CN108277196A - A kind of plasmid DNA transfection method of high-efficiency low-toxicity - Google Patents
A kind of plasmid DNA transfection method of high-efficiency low-toxicity Download PDFInfo
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Abstract
The invention discloses a kind of plasmid DNA transfection methods of high-efficiency low-toxicity, belong to biotechnology.The method of the present invention includes the following steps：(1) it with 20% 30% cell density inoculating cell, is cultivated；(2) when cell density grows to 40% 60%, secondary culture is carried out；(3) when the cell of secondary culture is in rigid adherent unstretched condition, with plasmid transfection agent composition transfectional cell.The present invention improves cell transfecting efficiency, while smaller to cytotoxicity, does not need additional equipment or reagent, is conveniently operated and grasps, easy to spread.
Description
Technical field
The invention belongs to biotechnologies, and in particular to the modified form plasmid that a kind of transfection efficiency is high, cytotoxicity is low
DNA transfection methods.
Background technology
Cell transfecting is an essential technology in molecular biology and RESEARCH ON CELL-BIOLOGY field.Cell transfecting
Common method is broadly divided into three classes：Physical transfection method, chemical transfection methods and biological transfection method.Physical transfection method packet
Include electroporation, microinjection etc..Chemical transfection methods include lipofection, calcium phosphate precipitation etc..Biology is situated between
The transfection method led includes virus-mediated transfection etc..Ideal transfection method should have that transfection efficiency is high, cytotoxicity is low and
Facilitate easy-operating feature.Physical transfection method and technology sensibility is high, but needs independent purchase of equipment, and generally to cell
Effect of vigor is all bigger.The transfection efficiency of virus transfection method is higher, but operating procedure is complicated, there is certain bio-safety
Risk may have an impact operating personnel, be not already ideal transfection method.The common transfection method in laboratory is chemistry at present
Transfection method, and it is the most commonly used with lipofection.The lipofectamine of commercialization is easy to buy and use.
In the cells such as HeLa, HEK 293, NIH/3T3, the transfection efficiency of liposome transfection method is higher and has the spy of low cytotoxicity
Point, but for difficulty turns cell, the transfection efficiency of liposome transfection method is simultaneously unsatisfactory, and which has limited liposome transfections
The use of method.
Liposome transfection method is divided into two kinds of different operation methods in concrete operations, one is positive transfection method, one
Kind is reverse transfection method.The operating guidance that most of companies are provided all uses positive transfection method.Positive transfection method is
Refer to 24 hours before transfection, cell inoculation to be transfected in cel culture plates and is ensured that cell density reaches before transfection
80%-90%.When transfection, plasmid is incubated a period of time after being mixed by the proportioning that company is provided with transfection reagent, then directly
It connects and is added in the culture medium of cell to be transfected.In difficulty turns cell, the method for forward direction transfection is often inefficient.Reverse transfection side
Method and the difference of positive transfection method are that cell to be transfected need not shift to an earlier date 24 hours and be inoculated with, first by plasmid and transfection before transfection
Reagent is incubated after being mixed by the proportioning that company is provided, while attached cell being digested, and then plasmid and transfection agents are mixed
Object is inoculated into together with the cell for the suspended state that digestion is got up in culture plate.Reverse transfection method have it is easy to operate, it can be achieved that
The characteristics of automation mechanized operation, is often used by high flux screening.However, mediating plasmid-transfected cells by reverse transfection method
When, often there is apparent toxicity to cell, which also limits the uses of reverse transfection method.From this, developing a kind of transfection
It is efficient, cytotoxicity is low and the transfection method that is conveniently operated is very necessary.
Invention content
It is an object of the invention to overcome shortcoming and deficiency of the existing technology, provide that a kind of transfection efficiency is high, cell
The low modified form plasmid DNA transfection method of toxicity.
The present invention is based on following principles：It is intracellular to hinder Plasmid DNA to enter cell and efficient table with many with extracellular
The obstacle reached.Plasmid-transfection reagent complexes, which enter in cell, to be first had to through cell membrane barrier, this process is mainly made by endocytosis
With mediation, cell layer tension internally gulps down effect and has a very big impact, and low cell layer tension can promote endocytosis.Cell exists
After passage in adherent stretching process, cell layer tension reduces, and may advantageously facilitate plasmid and enters cell, improves the expression of foreign gene
Effect.In addition, plasmid will must also enter nucleus ability transcript mRNA, and express corresponding albumen.In fission process
The middle temporary disintegration of nuclear membrane, can make plasmid be easily accessible nucleus.
The purpose of the invention is achieved by the following technical solution：
A kind of plasmid DNA transfection method of high-efficiency low-toxicity, includes the following steps：
(1) it with the cell density inoculating cell of 20%-30%, is cultivated；
(2) when cell density grows to 40%-60%, with trypsin digestion and cell, secondary culture is carried out；
(3) it when the cell of secondary culture is in rigid adherent unstretched condition, is transfected with plasmid-transfection reagent mixture thin
Born of the same parents.
Preferably, when transfecting 27 cells of CAL, described method includes following steps：
(1) 27 cells of CAL are inoculated into tissue culture plate by the density of 20%-30% before transfecting 24 hours, culture is added
Base culture；
(2) 1-2 hour cells density growth is to 40%-60% before transfecting, with 27 cells of trypsin digestion CAL, then general
It is cultivated in the cell renewed vaccination to tissue culture plate of digestion；
(3) after cultivating 1-2 hours, with plasmid-transfection reagent mixture transfectional cell.
The present invention is for transfecting 27 cells of CAL, and the cell density of inoculation in 24 hours is about 20%- before transfection
30%, before second day is transfected, the density of cell reaches about 40%-60%, and cell is in exponential phase of growth at this time,
In 24 hours after transfection, cell will generally be in exponential phase of growth, there is a large amount of cell division.Then, disappear before transfection
Change cell, then make cell again adherent, and transfected when cell is just adherent un-extended, is both conducive to cell during adherent
Cell layer tension reduces is conducive to transfected plasmids again, and avoids the influence adherent to cell to a certain extent, improves cell
Vigor.Testing result shows small toxicity of the improved transfection method to cell, and transfection efficiency is high, there is good application prospect.
Traditional positive transfection method is usually to be carried out after so that cell density is reached 80%-90%, and cell is soon at this time
Meeting stops division since cell density is excessive, is unfavorable for plasmid transfection.And the method for reverse transfection, although can be pasted in cell
Be conducive to plasmid in wall stretching process and enter cell, but may interfere with the adherent process of cell, cell viability is caused obviously to drop
It is low, finally affect transfection efficiency.The present invention has the following advantages compared with the prior art and advantageous effect：
(1) present invention cell density of inoculation in 24 hours before transfection is about 20%-30%, in transfection and after transfection
In 24 hours, usual cell will generally be in exponential phase of growth, there is a large amount of cell division, be conducive to Plasmid DNA and enter carefully
Karyon.
(2) present invention vitellophag before transfection, then make cell again adherent, and carried out when cell is just adherent un-extended
Transfection, not only using cell, the reduction of cell layer tension was conducive to transfected plasmids during adherent, but also avoided to a certain extent pair
The adherent influence of cell, improves the vigor of cell.
(3) present invention improves cell transfecting efficiency, while smaller to cytotoxicity.
(4) present invention uses now widely used transfection reagent, does not need additional equipment or reagent, is conveniently operated
And grasp, it is easy to spread.
Description of the drawings
Fig. 1 is present invention improvement transfection method schematic diagram.
Fig. 2 is fluorescence microscope result figure, and compared with conventional forward transfection method, improvement transfection method significantly improves
Transfection efficiency.27 cancer cell of oral cavity of CAL uses conventional forward transfection method and improvement transfection method to transfect pEGFP-N1 matter respectively
Grain is transfected successful cell enhanced green fluorescent protein in fluorescence microscopy microscopic observation after being cultivated 48 hours after transfection, in green
Color.A and D is difference visual field photo, and B and E are fluorescence photos, and C and F are the pictures of difference and fluorescence overlapping.A-C is improvement transfection
Method as a result, D-F is the result of traditional transfection methods.
Fig. 3 is flow cytometry analysis result figure, and compared with conventional forward transfection method, improvement transfection method significantly improves
Transfection efficiency.A and B is representative flow cytometry analysis result.A is 27 cells of CAL of conventional forward transfection method transfection
The quantity and intensity of enhanced green fluorescent protein, 45.3% cell is the green fluorescent protein positive.B is improvement transfection method
The quantity and intensity of the 27 cell enhanced green fluorescent proteins of CAL of transfection, 70.7% cell is green fluorescent protein sun
Property.C is the statistic analysis result of three repeated experiments, *：p<0.05.
Fig. 4 is Western results of hybridization figures, and compared with conventional forward transfection method, improvement transfection method significantly improves
Transfection efficiency.27 cells of CAL transfect pEGFP-N1 plasmids with two methods respectively, and western is used after being cultivated 48 hours after transfection
Expression of the hybridizing method through mouse antibodies green fluorescent protein (GFP) antibody test green fluorescent protein.Actin (actin)
It is the control of applied sample amount.
Fig. 5 is cell viability testing result figure, and compared with reverse transfection method, improvement transfection method significantly improves cell
Vigor.27 cells of CAL transfect pEGFP-N1 plasmids with two methods respectively, in collection supernatant after being cultivated 24 hours after transfection
And attached cell, check life or death cell quantity with Trypan Blue.Formula calculates：Cell viability=(total cell number-dead cell
Number)/total cell number.
Specific implementation mode
The content that following embodiment further illustrates the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.
1 modified form transfection method of embodiment
(1) detection of application and transfection efficiency of the modified form transfection method in transfecting 27 cells of CAL
1. transfecting 27 cells (Fig. 1) of cancer cell of oral cavity system CAL of more difficult transfection using modified form transfection method
1) 27 cells of CAL are inoculated in six well culture plates by the quantity in 200,000/hole before transfecting 24 hours, cell density is
About 20%-30%.The DMEM trainings that 2mL contains 5% fetal calf serum (HyClone) and 1% dual anti-(penicillin and streptomysin) are added
Support base, 37 DEG C, 5%CO2Stationary culture.
2) 1-2 hours before transfection, when cell density grows to 40%-60%, 0.25% trypsase (Thermo is used
Fisher Scientific) digestion 27 cells of CAL, then by whole cell renewed vaccinations to six well culture plates of digestion, add
Enter the DMEM culture mediums that 2mL contains 5% fetal calf serum and 1% dual anti-(penicillin and streptomysin), 37 DEG C, 5%CO2Stationary culture
To be transfected after 1-2 hours etc., at this point, most cells are attached to six orifice plate bottom surfaces, but cellular morphology is still rounded, not yet stretches
It opens.
3) it presses transfection agents product description and prepares Plasmid DNA and liposome transfection agent compound.It specifically includes：By 3.75 μ L
LipofectamineTM3000 (Thermo Fisher Scientific) are dissolved in 125 μ L Opti-MEMTM(Thermo
Fisher Scientific) in culture medium.Separately by 2.5 μ g pEGFP-N1 plasmids and 125 μ L Opti-MEMTMCulture medium mixing
Afterwards, 5 μ L P3000 reagent mixings are added.Then the mixture containing plasmid is added to containing LipofectamineTM 3000
Mixture in mixing, stand it is spare after ten minutes.
4) plasmid-transfection reagent mixture is added to mixing in 27 cell culture mediums of CAL to be transfected, 37 DEG C, 5%CO2It is quiet
Gene expression dose is detected after setting culture 24-48 hours.
2. using the detection of modified form forward direction transfection method transfection 27 cell transfecting efficiencies of CAL
1) expression of fluorescence microscope green fluorescent protein is used
After being cultivated 48 hours after transfection, the green fluorescent protein table of 27 cells of CAL after being transfected by fluorescence microscope
It reaches and takes pictures.
2) there is 27 cells of CAL of green fluorescence with Flow cytometry
27 cells of CAL after being transfected with trypsin digestion after being cultivated 48 hours after transfection are added containing fetal calf serum
DMEM culture mediums terminate digestion, blow and beat mixing cell, flow cytometer detection have green fluorescence 27 cell proportions of CAL and
Fluorescence intensity, using 27 cells of CAL without green fluorescence as negative control when detection.
3) 27 cell Green fluorescent protein expressions of CAL after using western hybridization checks to transfect
Culture medium is discarded after being cultivated 48 hours after transfection, SDS sample-loading buffers are added, and collects total protein of cell, 95 DEG C are boiled
Boil 2min.The separation in 10% SDS-PAGE glue (Thermofisher companies) by protein sample, instrument is transferred with Western
(Biorad companies) shifts under 60V voltages on 2 hours to nitrocellulose filter (Pall companies).The film transferred is used
5% hour of skim milk close membrane 1, with the anti-green fluorescent protein antibody of mouse (Santa Cruz companies, 1:1000 dilutions)
Be incubated and 4 spend night, with the goat anti-mouse igg antibody of horseradish peroxidase-labeled (Sigma companies, 1:10000) it is small to be incubated 1
When, with the expression of luminous substrate (Thermofisher) and the specific green fluorescent protein of X-ray film detection.
2 modified form transfection method of embodiment significantly improves the transfection efficiency of Plasmid DNA compared with conventional forward transfection method
(1) compare improvement transfection method and transfection efficiency of the conventional forward transfection method in transfecting 27 cells of CAL
1. the method using embodiment 1 transfects 27 cells of CAL using improvement transfection method.
2. transfecting 27 cells of CAL using conventional forward transfection method.The main distinction with improvement transfection method is that cell connects
Kind density is high, and is not passed on before transfection.The specific method is as follows：
1) 27 cells of CAL are inoculated in six well culture plates by the quantity in 500,000/hole before transfecting 24 hours, cell density is
The DMEM trainings that 2mL contains 5% fetal calf serum (HyClone) and 1% dual anti-(penicillin and streptomysin) are added in about 40%-60%
Support base, 37 DEG C, 5%CO2Stationary culture is to be transfected after 24 hours etc..
2) second day prepares Plasmid DNA and liposome transfection agent composition in the same manner as in Example 1.
3) plasmid-transfection reagent mixture is added to mixing in 27 cells of CAL to be transfected, 37 DEG C, 5%CO2Stand training
It supports.
3. the detection of transfection efficiency
In the same manner as in Example 1, there are 27 cell quantities of CAL of green fluorescence with fluorescence microscope detection record；It is thin with streaming
CAL 27 cell quantity of born of the same parents' art detection with green fluorescence；With 27 cell Greens of CAL after western Blot detection transfections
Fluorescent protein expression is horizontal.
It is above-mentioned the experimental results showed that, fluorescence microscope (Fig. 2) and FCM analysis (Fig. 3) all show that modified form turns
Dyeing method has the positive cell of greater proportion of enhanced green fluorescent protein compared with conventional forward transfection method.Western hybridization knots
Fruit shows that modified form transfection method improves the expression (figure of transfected green fluorescent protein compared with conventional forward transfection method
4).Therefore new transfection method provided by the present invention can significantly improve the transfection efficiency of cell.
3 modified form transfection method of embodiment is significantly reduced compared with the cytotoxicity of conventional counter transfection method
(1) compare improvement transfection method and conventional counter transfection method in transfecting 27 cells of CAL to the toxicity of cell
1. the method using embodiment 1 transfects 27 cells of CAL using improvement transfection method.
2. transfecting 27 cells of CAL using conventional counter transfection method, the specific method is as follows：
1) 27 cells of CAL are inoculated in six well culture plates by the quantity in 200,000/hole before transfecting 24 hours, cell density is
The DMEM cultures containing 5% fetal calf serum (HyClone) and 1% dual anti-(penicillin and streptomysin) are added in about 20%-30%
Base, 37 DEG C, 5%CO2Stationary culture.
2) Plasmid DNA and liposome transfection agent compound in the same manner as in Example 1, are prepared by transfection agents product description.
3) when cell density grows to 40%-60%, with 0.25% trypsase (Thermo Fisher
Scientific 27 cells of CAL) are digested, then are contained 2mL in whole cell renewed vaccinations to six well culture plates of digestion, is added
There are the DMEM culture mediums of 5% fetal calf serum and 1% dual anti-(penicillin and streptomysin), while Plasmid DNA and liposome turn is added
Stain compound, 37 DEG C, 5%CO2Stationary culture.
3. analyzing toxicity of both transfection methods to cell
After being cultivated 24 hours after transfection, culture supernatant is collected, is centrifuged 5 minutes with 2000 revs/min, is collected dead thin in supernatant
Born of the same parents.Again with 0.25% trypsin digestion attached cell, with containing pancreatin and closing in 5% fetal calf serum and with the dead cell in supernatant
And as sample.Same amount sample is taken, after being handled with trypan blue dye liquor, counts life or death cell quantity under the microscope.As a result, it has been found that
The cell viability of improvement transfection method is significantly higher than the method for reverse transfection, increases to about 80% (Fig. 5) from about 55%.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (2)
1. a kind of plasmid DNA transfection method of high-efficiency low-toxicity, it is characterised in that：Include the following steps：
(1) it with the cell density inoculating cell of 20%-30%, is cultivated；
(2) when cell density grows to 40%-60%, secondary culture is carried out；
(3) when the cell of secondary culture is in rigid adherent unstretched condition, with plasmid-transfection reagent mixture transfectional cell.
2. the plasmid DNA transfection method of high-efficiency low-toxicity according to claim 1, it is characterised in that：When transfection CAL 27 is thin
When born of the same parents, include the following steps：
(1) 27 cells of CAL are inoculated into tissue culture plate by the density of 20%-30% before transfecting 24 hours, culture medium training is added
It supports；
(2) 1-2 hour cells density growth is to 40%-60% before transfecting, with 27 cells of trypsin digestion CAL, then will digestion
Cell renewed vaccination to tissue culture plate in cultivate；
(3) after cultivating 1-2 hours, with plasmid-transfection reagent mixture transfectional cell.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457224A (en) * | 2008-12-17 | 2009-06-17 | 苏州吉玛基因药物科技有限公司 | MiR-21 antisense digonucleotides and use thereof |
| CN102174471A (en) * | 2010-12-30 | 2011-09-07 | 中国人民解放军军事医学科学院基础医学研究所 | Method for transfecting and marking iPS cells of three-fusion reporter genes mediated by lentivirus |
| CN103675262A (en) * | 2013-12-27 | 2014-03-26 | 步荣发 | Method of stable fluorescence labeling of living cells of oral squamous cell carcinoma |
| CN104419684A (en) * | 2013-09-01 | 2015-03-18 | 翁炳焕 | Construction of 18-trisomy syndrome cell model and cell bank of 18-trisomy syndrome cell by SV40LT gene transfection |
| CN105779395A (en) * | 2016-03-22 | 2016-07-20 | 西北农林科技大学 | Immortalized canine adipic mesenchymal stem cell line and constructing method thereof |
| CN106399485A (en) * | 2016-08-31 | 2017-02-15 | 北京泱深生物信息技术有限公司 | Genes highly expressed in tongue squamous carcinoma para-carcinoma tissue and applications of genes |
-
2018
- 2018-01-19 CN CN201810055524.9A patent/CN108277196B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457224A (en) * | 2008-12-17 | 2009-06-17 | 苏州吉玛基因药物科技有限公司 | MiR-21 antisense digonucleotides and use thereof |
| CN102174471A (en) * | 2010-12-30 | 2011-09-07 | 中国人民解放军军事医学科学院基础医学研究所 | Method for transfecting and marking iPS cells of three-fusion reporter genes mediated by lentivirus |
| CN104419684A (en) * | 2013-09-01 | 2015-03-18 | 翁炳焕 | Construction of 18-trisomy syndrome cell model and cell bank of 18-trisomy syndrome cell by SV40LT gene transfection |
| CN103675262A (en) * | 2013-12-27 | 2014-03-26 | 步荣发 | Method of stable fluorescence labeling of living cells of oral squamous cell carcinoma |
| CN105779395A (en) * | 2016-03-22 | 2016-07-20 | 西北农林科技大学 | Immortalized canine adipic mesenchymal stem cell line and constructing method thereof |
| CN106399485A (en) * | 2016-08-31 | 2017-02-15 | 北京泱深生物信息技术有限公司 | Genes highly expressed in tongue squamous carcinoma para-carcinoma tissue and applications of genes |
Non-Patent Citations (3)
| Title |
|---|
| MAKOTO KINOUCHI 等: "Involvement of miR-518c-5p to Growth and Metastasis in Oral Cancer", 《PLOS ONE》 * |
| 朴英实 等主编: "《分子病理生物学实验技术指南》", 31 May 2015, 人民军医出版社 * |
| 路晓薇 等: "靶向Cdc6 RNAi 抑制舌癌CAL-27细胞的增殖", 《中山大学学报（医学科学版）》 * |
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