CN106591339A - Transient transfection reagent and use thereof - Google Patents
Transient transfection reagent and use thereof Download PDFInfo
- Publication number
- CN106591339A CN106591339A CN201710029440.3A CN201710029440A CN106591339A CN 106591339 A CN106591339 A CN 106591339A CN 201710029440 A CN201710029440 A CN 201710029440A CN 106591339 A CN106591339 A CN 106591339A
- Authority
- CN
- China
- Prior art keywords
- transfection
- transfection reagent
- transient transfection
- reagent
- transient
- 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.)
- Pending
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
-
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a transient transfection reagent and a use thereof. The transfection efficiency of the transfection reagent is high and stable and is equivalent to the transfection efficiency of Lipofectamine 3000 (hereinafter referred to as lipo3000). The transient transfection reagent has high transfection efficiency both in plasmid single transfection and co-transfection and has very small cytotoxicity. The transient transfection reagent can be simply operated. Compared with lipo3000, the transient transfection reagent is free of plasmid dilution and reagent transfection, realizes transfection in 10min, has high solution stability, can be stored at a temperature of 4 DEG C for a long time, has a low cost, very high transfection efficiency and high stability, can be operated easily, is suitable for a wide range of host cells, is not influenced by antibiotics of the culture medium in transfection efficiency and is an excellent choice for a lab where a lot of transient transfection experiments are carried out.
Description
Technical field
The present invention relates to Gene transfer techniques field, and in particular to a kind of transient transfection reagent and its application process, are one
Planting can poison little transfection reagent with high-efficient simple.
Background technology
Transfection needs certain transfection reagent that the carrier with genes of interest is transported in host cell.At present, most often
Transfection reagent is cationic-liposome and cationic polymer, they the characteristics of it is similar with virus, readily penetrate through cell membrane.
Wherein, cationic-liposome has very high efficiency during gene is transfected in vitro, but in vivo, it is removed rapidly by serum,
Accumulation in lung tissue, induces strong anti-inflammatory response, and this will cause high-caliber toxicity, therefore, greatly limit
Its application.Due to the limitation of cationic-liposome, cationic polymer transfection reagent is increasingly subject to pay attention to.
This reagent adopts cationic polymer for main component, can form stable complex with DNA, protects DNA
From the degraded of nuclease, while Nucleic acid stabilization is increased, the effect that transfection reagent/DNA complex passes through cell membrane is improved
Rate, this unique design, significantly improves efficiency gene transfection.Such reagent is ultrahigh in efficiency in current non-viral mediated method
Transfection reagent（The transfection efficiency of variety classes cell can have notable difference）, can compare favourably with lipo3000.In addition this transfection
Reagent is not removed by serum, and serum and antibiotic do not affect its transfection, transfection reagent/DNA complex to be directly added into
Completely in cell culture medium.Its cytotoxicity very little, it is under appropriate conditions, real according to recommending consumption to carry out transfection using it
Test, cell survival rate is higher than 95%.
The content of the invention
For the deficiency of existing transfection reagent, the present invention provides a kind of transient transfection reagent and its application process, with turning
Dye efficiency high and stable, cytotoxicity it is little, it is easy to operate save time, it is solution-stabilized, adapt to host cell range it is wide the advantages of.
For achieving the above object, the present invention is adopted the following technical scheme that：
A kind of transient transfection reagent, its composition is：Efficient linear polyethyleneimine PEI-MAX and ddH2O, solution PH is 7.05.
When using the transient transfection reagent transfected plasmids, plasmid and transfection reagent ratio are between 1 microgram:1.5 microlitres to 1
Microgram:Between 4 microlitres, best proportion is 1 microgram:3 microlitres.
Another object of the present invention is to provide a kind of using method of above-mentioned transient transfection reagent, comprise the steps：
（1）Transfected plasmids and transfection reagent are proportionally mixed, with opti-MEM or ddH2O dilutes；
（2）It is added in DMEM culture medium after incubation at room temperature 5min；
（3）Can be detected after 24-36h.
The transient transfection reagent solution stability is high, can preserve for a long time under the conditions of 4 DEG C and -20 DEG C；Transfection efficiency
It is high；Cytotoxicity is low, and cell survival rate is higher than 95%.
It is an advantage of the current invention that with low cost, transfection efficiency is high, is adapted to host range extensively, and transfection is convenient and swift, to thin
Cellular toxicity is little, and stability of solution is high.
Description of the drawings
Fig. 1 DNA（3 μ g GFP plasmids）The ratio transfection different with transfection reagent, 24h microscopics albumen after transfection
Expression, it is recommended to use 1:3 transfections；
The each 1.5 μ g of Fig. 2 GFP and mcherry plasmids, transfect according to different ratios, 24h microscopics albumen after transfection
Expression, therefore plasmid and transfection reagent ratio are 1 when recommending cotransfection:4；
Fig. 3 the μ gGFP of cotransfection 3 and mcherry, PEI-max and lipo3000 comparison diagrams in HEK293T cells；
Fig. 4 the μ gGFP of cotransfection 3 and mcherry, PEI-max and lipo3000 comparison diagrams in HELA cells；
Fig. 5 transfects the plasmid and each 1 μ g of its activator protein plasmid of 500ng promoteres G in HEK293T cells, examines after transfection 24h
Survey the activity of luciferase；
Fig. 62 μ gCRY2 and SPA1 plasmids of each transfection in HEK293T cells, distinguish blue light and process 1.5h after 24h, do after 3.5h
Co-immunoprecipitation experiment.
Specific embodiment
Embodiment 1 is transfected
（1）150 μ LoptiMEM culture medium, then 3 μ gGFP plasmids of individual addition are added in centrifuge tube first, then according to plasmid（μ
g）：Transfection reagent（μL）1:1.5、1:2、1:3、1:4、1:6 add PEI-max transfection reagents.And with business-like transfection reagent
Lipofectamine 3000 carries out reference for control.
（2）It is added in DMEM cell culture fluids after incubation at room temperature 5min；
（3）Detected after 24-36h.
Experimental result is as shown in Figure 1, it is seen that the ratio of plasmid and transfection reagent is 1：3:When transfection it is optimal.Transfection effect
Really close commercialization transfection reagent lipofectamine 3000.
The cotransfection of embodiment 2
Cotransfection plasmid：The GFP and each 1.5 μ g of mcherry plasmids；Concrete transfection procedure is with embodiment 1.
Experimental result from GFP and mCherry cotransfection comparison diagrams as shown in Fig. 2 can be seen that three two kinds of transfection conditions
Albumen has substantial amounts of expression.
The albumen coexpression of embodiment 3 compares with commercialization transfection reagent：
6 μ g DNA (GFP plasmids and each 3 μ g of mcherry plasmids) are transfected in HEK293T cells, concrete transfection method is with enforcement
Example 1.
PEI-max reagents combination optimization transfection procedure in cotransfection efficiency is can be seen that from Fig. 3 results to be obtained in that and business
The similar effect of industry transfection reagent.
Albumen coexpression compares with commercialization transfection reagent in the Hela cells of embodiment 4：
Cotransfection 3 the μ gGFP and mcherry in HELA cells, concrete transfection method is with embodiment 1.
PEI-max reagents in cotransfection efficiency are can be seen that in Hela cells from Fig. 4 results combine optimization transfection procedure
It is obtained in that the effect similar to commercialization transfection reagent.
The LUC Photinus pyralis LUC Photinus pyralis FL of embodiment 5 detects functional transcription factor：
（1）According to 10 μ L/1 holes（96 orifice plates）Amount optiMEM culture medium is added in centrifuge tube, and backward its adds 0.05
μ g/1 holes（96 orifice plates）Promoter G fragment LUC Photinus pyralis LUC Photinus pyralis FL carrier and 0.1 μ g/1 holes CIB1 transcription factor and CO transcriptions
The factor, according to 1:3 ratios add PEI-max transfection reagents.
（2）Room temperature is placed and is added in 96 orifice plates according to the amount of the μ L of every hole 10 after 5min.
（3）After 24 hours, LUC Photinus pyralis LUC Photinus pyralis FL substrate is added, it is glimmering using microwell plate Chemiluminescence Apparatus detection Lampyridea
Light element expression of enzymes amount.
As seen from Figure 5, experimental result reflects well two transcription factor of CIB1, CO can promote reporter gene
Transcription.
The co-immunoprecipitation of embodiment 6 identifies interactions between protein：
（1）According to the scheme of embodiment 1 by CRY2/SPA1 cotransfections to HEK93T cells.
（2）Transfection carries out immunoprecipitation experiment in 24 hours using CRY2 antibody later.
（3）Western identifies CRY2 signals, and SPA1 signals.
As seen from Figure 6, experimental result reflects CRY2/SPA1 interactions and blue light illumination 1.5 hours（B1.5）With
And 3.5 hours（B3.5）CRY2/SPA1 interactions become strong, further demonstrate PEI-max transfection reagents and prioritization scheme reliability,
And Various Complex experiment can be carried out.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (5)
1. a kind of transient transfection reagent, it is characterised in that its composition is：Efficient linear polyethyleneimine PEI-MAX and ddH2O；
The concentration of PEI-MAX is 1ug/uL, and pH value of solution is 7.05.
2. a kind of transient transfection reagent according to claim 1, it is characterised in that using transient transfection reagent transfection
During plasmid, plasmid and transfection reagent ratio are between 1 microgram:1.5 microlitres to 1 microgram:Between 4 microlitres.
3. a kind of transient transfection reagent according to claim 1, it is characterised in that using transient transfection reagent transfection
During plasmid, singly turn best proportion for 1 microgram:3 microlitres of corotation best proportions are 1 microgram:4 microlitres.
4. the application process that transient transfection reagent described in a kind of claim 1 is transfected, it is characterised in that including following step
Suddenly：
（1）Transfected plasmids and the transfection reagent are proportionally mixed, with opti-MEM or ddH2O dilutes；
（2）It is added in DMEM culture medium after incubation at room temperature 5min；
（3）Can be detected after 24-36h.
5. a kind of transient transfection reagent according to claim 1, it is characterised in that stability of solution is high, under the conditions of 4 DEG C
Can preserve for a long time；Can preserve for a long time under the conditions of -20 DEG C, transfection efficiency is high；Cytotoxicity is low, and cell survival rate is higher than 95%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710029440.3A CN106591339A (en) | 2017-01-16 | 2017-01-16 | Transient transfection reagent and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710029440.3A CN106591339A (en) | 2017-01-16 | 2017-01-16 | Transient transfection reagent and use thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106591339A true CN106591339A (en) | 2017-04-26 |
Family
ID=58584899
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710029440.3A Pending CN106591339A (en) | 2017-01-16 | 2017-01-16 | Transient transfection reagent and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106591339A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110168093A (en) * | 2017-09-12 | 2019-08-23 | 广州中科蓝华生物科技有限公司 | It is a kind of transfect cytozoon kit and its application |
| CN111154805A (en) * | 2020-02-11 | 2020-05-15 | 苏州吉恒基因科技有限公司 | Cationic polymer DNA complex and method for promoting target plasmid to transfect cells and express |
| CN112695056A (en) * | 2020-12-29 | 2021-04-23 | 苏州汇桢生物技术有限公司 | Transfection reagent and transfection method for improving transient transfection efficiency |
| CN114561413A (en) * | 2022-03-24 | 2022-05-31 | 新乡医学院 | Transient transfection reagent and application thereof |
-
2017
- 2017-01-16 CN CN201710029440.3A patent/CN106591339A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| LAURENCE DELAFOSSE,ET AL.: "Comparative study of polyethylenimines for transient gene expression in mammalian HEK293 and CHO cells", 《JOURNAL OF BIOTECHNOLOGY》 * |
| LI WEI-WEI,ET AL.: "Effects of the dilution rate on cell cycle distribution and PEI-mediated transient gene expression by CHO cells in continuous culture", 《PROCESS BIOCHEMISTRY》 * |
| 丰干钧 等: "聚乙烯亚胺为载体的体内核酸转染研究进展", 《中国修复重建外科杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110168093A (en) * | 2017-09-12 | 2019-08-23 | 广州中科蓝华生物科技有限公司 | It is a kind of transfect cytozoon kit and its application |
| CN110168093B (en) * | 2017-09-12 | 2023-08-15 | 中科蓝华(广州)生物医药技术有限公司 | Kit for transfecting intracellular parasites and application thereof |
| CN111154805A (en) * | 2020-02-11 | 2020-05-15 | 苏州吉恒基因科技有限公司 | Cationic polymer DNA complex and method for promoting target plasmid to transfect cells and express |
| CN111154805B (en) * | 2020-02-11 | 2024-03-12 | 苏州吉恒基因科技有限公司 | Cationic multimeric DNA complexes and methods for promoting transfected cells and expression of plasmids of interest |
| CN112695056A (en) * | 2020-12-29 | 2021-04-23 | 苏州汇桢生物技术有限公司 | Transfection reagent and transfection method for improving transient transfection efficiency |
| CN114561413A (en) * | 2022-03-24 | 2022-05-31 | 新乡医学院 | Transient transfection reagent and application thereof |
| CN114561413B (en) * | 2022-03-24 | 2023-09-29 | 新乡医学院 | Transient transfection reagent and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106591339A (en) | Transient transfection reagent and use thereof | |
| Dalby et al. | Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications | |
| US9493768B2 (en) | Translational enhancer-element dependent vector systems | |
| Moore et al. | Efficient, high-throughput transfection of human embryonic stem cells | |
| Chen et al. | Messenger RNA half‐life measurements in mammalian cells | |
| Stein et al. | Translation of vascular endothelial growth factor mRNA by internal ribosome entry: implications for translation under hypoxia | |
| Elango et al. | Optimized transfection of mRNA transcribed from ad (A/T) 100 tail-containing vector | |
| US20190185856A1 (en) | Synthetic Near-Threshold Translational Repressors | |
| WO2017066943A1 (en) | Method and kit for dna fragment assembling in vitro | |
| Mehta et al. | Derepression of the Her-2 uORF is mediated by a novel post-transcriptional control mechanism in cancer cells | |
| AU2021325586A1 (en) | KRAB fusion repressors and methods and compositions for repressing gene expression | |
| Lee et al. | Exon junction complex enhances translation of spliced mRNAs at multiple steps | |
| CN111088251B (en) | Gene expression cassette and application thereof in Cre-lox recombination efficiency detection | |
| CN103898158B (en) | A kind of carrier containing MALAT1 promoter sequence and reporter gene and its construction method and purposes | |
| Rauch et al. | Targeted m6A reader proteins to study the epitranscriptome | |
| CA3182247A1 (en) | Riboswitch modules and methods for controlling eukaryotic protein translation | |
| Gong et al. | Regulation of isoform expression by blocking polyadenylation signal sequences with morpholinos | |
| Mariati et al. | Post-transcriptional regulatory elements for enhancing transient gene expression levels in mammalian cells | |
| Feng et al. | A novel conditional gene silencing method using a tumor-specific and heat-inducible siRNA system | |
| CN114807145B (en) | Leader sequence for improving cap-independent translation efficiency and application thereof | |
| Wan et al. | Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting | |
| Misra et al. | Fluorescence reporter-based genome-wide RNA interference screening to identify alternative splicing regulators | |
| CN112126645B (en) | Ring RNA (ribonucleic acid) knocking-down method and application thereof | |
| Kaźmierczak et al. | Construction of a full-length 3′ UTR reporter system for identification of cell-cycle regulating MicroRNAs | |
| EP3305903A1 (en) | Rna toolbox |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170426 |
|
| RJ01 | Rejection of invention patent application after publication |