CN1687427A - Gene transduction method with nano granule as carrier based on ultrasonic intervention guiding - Google Patents
Gene transduction method with nano granule as carrier based on ultrasonic intervention guiding Download PDFInfo
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- CN1687427A CN1687427A CNA2005100313971A CN200510031397A CN1687427A CN 1687427 A CN1687427 A CN 1687427A CN A2005100313971 A CNA2005100313971 A CN A2005100313971A CN 200510031397 A CN200510031397 A CN 200510031397A CN 1687427 A CN1687427 A CN 1687427A
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Abstract
A gene transduction method ultrasonic-based, which using the nano particulate as the carrier. It consists of animal transgenic gene method and plant transgenic gene method. The outer gene joins with the particulate with the effect of static form together to form the gene carrier, which can protect the DNA being broken by ultrasonic. The ultrasonic synchronously give effect to particulate gene carrier, cells, organize and apparatus, the carrier can run efficiently through the short period alleyway of cell wall, cell film, core film cell, enables the outer gene constructs with the gene group in cell. This method has a high efficiency, and is provided with convenient manipulation, high efficiency, no request of differential function e.g. other merits. It can be applied widely to these range, animal and plant transgenic technology and genic cure of human being.
Description
Technical field
The present invention relates to fields such as biotechnology, nanobiology and the application of biological nano material, refer in particular to a kind of animal and plant transgenic method of making genophore with nano particle.
Background technology
Nano particle is made genophore and obtained success in the somatic gene transfer of animal and human, Chang Yong genophore relatively, advantages such as having bioaffinity height, good stability, be easy to control, target is strong shows great application prospect at aspects such as gene transfer and gene therapies.But when this nano particle gene carrier imports animal recipient cell with foreign gene; still there is the challenge of following aspect: when gene-nano-particle complex and cell carry out common cultivation; it is many that the barrier of cytolemma makes exogenous origin gene integrator arrive the middle-chain of cellular genome; the nano particle that makes major part carry DNA is difficult to directly enter cell; be difficult to directly to take DNA and be diffused on the nuclear membrane, and pass Nuclear pore or in fission process, enter nucleus with expression alien gene.Therefore, seek the novel method that improves the nano-gene carrier transduction efficiency and still become the task of top priority.Studies show that in the past, the instant channel that ultrasonication can make cell walls, cytolemma produce down to nuclear membrane, foreign DNA can directly enter cell whereby and express, yet, dna direct is when ultrasonic-mediated, and ultrasonic wave has very big destruction to foreign DNA.Thereby the researchist seeking always new method make foreign DNA more direct, enter recipient cell effectively.
Its transduction efficiency of traditional plant transgenic method is lower, because plant cell wall has stoped foreign gene to enter cell.In plant transgenic technology, two kinds of transgenic methods of main now use: particle bombardment and agrobacterium-mediated transformation, but the particle bombardment transduction efficiency is lower, funds are higher, and agrobacterium-mediated transformation is mainly used in the conversion of dicotyledons, uses less in monocotyledonous conversion.Can make genophore with nano particle, under hyperacoustic mediation, change foreign gene over to vegetable cell, overcome the deficiency of these two kinds of methods? never the someone carried out research in the past.For solving this difficult problem, we change foreign gene over to vegetable cell by the nano particle carrier down ultrasonic-mediated at imagination, because ultrasonication can make cell walls, cytolemma, nuclear membrane produce instant channel, and the size of nano particle carrier less (10-100nm) can easily be passed through these passages.Can but ultrasonic wave have destruction to the DNA on the nano particle gene carrier?
Summary of the invention
Above-mentioned puzzlement at prior art; the present invention is by selecting ultrasonication; being combined in DNA on the nano particle is not destroyed and is subjected to effective protection; nano particle gene carrier has significantly improved the genetic transformation efficiency of zooblast, thereby has set up the transgenic method of animal efficiently of making genophore based on nano particle under the ultrasonic wave.Another important content of the present invention is the gene transfer that first nano particle gene carrier is applied to vegetable cell, simultaneously in conjunction with ultrasonication, has set up a kind of brand-new plant transgenic method of making genophore based on nano particle under the ultrasonic wave.
One of purpose of the present invention aims to provide a kind of method that can effectively improve nano particle gene carrier transgene efficiency in zooblast.It is transgenic method in the vegetable cell of genophore with the nano particle that another object of the present invention aims to provide a kind of, first the nano particle of biocompatibility is applied to the gene transformation of plant.That the inventive method has is easy and simple to handle, transduction efficiency is high, do not required advantages such as recipient cell specificity, can be widely used in fields such as animal and plant transgenic technology and human gene therapy.Transgenic method of the present invention yet there are no relevant report.
The objective of the invention is to realize by following manner:
Nano particle is combined with goal gene; formed the gene-nano-particle complex of zooblast; promptly made up nano particle gene carrier; in animal recipient cell, add this gene-nano-particle complex; and with ultrasonic-mediated; by ultrasonic wave gene-nano-particle complex is imported animal recipient cell, realize the importing of goal gene.
The bonding state of DNA and nano particle in the mixture after electrophoretic analysis is handled is found the ultrasonic wave destruction that nano particle can effectively protect DNA to prevent certain intensity, the DNA in the eluting cmp, and its biological property does not change; With the ultrasonication zooblast of varying strength, detect the surviving rate of cell with mtt assay, with reference to the finally selected cell survival rate height of the ultrasonication intensity of DNA protection, ultrasonic intensity that the DNA protectiveness is good.The present invention is owing to selected the ultrasonic wave aided nano particle of certain intensity to enter zooblast; nano particle has good protective action to DNA again; so, not compare with there being the auxiliary nano particle transduction of ultrasonic wave method, the inventive method has obviously improved the gene transfer efficient of zooblast.
Another object of the present invention realizes by following manner:
The goal gene that needs are changed in the vegetable cell combines with nano particle; made up the gene-nano particle gene carrier of vegetable cell; in the plant recipient cell, add this gene-nano-particle complex; and with ultrasonic-mediated; by ultrasonic wave gene-nano-particle complex is imported the plant recipient cell, realize the importing of goal gene.
Poly-lysine starch nanometer granule and plant suspension cell that the ruthenium pyridine is modified are cultivated altogether, with this mixing solutions of ultrasonication, cell after wash-out is handled, observe fluorescent nano particle and entered vegetable cell under fluorescent microscope, this cell still can carry out normal merisis when succeeding transfer culture.The present invention has set up a kind of method that nano particle gene carrier is changed over to vegetable cell first.
The goal gene (pEGAD gene) that needs are changed in the vegetable cell combines with nano particle; made up the nano particle gene carrier of vegetable cell; then; add vegetable cell suspension; mix,, in fresh culture, cultivate with this mixing solutions of ultrasonication; observe under fluorescent microscope, most of cell sends the intensive green fluorescence.Presentation of results nano particle gene carrier has successfully changed the pEGAD gene over to vegetable cell, and realizes egfp expression in cell.Therefore, we have set up first with the nano particle is the plant transgenic method of genophore, has filled up the blank that nano particle is made the plant transgene carrier.
Describedly ultrasonic-mediatedly be meant that this ultrasonic wave acts on gene-nano-particle complex and biomass cells or tissue or organ simultaneously.
The nano particle of indication of the present invention be meant can be effectively in conjunction with the nano particle of DNA.Described nano particle is for modifying through amination or modifying through polylysine modification or other biological.
Described goal gene is dna segment or RNAi or sense-rna, and its bonded gene can be a kind of gene or be several genes simultaneously.The copy number of gene is regulated by the mass ratio of control DNA or RNA and nano particle.
Described nano particle is in conjunction with the amount of DNA, according to needing the number of cell transformed artificially to regulate.
Described ultrasonic wave is produced by ultrasonic generator.
Producing hyperacoustic method comprises by solution and is transmitted to the mixed solution of nano particle and recipient cell or directly acts on this solution or tissue or organ by ultrasonic probe.
The present invention combines by electrostatic interaction foreign gene and is built into genophore with nano particle, can prevent effectively that ultrasonic wave is to the destruction in conjunction with DNA; Ultrasonic wave acts on nano particle gene carrier and cell, tissue or organ simultaneously; make nano particle gene carrier can pass the instant channel that produces on cell walls, cytolemma and the nuclear membrane effectively; realize that foreign gene is integrated directly in the cellular genome, significantly improved gene transfer efficient.
Advantage of the present invention is:
One. the ultrasonic generator low price that the present invention is used, operation is very easy, is easy to the popularization of user's acceptance and grasp and this method.
Two. the present invention has not required the recipient cell specificity, can be widely used in animal, plant transgenic technology and human gene therapy.
Three. the present invention makes genophore with nano particle, under hyperacoustic mediation, makes foreign gene directly pass cell walls, cytolemma and nuclear membrane, is incorporated into cellular genome, has significantly improved genetic transformation efficiency.Nano particle gene carrier effectively protection prevents hyperacoustic destruction with its bonded DNA or RNA, and this provide protection is to be caused by the volume effect of nano particle and locus effect.
Four. the present invention is by the amount of control with nano particle bonded DNA, thus the copy number of the foreign gene that effective control changes over to, and in other transgenic method, it is at random that foreign gene enters cell, can not control the copy number of transgene.
Five. the present invention can will load heterogeneic nano particle gene carrier simultaneously; under hyperacoustic mediation, directly change cell over to; avoided vector construction process loaded down with trivial details in the existing method, thereby made and in same cell, once change a plurality of genes over to and become simple and easy to do.
Six. the present invention can directly apply in the organism or external cell, tissue and organ, applied range.
Description of drawings
Accompanying drawing 1 effectively protects DNA to prevent hyperacoustic destruction for embodiments of the invention 1 nano particle;
Accompanying drawing 2 changes foreign gene over to zooblast and genetic expression for the embodiment of the invention 2 based on nano particle gene carrier under the ultrasonic wave; Can effectively change external source GFP gene over to acceptor COS-7 cell based on ultrasonic-mediated nano particle gene carrier, and can obtain efficiently expressing green fluorescent protein.
A figure optical imagery B figure fluorescence imaging (200 *)
Accompanying drawing 3 changes vegetable cell for embodiments of the invention 3 over to based on fluorescent nano particle under the ultrasonic wave; The nano particle that the ruthenium pyridine is modified enters arabidopsis cell under hyperacoustic mediation
A figure optical imagery B figure fluorescence imaging (400 *)
Accompanying drawing 4 changes foreign gene over to vegetable cell and genetic expression for embodiments of the invention 4 based on nano particle gene carrier under the ultrasonic wave.Ultrasonic-mediated nano particle changes the GFP gene over to arabidopsis cell
A figure optical imagery B figure fluorescence imaging (200 *)
Embodiment:
Following examples are intended to illustrate the present invention rather than the present invention are limited.
Embodiment 1:
Effectively protect the ultrasonic processing method of nano particle DNA
1, ultrasonication DNA-nano-particle complex.Get the centrifuge tube of six 150 μ l, in centrifuge tube, add nanoparticles solution respectively, add plasmid DNA solution again, mixing, after room temperature left standstill 30min, experiment divided two groups, first group: get three centrifuge tubes, on ultrasonic generator, under the intensity of 120W, 40KHz, respectively through ultrasonication 30min, 20min, 10min; Second group: the sample in three centrifuge tubes through and first group of identical processing;
2, in second group of sample, add NaOH solution, shake up, by the method for extracting DNA, the DNA in eluted dna-nano-particle complex;
3, ultrasonication DNA.In the centrifuge tube of one 150 μ l, add plasmid DNA, on ultrasonic generator, under the intensity of 120W, 40KHz, ultrasonication 5min;
4, from every pipe, take a sample, add gel point sample hole, carry out electrophoretic analysis.Be not protected with nano particle bonded DNA and destroyed, and do not have combining nano particulate naked DNA to be destroyed by ultrasonic wave by ultrasonic wave.Experimental result is seen accompanying drawing 1.Nano particle is combined with DNA, and 1% gel electrophoresis finds that nano particle has very strong DNA accumulation ability (the 2nd road among Fig. 1).After the DNA-nano-particle complex passes through ultrasonication 10～30min respectively, the electrophoretic band identical (the 2nd road among Fig. 1) of its electrophoretic band (the 3rd～5 road among Fig. 1) and the mixture that does not carry out ultrasonication, to elute through the DNA in the DNA-nano-particle complex of ultrasonication and investigate (the 6th～8 road among Fig. 1), find identical with control plasmid DNA band (the 1st road among Fig. 1) with gel electrophoresis.Show that ultrasonication does not influence the combination of nano particle to DNA, nano particle has protected DNA to prevent hyperacoustic destruction.And not with nano particle bonded DNA behind ultrasonication 5min, electrophoresis finds that track does not have tangible band and is related shape (the 9th road among Fig. 1), shows that DNA is destroyed by ultrasonic wave.
Embodiment 2:
Based on the gene transfer of zooblast efficiently of making genophore under the ultrasonic wave with the poly-lysine starch nanometer granule
1, gets the COS-7 cell inoculation in six well culture plates, at 5%CO
2, 37 ℃ of conditions incubator in cultivate, after the cell fraction of coverage reaches 50%～70%, outwell substratum, behind D-Hanks liquid washed cell, use trysinization 5min, add fresh culture and make cell suspending liquid, under different ultrasonic intensities, handle cell suspending liquid, detect the surviving rate of cell with mtt assay.The results are shown in Table 1, thereby, select 120W, handle 2min under the 40KHz intensity as optimal treatment condition.
Different ultrasonic intensities of table 1 and treatment time are to the influence of COS-7 cell survival rate
A)
| The ultrasonic intensity treatment time | ????15min | ??10min | ??5min | ??2min | ??1min |
| ????120W、40KHz ????180W、40KHz ????300W、40KHz | ????10.6％ ????0％ ????0％ | ??49.8％ ??11.4％ ??0％ | ??95.7％ ??51.3％ ??10.8％ | ??98.1％ ??97.6％ ??65.5％ | ??98.6％ ??98.2％ ??98.1％ |
A) on microplate reader, detect the surviving rate of COS-7 cell with mtt assay.
2, by 1 method, with trysinization 5min, and added in the cell suspending liquid of fresh culture and added pIRGFP plasmid DNA-nanoparticles solution, ultrasonication 2min, cultivate 24h after, fluorescent microscope is observed down;
3, control experiment is designed to: the substratum that adds no calf serum in the cell suspending liquid after trysinization, add pIRGFP plasmid DNA-nanoparticles solution again, carry out common cultivation with cell, behind the 6h, replace with the perfect medium that contains calf serum, after cultivating 24h, fluorescent microscope is observed down.Through the counting statistics analysis; the resultant gene transfer rate of gene transfer method that the present invention sets up is about 70%; and contrast is not about 40% through the gene transfer rate of ultrasonic-mediated nano particle carrier; the result shows, can significantly improve the gene transfer rate of poly-lysine starch nanometer granule genophore by action of ultrasonic waves.Experimental result is seen accompanying drawing 2.Can effectively change external source GFP gene over to acceptor COS-7 cell based on ultrasonic-mediated nano particle gene carrier, and can obtain efficiently expressing green fluorescent protein.A figure is fluorescence imaging (200 *) for optical imagery B figure
Embodiment 3:
Change vegetable cell over to based on fluorescent nano particle under the ultrasonic wave
1, the foundation of Arabidopis thaliana suspension cell line: well-grown Arabidopis thaliana callus is transferred in the fresh culture of MS+BA0.1mg/l+NAA 0.5mg/l.After one week, select the topmost callus and change in the liquid nutrient medium, camera bellows was cultivated 48 hours on the shaking table of 110rpm;
2, get above-mentioned solution 1ml, add the poly-lysine starch nanometer granule that the ruthenium pyridine is modified, mix on miniature oscillator, at 120W, the ultrasonic intensity of 40KHz is handled 5min down;
3, with cell suspending liquid centrifugal 1min under the 500rpm/min rotating speed of above-mentioned processing, inhale and remove supernatant liquor, wash suspension cell repeatedly with the MS liquid nutrient medium, centrifugal again, remove supernatant liquor again, so repeat 5 times;
4, suspension cell camera bellows on the shaking table of 110rpm of above-mentioned processing was cultivated 48 hours;
5, observe under fluorescent microscope, fluorescent nano particle has entered arabidopsis cell, and, through succeeding transfer culture, these cells can continue merisis, illustrate under hyperacoustic mediation, nano particle can effectively enter vegetable cell, and nano particle enter the growth that does not influence cell.Experimental result is seen accompanying drawing 3.Ultrasonic-mediated nano particle changes the GFP gene over to arabidopsis cell
A figure is fluorescence imaging (200 *) for optical imagery B figure
Make the vegetable cell gene transfer of genophore based on nano particle under the ultrasonic wave
1, the structure of nano particle gene carrier: get the nano particle aqueous suspensions that 10 μ l concentration are 1 μ g/ μ l, add 3 μ gpEGAD plasmid DNA, mix, room temperature was placed 30 minutes, obtained the DNA-nano-particle complex;
2, get arabidopsis cell suspension 1ml among the embodiment 3, add the DNA-nano-particle complex in 1, mix on miniature oscillator, at 120W, the ultrasonic intensity of 40KHz is handled 5min down;
3, with cell suspending liquid centrifugal 1min under the 500rpm/min rotating speed of above-mentioned processing, inhale and remove supernatant liquor, wash suspension cell repeatedly with the MS liquid nutrient medium, centrifugal again, remove supernatant liquor again, so repeat 5 times;
4, suspension cell camera bellows on the shaking table of 110rpm of above-mentioned processing was cultivated 48 hours;
5, under fluorescent microscope, observe, the arabidopsis cell high level expression green fluorescent protein, the used transgenic method transduction efficiency of the present invention is about 20%-30%, is higher than conventional transgenic method.Experimental result is seen accompanying drawing 4.
Claims (10)
1, based on the transgenic method of ultrasonic-mediated nano granule as carrier; it is characterized in that: nano particle is combined with goal gene; formed gene-nano-particle complex; promptly made up nano particle gene carrier; in animal recipient cell, add this gene-nano-particle complex; and, gene-nano-particle complex is imported animal recipient cell, thereby realize the importing of goal gene with ultrasonic-mediated.
2, based on the transgenic method of ultrasonic-mediated nano granule as carrier; it is characterized in that: the goal gene that needs are changed in the vegetable cell combines with nano particle; made up gene-nano particle gene carrier; then; adding vegetable cell mixes; and, gene-nano-particle complex is imported the plant recipient cell, thereby realize the importing of goal gene with ultrasonic-mediated.
3, the transgenic method based on ultrasonic-mediated nano granule as carrier according to claim 1 and 2, it is characterized in that: described goal gene can be DNA, also can be RNAi or sense-rna, its bonded gene can be a kind of gene or is several genes simultaneously.
4, the transgenic method based on ultrasonic-mediated nano granule as carrier according to claim 1 and 2 is characterized in that: ultrasonic-mediatedly be meant that this ultrasonic wave acts on gene-nano-particle complex and biomass cells or tissue or organ simultaneously.
5, the transgenic method based on ultrasonic-mediated nano granule as carrier according to claim 4 is characterized in that: produce hyperacoustic method and comprise by solution and be transmitted to the mixed solution of nano particle and recipient cell or directly act on this solution or tissue or organ by ultrasonic probe.
6, the transgenic method based on ultrasonic-mediated nano granule as carrier according to claim 1 and 2 is characterized in that: nano particle is meant through biology and modifies, and can be effectively in conjunction with the nano particle of DNA.
7, the transgenic method based on ultrasonic-mediated nano granule as carrier according to claim 6 is characterized in that: the biology of nano particle is modified and is mainly amination modification or polylysine modification.
8, the animal transgenic method based on ultrasonic-mediated nano granule as carrier according to claim 1 is characterized in that: at 120W, the 40KHz ultrasonic intensity is handled the zooblast 2min that adds gene-nano-particle complex down.
9, the plant transgenic method based on ultrasonic-mediated nano granule as carrier according to claim 2, it is characterized in that: behind the poly-lysine starch nanometer granule binding purposes gene with the modification of ruthenium pyridine, cultivate altogether with plant suspension cell, with this mixing solutions of ultrasonication.
10, according to claim 2 or 9 described plant transgenic methods based on ultrasonic-mediated nano granule as carrier, it is characterized in that: at 120W, the 40Hz ultrasonic intensity is handled the vegetable cell 5min that adds gene-nano-particle complex down.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100311168A1 (en) * | 2009-04-07 | 2010-12-09 | Dow Agrosciences Llc | Nanoparticle mediated delivery of sequence specific nucleases |
| CN102260704A (en) * | 2011-06-08 | 2011-11-30 | 吉林农业大学 | Plant transgene method based on taking silicon dioxide nano particles as gene vector |
| US8722410B2 (en) * | 2007-10-05 | 2014-05-13 | Dow Agrosciences, Llc. | Methods for transferring molecular substances into plant cells |
| CN111956947A (en) * | 2019-05-20 | 2020-11-20 | 安行生物技术有限公司 | Method and device for intracellular delivery of nanoparticles |
-
2005
- 2005-03-30 CN CNA2005100313971A patent/CN1687427A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8722410B2 (en) * | 2007-10-05 | 2014-05-13 | Dow Agrosciences, Llc. | Methods for transferring molecular substances into plant cells |
| US9476057B2 (en) | 2007-10-05 | 2016-10-25 | Dow Agrosciences Llc | Methods for transferring molecular substances into plant cells |
| US9719108B2 (en) | 2007-10-05 | 2017-08-01 | Dow Agrosciences Llc | Nanoparticle mediated delivery of sequence specific nucleases |
| US20100311168A1 (en) * | 2009-04-07 | 2010-12-09 | Dow Agrosciences Llc | Nanoparticle mediated delivery of sequence specific nucleases |
| CN102369287A (en) * | 2009-04-07 | 2012-03-07 | 陶氏益农公司 | Nanoparticle mediated delivery of sequence specific nucleases |
| CN102369287B (en) * | 2009-04-07 | 2014-09-17 | 陶氏益农公司 | Nanoparticle mediated delivery of sequence specific nucleases |
| US9187755B2 (en) * | 2009-04-07 | 2015-11-17 | Dow Agrosciences Llc | Nanoparticle mediated delivery of sequence specific nucleases |
| CN102260704A (en) * | 2011-06-08 | 2011-11-30 | 吉林农业大学 | Plant transgene method based on taking silicon dioxide nano particles as gene vector |
| CN111956947A (en) * | 2019-05-20 | 2020-11-20 | 安行生物技术有限公司 | Method and device for intracellular delivery of nanoparticles |
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