CN103981218A - Optimizing method for transfecting suspension cell by electroporation technology - Google Patents
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Abstract
The invention belongs to mammal cell gene engineering field, and concretely relates to an optimizing method for transfecting suspension cell by an electroporation technology, especially suitable for a difficultly transfected CEMx174 cell line. The method is characterized in that a 10% of serum-containing RPMI-1640 cell medium is taken as an electroporation buffer, and is mixed with cell and exogenous DNA with an appropriate concentration, then an electroporation apparatus is employed for electrotransfection, and the exogenous DNA is introduced into cell. According to the invention, a special prepared electrotransfection buffer and other transfection reagents are not required for conveniently and rapidly introducing the exogenous DNA to the cell, and simultaneously acquiring the high suspension cell transfection efficiency.
Description
Technical field
The invention belongs to mammalian cell genetically engineered field, a kind of specifically method of electroporation technology transfection suspension cell of optimization, is particularly applicable to the CEMx174 clone of difficult transfection.
Background technology
Transfection is a kind of technology that exogenous nucleic acid is imported in eukaryotic cell and bring into play its biological action, and the nucleic acid of importing comprises DNA (plasmid and linear dsdna), antisense oligonucleotide and RNAi (RNA interference).At present, Gene transfer techniques has been widely used in genome functions research (gene expression regulation, gene function, signal transduction and drug screening research) and gene therapy research.
Rotaring dyeing technology can be divided into transient transfection and the large class of stable transfection two according to timeliness.The former foreign DNA/RNA unconformability, in host chromosome, can import multiple copies, obtains the temporary transient high level expression of goal gene, is applicable to after transfection harvested cell in 1～4 day, the experiment of testing goal gene expression results.Stable transfection is for setting up monoclonal clone, and goal gene is incorporated in target cell karyomit(e) or with free form stable existence in continuing to go down to posterity in cultured cells, instructs the appropriate expression of goal gene.In general, the expression efficiency of stable transfection is than low 1～2 order of magnitude of transient transfection, but effect is more continual and steady, reproducible.Conventionally utilize selectable genetic marker medicine to filter out the successful cell of transfection; set up monoclonal cell system; as G418 (neomycin resistance), aminopterinum (thymidine kinase resistance), tetracycline (tetracycline-N-acetyltransferase resistance).
Conventional transfection method can be classified as three classes now: biochemical method, virus-mediated method and physical method.
Biochemical method is one the most conventional in laboratory, that after forming mixture by transfection reagent and nucleic acid, facilitation nucleic acid approaches adherent cell and promotes the endocytosis of cell that nucleic acid is entered in cell, conventional DEAE-dextran method, calcium phosphate method, liposome method etc.But due to the difference of membrane structure, or the mode of suspension cluster-shaped growth has reduced the probability of mixture and cells contacting, and this class transfection method is very undesirable for suspension cell effect.Although have be much known as can transfection suspension cell new and improved reagent, its result is still barely satisfactory.
Retroviral vector is normally used in virus-mediated transfection.It belongs to RNA viruses, can in target cell, reverse transcription produce DNA complementary strand, and this DNA single chain can be used as the synthetic Article 2 DNA chain of template, and Article 2 DNA chain can mix in cell genomic dna.This virus can be utilized the enzyme Transcription and replication voluntarily of host cell, and RNA can synthetic proteins, then packaging virus, in born of the same parents, discharges, and becomes infectious virus, and the process of its mediation can make viral single copy gene group stably enter cell.In theory, the transfection method of this retrovirus-mediated can obtain higher transfection efficiency, but the preparation work very complicated in its early stage, and there is certain security risk, be not suitable for applying widely.And according to the practical studies of this chamber, the result of the method transfection suspension cell is also unsatisfactory.
And conventional physical method has two kinds: by membrane perforation and import nucleic acid, or utilize of short duration impulse of current instantaneous formation on plasma membrane can allow micropore that nucleic acid passes through make nucleic acid initiatively enter cell under electric field action by direct microinjection.The former is mainly used in the preparation of transgenic animal, is unsuitable for the transfection of a large amount of cells.The range of application of electroporation technology is more extensive, can quick, easy, effective DNA be imported and be comprised in the mammalian cell of bacterium, yeast, vegetable cell and multiple cultivation.
CEMx174 clone is people T, B lymphoblast hybrid strain, get (Salter RD by 721.174 (mutation of LCL721B lymph matricyte system) and CEMR.3 (azaguanine of CEM T lymph matricyte system and Wu Bayin opposing clone strain) hybridization, Howell DN, 1985).This cell strain is expressed CXCR4 (Strizki JM et a1,1997), CCR5 (Miyagi T et al, 2000), orphan receptor GPR15 (Kiene M et al, 2012) etc. are important SIV and the cell model of HIV infection, be widely used in the mechanism of virus infection and the research of clinical medicine effect (Dong MX et al, 2012; Lim HG et al, 2008; Newman JT et al, 2007).Meanwhile, CEMx174 clone is also expressed opiate receptor (μ/κ/Δ), therefore through being usually used in Study on mechanism and clinical application research (Miyagi T et al, 2000 of the opioid drugs such as morphine; Jin Xu et al, 2004; Han Liu et al, 2009).But CEMx174 clone has identical growth characteristics with other lymphocyte series (as Jurkat, U937 etc.), be suspension cluster-shaped and be grown in nutrient solution, same, transfection efficiency is very low, often the carrying out of serious obstruction experimental study.Conventional lipofectamine or positively charged ion transfection reagent all can not reach satisfactory effect, the low and poor repeatability of transfection efficiency, direct interference the carrying out of subsequent experimental, confidence level and reliability to experimental result cause interference.
For the conventional transfection method of this class suspension cell clone at one's wit's end, electroporation is a kind of method of simple and effective.But the condition of electroporation is very large for result impact, the intensity of impulse of current and time, and the transfection efficiency that affects cell and survival rate that the electric selection that turns damping fluid all can be serious.But due to the difference of membrane structure, the electroporation conditions of different clones has very large difference sometimes, and the good performance of electroporation apparatus tends to provide very wide in range electric shock intensity and time range, therefore the screening of optimal conditions is a great problem of electrotransfection.Certainly concrete electroporation conditions has been drafted for modal clone by the company of some manufacture bio-instruments, or preset relevant program at machine intimate, can obtain higher transfection efficiency, but the consumptive material that it is supporting or damping fluid are often expensive, default condition is fixed on particular instrument by scope especially, is unfavorable for widespread use.
The present invention is in order to change this present situation; obtain one group of suspension cell transfection conditions scope of optimizing through screening; and can be widely used in multiple electroporation device; and required consumptive material is all conventional consumptive material in laboratory; electricity turns damping fluid and uses the RPMI-1640 nutrient solution that ionic concn is lower, meanwhile, adds 10% serum after biliographic data; play the effect of Cell protection, the survival rate of cell after raising electricity irritation.And then carried out finer optimization for CEMx174 system again, electroporation conditions is fixed as to 4mm pole cup, taking the RPMI-1640 nutrient solution (containing 10% serum) of preheating for electricity turns damping fluid, pulse strength 210v, burst length 30ms, shocks by electricity once.Through multiple authentication, this condition can obtain at least transfection efficiency of > 50%.
Summary of the invention
The present invention has developed a kind of by general commercial the electroporation apparatus rapidly and efficiently method of transfection suspension cell, particularly lymphocyte series CEMx174 clone.
CEMx174 clone is people T, B lymphoblast hybrid strain, is got by 721.174 (mutation of LCL721B lymph matricyte system) and CEMR.3 (azaguanine of CEM T lymph matricyte system and Wu Bayin opposing clone strain) hybridization.Cell is cultivated in the RPMI-1640 substratum that contains 10% serum, with cluster form suspension growth.
The present invention is taking the RPMI-1640 cell culture medium of 37 DEG C of preheatings (containing 10% serum) as electroporation damping fluid, with suitable concentration re-suspended cell and foreign DNA respectively, after mixing, transfer in the pole cup of 4mm, use common commercial electroporation apparatus for the ECM830 of BTX company cell electricity fusion instrument, electric shock condition be under LV pattern with 210mV pulse electric shock once, time length 30ms, mainly comprises the following steps:
1. cell cultures.Cell cultures (containing 10% serum) in RPMI-1640 complete culture solution, collects as experiment during in logarithmic phase in good condition.
2. nutrient solution preheating.Experimental demand is got appropriate nutrient solution and is added in orifice plate or plate, puts into the CO2 incubator preheating of 37 DEG C before electrotransfection experimental implementation starts.
3. collect counting cells.By cell harvesting together, softly mix, take out a small amount of cell counting, measure appropriate cell suspension according to count results, the centrifugal 10min of 800rpm (90g), the supernatant that as far as possible exhausts, with the perfect medium re-suspended cell of a certain amount of preheating, makes cell and the mixed final concentration of DNA diluent reach 1 × 107.
4. dilution DNA.When collecting cell, get appropriate transfection level DNA equally with appropriate preheating substratum dilution, volume is no more than 1/6 of cumulative volume.The quality of required plasmid is directly proportional to plasmid size, and to need the plasmid amount of 1kb be 1 μ g to every 100 μ l systems.
5. electrotransfection.Softly mix cell suspension and DNA diluent, transfer in the BTX pole cup of 4mm, the bowl cover that closes, puts into slot electrode.Electricity turns condition: 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 1 time.
6. after transfection, recover.Softly remove the electricity raw cell debris of changing the line of production, remaining part is transferred in the nutrient solution of preheating, put back in the constant temperature CO2gas incubator of 37 DEG C.After 18h～48h, detect, or the strain of screening stable transfected cells.
7. according to above step, transfection CEM × 174 cell is that those skilled in the art can realize.The method of transfection CEM × 174 cell of this experimental development, based on the ultimate principle of electroporation transfection cell, used the perfect medium of preheating as damping fluid in whole process, be cell under gentle condition, improved electricity and turned the survival rate of rear cell.In the condition and range that can produce at electroporation apparatus, carry out careful screening simultaneously, optimize the condition that electricity turns, under 210v, the impulse stimulation of 30ms can ensure that the opening of cell hole does not affect the survival of cell, can ensure again time enough space and allow foreign DNA can effectively enter cell.Whole transfection process is simple to operation, and has broken through difficult this technology barrier of transfection of CEM × 174 cells, makes transfection efficiency have very large raising, for follow-up research and application are laid a good foundation.
Brief description of the drawings
Fig. 1 uses the method for the invention transfection pEGFP-N1 plasmid in CEMx174, after 24h under fluorescent microscope observations.
Fig. 2 uses lipofectamine Lipofetmiane2000 (Invitrogen) transfection pEGFP-N1 plasmid in CEMx174, after 24h under fluorescent microscope observations.
Fig. 3 uses non-lipofectamine MegaTran1.0 (Origene) transfection pEGFP-N1 plasmid in CEMx174, after 24h under fluorescent microscope observations.
Fig. 4 is using on the basis of the described method of invention, is using respectively preheating increase serum, and preheating not increase serum and not preheating of increase serum nutrient solution turns damping fluid and carry out the result of electrotransfection as electricity.
Fig. 5 is using on invention described method basis, uses respectively the plasmid of different amounts to carry out the result of electrotransfection, is respectively from left to right 3 μ g, 6 μ g, 9 μ g, 14.1 μ g, 30 μ g.
Embodiment
Embodiment 1: with three kinds of different methods transfection pEGFP-N1 plasmid in CEMx174 clone
1. to invent described method transfection.
1) cell cultures (containing 10% serum) in RPMI-1640 complete culture solution, with 1 × 10
5～2 × 10
5starting point concentration go down to posterity, after going down to posterity at 37 DEG C of CO
2in incubator, cultivate after 24h, under the good condition of cell state, test for electrotransfection.
2) get 2ml RPMI-1640 complete culture solution (containing 10% serum) and add in six orifice plates, separately get a 1.5ml ep pipe, add wherein (containing 10% serum) in 1ml RPMI-1640 complete culture solution.Six orifice plates and ep pipe are put into the CO of 37 DEG C
2preheating in incubator.
3) after putting the nutrient solution that needs preheating well, take out the logarithmic phase cell of cultivating, be collected together, softly mix, take out 10 μ l cell culture fluid countings, measure appropriate cell suspension according to count results, make its total cell concentration reach 3 × 10
6, the centrifugal 10min of 800rpm (90g), supernatant exhausts as far as possible.Take out 1.5ml ep pipe, with the nutrient solution 250 μ l re-suspended cells of preheating in pipe, make cell and the mixed final concentration of DNA diluent reach 1 × 10
7.
4) when collecting cell, remove endotoxic transfection level pEGFP-N1 plasmid 14.1 μ g, equally with the dilution of preheating substratum, final volume 50 μ l.(pEGFP-N1 plasmid is 4.7kb)
5) softly mix cell suspension and DNA diluent, transfer in the BTX pole cup of 4mm, the bowl cover that closes, puts into slot electrode.Electricity turns condition: 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 1 time.
6) softly remove the electricity raw cell debris of changing the line of production, remaining part is transferred in the nutrient solution of preheating, put back to 37 DEG C of CO
2in incubator, cultivate 24h, just putting fluorescence microscopy Microscopic observation transfection effect (Fig. 1).Cell survival rate is about 80%, and after transfection, the cell of successful expression target protein is greater than 70%.
2. use Lipofetmiane2000 (Invitrogen) reagent to carry out transfection
1) cell cultures (containing 10% serum) in RPMI-1640 complete culture solution, with 1 × 10
5～2 × 10
5starting point concentration go down to posterity, after going down to posterity at 37 DEG C of CO
2in incubator, cultivate after 24h, under the good condition of cell state for transfection.
2) take out the logarithmic phase cell of cultivating, the centrifugal 10min of 800rpm (90g) is collected together, softly resuspended with the complete culture solution of a certain amount of preheating, takes out 10ul cell culture fluid counting, 4-8 × 10, every hole before preparation transfection liquid
5individual cell is inoculated in 500 μ l not containing (with 24 orifice plate transfections) in antibiotic perfect medium.
3) with 50 μ l Opti-MEM serum free medium dilution plasmid DNA 0.8ug, mix gently.
4) before use, shake up gently Lipofectamine2000, then get appropriate 2ul Lipofectamine2000 and dilute in 50 μ l Opti-MEM substratum, incubated at room 5 minutes.
5) DNA first two steps being diluted and Lipofectamine2000 mix (make cumulative volume be 100 μ l), mix gently, room temperature is placed 20 minutes (muddiness can appear in solution).
6) in every porocyte, add 100 μ l transfection liquid, shake up gently.
7) 37 DEG C of cultivations were just put fluorescence microscopy Microscopic observation transfection effect after 24 hours, and cell survival rate is less than 10%, and after transfection, the cell of successful expression target protein is less than 50% (Fig. 2).After optimizing transfection conditions according to manufacturer's specification sheets, test, after transfection, successful expression rate is still less than 50%, and no longer accompanying drawing repeats.
3. use a kind of non-liposome polymkeric substance transfection reagent MegaTran1.0 (Origene) to carry out transfection:
1) cell cultures (containing 10% serum) in RPMI-1640 complete culture solution, with 1 × 10
5～2 × 10
5starting point concentration go down to posterity, after going down to posterity at 37 DEG C of CO
2in incubator, cultivate after 24h, under the good condition of cell state for transfection.
2) take out the logarithmic phase cell of cultivating, the centrifugal 10min of 800rpm (90g) is collected together, softly resuspended with the complete culture solution of a certain amount of preheating, takes out 10ul cell culture fluid counting, every hole 5 × 10 before preparation transfection liquid
5individual cell is inoculated in 900 μ l not containing (with 24 orifice plate transfections) in antibiotic perfect medium.
3) with 100 μ l Opti-MEM serum free medium dilution plasmid DNA 1ug, mix gently.Add 3ul MegaTran1.0, mix immediately 10s, incubated at room 10 minutes.
4) in every hole, add the transfection liquid of above-mentioned 100 μ l, shake up gently.
5) 37 DEG C of cultivations were just put fluorescence microscopy Microscopic observation transfection effect after 24 hours, and cell survival rate is less than 30%, and after transfection, the cell of successful expression target protein is less than 50% (Fig. 3).After optimizing transfection conditions according to manufacturer's specification sheets, test, after transfection, successful expression rate is still less than 50%, and no longer accompanying drawing repeats.
Embodiment 2:pEGFP-N1 plasmid electrotransfection CEMx174 clone.
1. cell cultures (containing 10% serum) in RPMI-1640 complete culture solution, with 1 × 10
5～2 × 10
5starting point concentration go down to posterity, after going down to posterity at 37 DEG C of CO
2in incubator, cultivate after 24h, under the good condition of cell state, test for electrotransfection.
2. get the Tissue Culture Dish of three 35mm, each 2ml RPMI-1640 complete culture solution (containing 10% serum) that adds, puts into the CO of 37 DEG C for two
2preheating in incubator, another room temperature is placed.
3. three 1.5ml ep pipes of another taking-up, two add (containing 10% serum) in 1ml RPMI-1640 complete culture solution wherein, and another adds the not RPMI-1640 nutrient solution of increase serum.Two ep pipes (increase serum, does not add) are put into the CO of 37 DEG C
2preheating in incubator, the ep pipe room temperature of another increase serum is placed.
4. after putting nutrient solution well, take out the logarithmic phase cell of cultivating, be collected together, softly mix, take out 10ul cell culture fluid counting, measure three parts of cell suspensions according to count results, make its separately total cell concentration reach 3 × 10
6, the centrifugal 10min of 800rpm (90g), supernatant exhausts as far as possible.Take out three 1.5ml ep pipes, use respectively preheating increase serum in pipe, preheating is increase serum and the nutrient solution 250ul re-suspended cell without preheating not, makes cell and the mixed final concentration of DNA diluent reach 1 × 10
7.
5. when collecting cell, remove endotoxic transfection level pEGFP-N1 plasmid 14.1 μ g for every part, respectively with above-mentioned three kinds of nutrient solutions dilution, final volume 50 μ l.(pEGFP-N1 plasmid is 4.7kb)
6. difference corresponding mixed cell suspension and DNA diluent one by one, softly mixes, and transfers in the BTX pole cup of 4mm, and the bowl cover that closes, puts into slot electrode.Electricity turns condition: 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 1 time.
7. softly remove the electricity raw cell debris of changing the line of production, remaining part is transferred in the nutrient solution of preheating, wherein use a group of preheating damping fluid not to add not in preheating nutrient solution, put back to together 37 DEG C of CO
2in incubator, cultivate 24h, just putting fluorescence microscopy Microscopic observation transfection effect.Result demonstration, it is the highest that the increase serum nutrient solution of the described preheating of use invention turns damping fluid transfection efficiency as electricity, and cell mortality is low; Use the nutrient solution of increase serum not or without the nutrient solution of preheating as damping fluid, cell mortality is all apparently higher than the former, and transfection efficiency is low, result is (Fig. 4) as shown in Figure of description.
Embodiment 3:pEGFP-N1 plasmid electrotransfection CEMx174 clone.
1. cell cultures (containing 10% serum) in RPMI-1640 complete culture solution, with 1 × 10
5～2 × 10
5starting point concentration go down to posterity, after going down to posterity at 37 DEG C of CO
2in incubator, cultivate after 24h, under the good condition of cell state, test for electrotransfection.
2. getting 2ml RPMI-1640 complete culture solution (containing 10% serum) adds in six orifice plates, 32 holes altogether, separately get (containing 10% serum) in 10ml RPMI-1640 complete culture solution and, for dilution DNA and re-suspended cell, above-mentioned nutrient solution is put into the CO of 37 DEG C
2preheating in incubator.
3. after putting the nutrient solution that needs preheating well, take out the logarithmic phase cell of cultivating, be collected together, softly mix, take out 10 μ l cell culture fluid countings, measure appropriate cell suspension according to count results, make every part of cell total amount reach 3 × 10
6, altogether prepare 32 parts of cells, the centrifugal 10min of 800rpm (90g) respectively, supernatant exhausts as far as possible.Every part of 250 μ l of the nutrient solution re-suspended cell respectively that takes out preheating, makes cell and the mixed final concentration of DNA diluent reach 1 × 10
7.
4. when collecting cell, remove endotoxic transfection level pEGFP-N1 plasmid 14.1 μ g for every part, equally with the dilution of preheating substratum, final volume 50 μ l.(pEGFP-N1 plasmid is 4.7kb)
5. softly mix cell suspension and DNA diluent, transfer in the BTX pole cup of different size, the bowl cover that closes, puts into slot electrode.Carrying out electricity according to different electric shock conditions turns:
1) 4mm pole cup, voltage 110v, pulse length 10ms, pulse number 1 time.
2) 4mm pole cup, voltage 210v, pulse length 10ms, pulse number 1 time.
3) 4mm pole cup, voltage 310v, pulse length 10ms, pulse number 1 time.
4) 4mm pole cup, voltage 410v, pulse length 10ms, pulse number 1 time.
5) 4mm pole cup, voltage 110v, pulse length 20ms, pulse number 1 time.
6) 4mm pole cup, voltage 210v, pulse length 20ms, pulse number 1 time.
7) 4mm pole cup, voltage 310v, pulse length 20ms, pulse number 1 time.
8) 4mm pole cup, voltage 410v, pulse length 20ms, pulse number 1 time.
9) 4mm pole cup, voltage 110v, pulse length 30ms, pulse number 1 time.
10) 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 1 time.
11) 4mm pole cup, voltage 310v, pulse length 30ms, pulse number 1 time.
12) 4mm pole cup, voltage 410v, pulse length 30ms, pulse number 1 time.
13) 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 1 time.
14) 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 2 times.
15) 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 3 times.
16) 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 4 times.
17) 2mm pole cup, voltage 110v, pulse length 10ms, pulse number 1 time.
18) 2mm pole cup, voltage 210v, pulse length 10ms, pulse number 1 time.
19) 2mm pole cup, voltage 310v, pulse length 10ms, pulse number 1 time.
20) 2mm pole cup, voltage 410v, pulse length 10ms, pulse number 1 time.
21) 2mm pole cup, voltage 110v, pulse length 20ms, pulse number 1 time.
22) 2mm pole cup, voltage 210v, pulse length 20ms, pulse number 1 time.
23) 2mm pole cup, voltage 310v, pulse length 20ms, pulse number 1 time.
24) 2mm pole cup, voltage 410v, pulse length 20ms, pulse number 1 time.
25) 2mm pole cup, voltage 110v, pulse length 30ms, pulse number 1 time.
26) 2mm pole cup, voltage 210v, pulse length 30ms, pulse number 1 time.
27) 2mm pole cup, voltage 310v, pulse length 30ms, pulse number 1 time.
28) 2mm pole cup, voltage 410v, pulse length 30ms, pulse number 1 time.
29) 2mm pole cup, voltage 210v, pulse length 30ms, pulse number 1 time.
30) 2mm pole cup, voltage 210v, pulse length 30ms, pulse number 2 times.
31) 2mm pole cup, voltage 210v, pulse length 30ms, pulse number 3 times.
32) 2mm pole cup, voltage 210v, pulse length 30ms, pulse number 4 times.
6. softly remove the electricity raw cell debris of changing the line of production, remaining part is transferred in the nutrient solution of preheating, put back to 37 DEG C of CO
2in incubator, cultivate 24h, just putting fluorescence microscopy Microscopic observation transfection effect.Result demonstration, condition of the present invention has the transfection efficiency that is obviously better than other conditions in ensureing enough cell survival rates.Concrete outcome is too numerous and jumbled is not listed as figure explanation.
Embodiment 4:pEGFP-N1 plasmid electrotransfection CEMx174 clone.
1. cell cultures (containing 10% serum) in RPMI-1640 complete culture solution, with 1 × 10
5～2 × 10
5starting point concentration go down to posterity, after going down to posterity at 37 DEG C of CO
2in incubator, cultivate after 24h, under the good condition of cell state, test for electrotransfection.
2. get 2ml RPMI-1640 complete culture solution (containing 10% serum) and add in six orifice plates, totally five holes, separately get a 1.5ml ep pipe, add wherein (containing 10% serum) in 1.5ml RPMI-1640 complete culture solution.Six orifice plates and ep pipe are put into the CO of 37 DEG C
2preheating in incubator.
3. after putting nutrient solution well, take out the logarithmic phase cell of cultivating, be collected together, softly mix, take out 10ul cell culture fluid counting, measure cell suspension according to count results, make every part of cell total amount reach 3 × 10
6, the centrifugal 10min of 800rpm (90g), supernatant exhausts as far as possible.Take out five 1.5ml ep pipes, use respectively preheating increase serum nutrient solution 250u1 re-suspended cell in pipe, make cell and the mixed final concentration of DNA diluent reach 1 × 10
7.
4. when collecting cell, get the endotoxic transfection level pEGFP-N1 plasmid that goes of 5 groups of different concns, with above-mentioned nutrient solution dilution, final volume 50 μ l.(pEGFP-N1 plasmid is 4.7kb) five groups got respectively: 3 μ g, 6 μ g, 9 μ g, 14.1 μ g, 30 μ g.
5. softly mix respectively cell suspension and DNA diluent, transfer in the BTX pole cup of 4mm, the bowl cover that closes, puts into slot electrode.Electricity turns condition: 4mm pole cup, voltage 210v, pulse length 30ms, pulse number 1 time.
6. softly remove the electricity raw cell debris of changing the line of production, remaining part is transferred in the nutrient solution of preheating, wherein use a group of preheating damping fluid not to add not in preheating nutrient solution, put back to together 37 DEG C of CO
2in incubator, cultivate 24h, just putting fluorescence microscopy Microscopic observation transfection effect.Result shows, uses the plasmid amount of ratio of the present invention can reach best transfection effect; After the transfection of use 30ug plasmid (being 100ug/ml), the cell proportion of expressing green fluorescent protein is not significantly increased, but cell mortality increases; Use the plasmid amount transfection efficiency of other ratios to be starkly lower than the described experimental group of invention, result is (Fig. 5) as shown in Figure of description.
Claims (7)
1. a method for transfection suspension cell, it is characterized in that taking preheating containing the RPMI-1640 cell culture medium of 10% serum as electroporation damping fluid, cell mixing mixes with foreign DNA, then electricity consumption conversion system carries out electrotransfection, in foreign DNA transfered cell.
2. method according to claim 1, is characterized in that described electrotransfection damping fluid is not containing microbiotic.
3. method according to claim 1, is characterized in that described preheating environment is 37 DEG C of constant temperature cell culture incubators.
4. the method for transfected with human CEM × 174 clone, CEM × 174 clone behaviour T, B lymphoblast hybridization system, it is characterized in that, taking preheating containing the RPMI-1640 cell culture medium of 10% serum as electroporation damping fluid, cell is mixed with foreign DNA, and then electricity consumption conversion system carries out electrotransfection, in foreign DNA transfered cell, preheating temperature is 37 DEG C, and described cell culture medium is not containing microbiotic.
5. according to the arbitrary described method of claim 1-4, it is characterized in that it is that middle cell concn is 5 × 10 that electricity is turned
6～1 × 10
7individual cell/ml.
6. method according to claim 5, is characterized in that electrotransfection condition is, in 4mm pole cup with 210mV pulse electric shock once, time length 30ms.
7. method according to claim 6, is characterized in that in transfection process cell concn: plasmid lineal measure: plasmid weight ratio is 10
6: 1kb: 1 μ g.
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| CN105154472A (en) * | 2015-09-28 | 2015-12-16 | 重庆高圣生物医药有限责任公司 | Mammalian cell efficient electrotransfection buffer solution and preparation method thereof |
| CN111690685A (en) * | 2020-06-29 | 2020-09-22 | 深圳赛桥生物创新技术有限公司 | Method for improving transfection efficiency of electroporation cell |
| CN112824530A (en) * | 2019-11-20 | 2021-05-21 | 中国科学院深圳先进技术研究院 | HEK293F suspension cell high-efficiency electrotransfection method |
| CN116042713A (en) * | 2022-12-30 | 2023-05-02 | 四川阿思科力生物科技有限公司 | Electrotransfection method of NK-92 cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105154472A (en) * | 2015-09-28 | 2015-12-16 | 重庆高圣生物医药有限责任公司 | Mammalian cell efficient electrotransfection buffer solution and preparation method thereof |
| CN105154472B (en) * | 2015-09-28 | 2020-01-07 | 重庆高圣生物医药有限责任公司 | High-efficiency electrotransfection buffer solution for mammalian cells and preparation method thereof |
| CN112824530A (en) * | 2019-11-20 | 2021-05-21 | 中国科学院深圳先进技术研究院 | HEK293F suspension cell high-efficiency electrotransfection method |
| CN111690685A (en) * | 2020-06-29 | 2020-09-22 | 深圳赛桥生物创新技术有限公司 | Method for improving transfection efficiency of electroporation cell |
| CN116042713A (en) * | 2022-12-30 | 2023-05-02 | 四川阿思科力生物科技有限公司 | Electrotransfection method of NK-92 cells |
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