acquisition is stored in -80 DEG C of refrigerator.
3）The infection of T cell：The preparation of tablet needed for infection：The previous day to be infected, use Retronectin（TAKARA,
100T-A）12 orifice plates of the not adherent processing of coating.Retronectin is diluted to 20 μ g/ml with PBS, 1 ml of addition per hole, 4
DEG C overnight.Before use, drawing liquid in hole, and the PBS containing 2% BSA is added, is placed at room temperature for 30 minutes.It draws in culture bottle
The PBMC of culture is centrifuged and is counted.When infection, 2 × 10 are added per hole6A cell and contain 500 U/ml IL-2,20% FBS
1640 culture mediums and virus liquid, final volume is in 2 ml.Culture 5 days or so contains 500 U/ml IL- according to growing state supplement
2,1640 culture mediums of 20% FBS can observe apparent fluorescence under fluorescence microscope after 4 days.
4）The addition of mixed liquor A：Before T cell infection, above-mentioned mixed liquor A, mixed liquor and culture are added in the medium
The ratio of base is 1:1000；Final concentration of 5 μM of BX795 of mixed liquor A, 5 μM of Ruxolitinib, 5 μM
Tofacitinib Citrate,5 nM Actinomycin D.In addition, during slow-virus infection, need to keep in culture medium
The concentration of these drugs.
Test result is as follows：
As shown in fig. 6, after the T cell slow-virus infection of people, part fluorescence can be presented, illustrate that slow virus successfully infects people's
T cell, the T cell of the pretreated people of mixed liquor A is after slow-virus infection, and fluorescence is significantly increased compared with control group, explanation
Mixed liquor A has significant enhancement effect for the slow-virus infection of T cell and exogenous gene expression efficiency.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Claims (3)
1. the reagent composition for improving cell transfecting efficiency, it is characterised in that：The reagent composition includes：BX795, Shandong
Suo Li is for Buddhist nun, support method for cloth, actinomycin D.
2. reagent composition according to claim 1, it is characterised in that：The reagent composition is replaced by BX795, Luso profit
Buddhist nun, support method are for 2 kinds or two or more material composition in cloth and actinomycin D.
3. reagent composition according to claim 1 or 2, which is characterized in that each material concentration is as follows in composition：
BX795 is 0.01-10 μM, and Luso profit is 0.11-100 μM for Buddhist nun, and support method is 0.1-100 μM for cloth, actinomycin D 0.1-
100 nM。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810525471.2A CN108715865B (en) | 2018-05-28 | 2018-05-28 | Reagent composition for improving cell transfection efficiency |
| PCT/CN2019/084394 WO2019228108A1 (en) | 2018-05-28 | 2019-04-25 | Reagent composition used for increasing cell transfection efficiency |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810525471.2A CN108715865B (en) | 2018-05-28 | 2018-05-28 | Reagent composition for improving cell transfection efficiency |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108715865A true CN108715865A (en) | 2018-10-30 |
| CN108715865B CN108715865B (en) | 2021-09-21 |
Family
ID=63911566
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810525471.2A Active CN108715865B (en) | 2018-05-28 | 2018-05-28 | Reagent composition for improving cell transfection efficiency |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN108715865B (en) |
| WO (1) | WO2019228108A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109868260A (en) * | 2019-04-16 | 2019-06-11 | 上海汉尼生物细胞技术有限公司 | A kind of preparation method of CAR-T cell |
| WO2019228108A1 (en) * | 2018-05-28 | 2019-12-05 | 福建师范大学 | Reagent composition used for increasing cell transfection efficiency |
| CN115702899A (en) * | 2021-08-03 | 2023-02-17 | 上海优卡迪生物医药科技有限公司 | Application of luccotinib to preparation of CAR-T medicine |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2023015419A (en) * | 2020-01-06 | 2023-02-01 | 国立大学法人大阪大学 | NUCLEIC ACID INTRODUCTION USING TWO TBK1/IKKe INHIBITORS |
| EP4071248A1 (en) * | 2021-04-07 | 2022-10-12 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Means and methods for enhancing receptor-targeted gene transfer |
| CN114686502A (en) * | 2022-04-28 | 2022-07-01 | 广州市花都区人民医院 | Method for quickly constructing stable high-expression cell strain in Raw264.7 cell |
| CN115873787A (en) * | 2022-12-28 | 2023-03-31 | 云舟生物科技(广州)股份有限公司 | Efficient transfection cell strain and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014199166A1 (en) * | 2013-06-12 | 2014-12-18 | University Court Of The University Of St Andrews | Method of increasing viral growth rate and/or titre in cells |
| CN105792831A (en) * | 2013-10-08 | 2016-07-20 | 普罗麦迪奥股份有限公司 | Methods for treating fibrotic cancers |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5397692B2 (en) * | 2007-11-28 | 2014-01-22 | 国立大学法人名古屋大学 | Malignant melanoma antigen expression increasing agent and use thereof |
| JP6754761B2 (en) * | 2014-07-11 | 2020-09-16 | セルジーン コーポレイション | How to improve the efficiency of vector introduction into T lymphocytes |
| DE102016113714A1 (en) * | 2016-07-26 | 2018-02-01 | Rosa Karl | Transfection procedure with non-viral gene delivery systems |
| CN108715865B (en) * | 2018-05-28 | 2021-09-21 | 福建师范大学 | Reagent composition for improving cell transfection efficiency |
-
2018
- 2018-05-28 CN CN201810525471.2A patent/CN108715865B/en active Active
-
2019
- 2019-04-25 WO PCT/CN2019/084394 patent/WO2019228108A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014199166A1 (en) * | 2013-06-12 | 2014-12-18 | University Court Of The University Of St Andrews | Method of increasing viral growth rate and/or titre in cells |
| CN105792831A (en) * | 2013-10-08 | 2016-07-20 | 普罗麦迪奥股份有限公司 | Methods for treating fibrotic cancers |
Non-Patent Citations (1)
| Title |
|---|
| 靳翠红 等: "放线菌素D对仓鼠成纤维细胞V79靶细胞和旁观者细胞活力和染色体畸变的影响", 《CARCINOGENESIS,TERATOGENESIS & MUTAGENESIS》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019228108A1 (en) * | 2018-05-28 | 2019-12-05 | 福建师范大学 | Reagent composition used for increasing cell transfection efficiency |
| CN109868260A (en) * | 2019-04-16 | 2019-06-11 | 上海汉尼生物细胞技术有限公司 | A kind of preparation method of CAR-T cell |
| CN115702899A (en) * | 2021-08-03 | 2023-02-17 | 上海优卡迪生物医药科技有限公司 | Application of luccotinib to preparation of CAR-T medicine |
| CN115702899B (en) * | 2021-08-03 | 2024-05-28 | 上海优卡迪生物医药科技有限公司 | Application of ponatinib in preparing CAR-T medicine |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019228108A1 (en) | 2019-12-05 |
| CN108715865B (en) | 2021-09-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108715865A (en) | Reagent composition for improving cell transfecting efficiency | |
| Xue et al. | Exosomes derived from hypoxia-treated human adipose mesenchymal stem cells enhance angiogenesis through the PKA signaling pathway | |
| CN105602992B (en) | It is a kind of based on the CAR-T transgene carrier and its construction method of replication defective recombinant slow virus and application | |
| JP2021530985A (en) | Fososome composition and its use | |
| US20190127696A1 (en) | Anti-robo1 car-t cell, and preparation and application thereof | |
| CN105753991B (en) | Chimeric antigen receptor for resisting placenta-like chondroitin sulfate and application thereof | |
| CN105671082A (en) | Lentivirus vectors expressing exosome markers and building method and application of lentivirus vectors | |
| CN115029320B (en) | Engineering exosome for tumor radiotherapy sensitization, preparation method and application | |
| Guo et al. | The application of mRNA-based gene transfer in mesenchymal stem cell-mediated cytotoxicity of glioma cells | |
| CN111032869B (en) | Virus transfection synergist and application of virus transfection based on click chemistry | |
| CN112410304A (en) | Gene-modified exosome and preparation method and application thereof | |
| CN109576227B (en) | Luciferase reporter virus-based autophagy cell line construction method | |
| CN115011561A (en) | Chimeric antigen receptor macrophage and preparation method and application thereof | |
| Kenarkoohi et al. | Efficient lentiviral transduction of adipose tissue-derived mouse mesenchymal stem cells and assessment of their penetration in female mice cervical tumor model | |
| CN116196334A (en) | Application of mesenchymal stem cell-derived small extracellular vesicles in preparation of drugs for treating diabetic retinopathy | |
| CN112430575B (en) | Universal CAR-T cell, preparation method and application thereof, and anti-tumor drug | |
| CN114672460A (en) | Preparation method and application of CD 44-targeted heterogeneous CIC cell model | |
| CN104212766B (en) | Mesenchymal stem cell as well as application of mesenchymal stem cell | |
| CN106977607A (en) | A kind of Chimeric antigen receptor of anti-placenta sample chondroitin sulfate and its application | |
| CN103756970B (en) | Optimize episomal technology and set up the method for people ips | |
| CN116514992B (en) | Chimeric antigen receptor targeting CD19 with optimized signal peptide sequence and application thereof | |
| Pearson et al. | Lentiviral-mediated ectopic expression of YAP and TAZ in YAPoff cancer cell lines | |
| CN114410689B (en) | Preparation method for enhancing lethality of tumor infiltrating lymphocytes | |
| CN118126201B (en) | Cell membrane targeting GRP94 chimeric antigen receptor, engineering cell and application | |
| Ipinmoroti | Modulatory Roles of Human Adenovirus Type 3 in Extracellular Vesicle Development, Trafficking, and Inhibition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |