CN103233042A - Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism - Google Patents
Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism Download PDFInfo
- Publication number
- CN103233042A CN103233042A CN201310153676XA CN201310153676A CN103233042A CN 103233042 A CN103233042 A CN 103233042A CN 201310153676X A CN201310153676X A CN 201310153676XA CN 201310153676 A CN201310153676 A CN 201310153676A CN 103233042 A CN103233042 A CN 103233042A
- Authority
- CN
- China
- Prior art keywords
- pei
- magnetic nano
- gene
- nanometer
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a magnetic nano-gene vector for transporting a gene, and a preparation technology and application thereof. The invention also provides a method for transforming animal and plant cells by a foreign gene loaded by the magnetic nano-gene vector. In the method, the magnetic nano-gene vector is nano Fe3O4/PEI particles, and enters the animal and plant cells and/or generative cell with the foreign gene under drive of an external magnetic field; genetic transformation and expression are realized; and a new variety with excellent performance can be obtained through transgenosis cultivation. The preparation method and the transformation method disclosed by the invention are simple to operate, easy to control, and convenient to popularize. The transformation method disclosed by the invention is not limited by species; the receptor cell specificity is not required; and the transformation method can be widely applied to a transgenic technology of animals and plants. In the transformation method disclosed by the invention, the transformation efficiency of the gene can be significantly improved; and the copy number of the transferred foreign gene can be effectively controlled by controlling the quantity of the foreign genes combined with the magnetic nano-gene vector.
Description
Technical field
The present invention relates to a kind of genetically modified organism and cultivate the preparation method and application that use the magnetic Nano genophore, this carrier can transform animal and plant cell by mediate foreign gene, can be applicable to the preparation of transgenosis germplasm and rearing new variety.
Background technology
Nanoparticle is because its unique physical and chemical properties has been widely used in field of biology.Nano material has following advantage as genophore: can wrap up, concentrate and protection nucleic acid is avoided the degraded of nuclease; Specific surface area is big, has bioaffinity; Have controlled degradation, can realize the controllable release of gene, prolong action time effectively; Avoided the potentially dangerous of using conventional virus vector to cause.Nanometer Fe
3O
4Except the general advantage that possesses nano material, also has superparamagnetism, under the driving of externally-applied magnetic field, make solid support material to the purpose cell enrichment, improve it significantly as the efficient of genophore, studied widely in fields such as medical science, targeted drug, gene therapy, immunodetection, immobilized enzyme.
In recent years, transgenic technology has obtained remarkable achievement at aspects such as cultivating animals and plants new variety, gene diagnosis and treatment, has caused the concern of various fields researchers such as medical science, agricultural and environment.Good gene vector material is the key link that successfully realizes transgenic technology.At present, non-viral gene vector has overcome the limitations such as immunogenicity of viral genetic vector because of hypotoxicity, reduced immunogenicity and advantage such as easy and simple to handle, becomes the research focus in transgenosis field.Cationic-liposome is widely used as non-viral gene vector, but because it has certain cell-specific, is suppressed reasons such as inactivation easily by serum protein, has also limited its range of application.
At present about nanometer Fe
3O
4Preparation mainly contain coprecipitation method, high-temperature decomposition, microemulsion method and hydrothermal method etc.Though the Fe of high quality, good dispersity that at present a lot of bibliographical informations arranged
3O
4Nanometer particle process method, but this class material all can not directly apply to the zooblast gene transformation mostly, because these Fe
3O
4Bad, the problems such as the carrier capacity is low, magnetic instability of magnetic nano-particle ubiquity bio-compatibility, thereby cause transformation efficiency low.Xu Yuhong has reported " preparation method of the genophore of direct compound PEI " in patent application CN101748150A, it utilizes Fe
3O
4/ PEI is carrier, carry the pGL-3control plasmid transforms the COS-7 cell.
The invention provides a kind of preparation method and application thereof of novel gene vector, this genophore can transform the animal and plant cell by mediate foreign gene.
Summary of the invention
The present invention at first provides a kind of genetically modified organism to cultivate the preparation method who uses the magnetic Nano genophore, comprises steps A, preparation nanometer Fe
3O
4Colloidal sol: be that 1～3:1 takes by weighing molysite and ferrous salt and adds in the dilute acid soln by the ratio of amount of substance, the back is stirred in dissolving makes the pH value of solution value be alkalescence to wherein adding weak caustic solution, continues to be stirred to reaction and finishes, with sour neutralization solution pH value to 6～8; Aforementioned preparation process is carried out under nitrogen or argon shield; With the reaction system solid-liquid separation, clean solid repeatedly to remove the negatively charged ion of bringing in the diluted acid; Add deionized water or ethanol in the gained solid, be stirred well to even dispersion, add H then
2O
2And be heated to 70～100 ℃ of reaction 2～24h, and obtain brown transparence solution, be nanometer Fe
3O
4Colloidal sol; Step B, preparation PEI-CH
2Cl
2Solution: get PEI and sodium lauryl sulphate joins CH
2Cl
2In the solution, be made into PEI-CH
2Cl
2Solution; Step C is compound: with above-mentioned nanometer Fe
3O
4Colloidal sol adds PEI-CH
2Cl
2In the solution, carry out complex reaction; Step D cleans and drying: after the composite solution solid-liquid separation that step C obtains, add ethanol or deionized water wash in the solid phase to remove magnetic Nano Fe
3O
4The CH of/PEI remained on surface
2Cl
2And PEI, reclaim throw out and lyophilize, obtain the nanometer Fe of PEI coating
3O
4/ PEI particle is described magnetic Nano genophore.
In the methods of the invention, among the step C complex reaction for example for reacting 0.3～3h at normal temperatures.
The magnetic Nano genophore that the method for the invention prepares has paramagnetism, under the driving in magnetic field, can carry the external source goal gene and carry out directed driven.Compare with traditional transgene carrier, the present invention can realize the directional transmissions of large fragment DNA, shortens transformation time, improves transformation efficiency greatly.
In above-mentioned preparation method, also be included among the preferred described step D and clean the residual CH of nanoparticle surface
2Cl
2Behind PEI, with nanometer Fe
3O
4/ PEI particle is scattered in ethanol or the deionized water again, and to wherein adding fluorescent substance, fully the reaction back forms phosphor layer at particle surface, obtains the magnetic Nano genophore with fluorescent layer after the lyophilize.The existence of fluorescent layer on the carrier makes the expressing gene that needn't carry fluorescin in the foreign gene, still can make foreign gene detection of changing effect and efficient in zooblast to carry out easily.Described fluorescent substance is for example for being selected from red fluorescent protein RFP, green fluorescent protein GFP, fluorochrome, the fluorescence quantum one or more; More preferably described fluorescent substance is RFP and/or GFP.
The present invention also provides the application of a kind of magnetic Nano genophore for preparing according to the method described above in zooblast or vegetable cell conversion.For example, described zooblast comprises animal fibroblast cell, embryonic stem cell and spermatid; Described vegetable cell comprises protoplastis, somatocyte and pollen.
In above-mentioned application, preferably, transformed animal cell or vegetable cell after described magnetic Nano genophore links to each other with foreign gene, under the external source field drives of 0.1～0.6T, preferred described magnetic Nano genophore links to each other with foreign gene by electrostatic interaction or chemical coupling effect.Wherein, more preferably, carrier particle links to each other with foreign gene by electrostatic interaction; The connection of electrostatic interaction more is conducive in cell dissociating of carrier after the external source gene transformation.
In above-mentioned application, contain fluorescence protein gene in the preferred described foreign gene, can realize the expression of fluorescin; So foreign gene does not need its carrier to have phosphor layer when transforming detection.
During the present invention used, particularly, described magnetic Nano genophore had superparamagnetism, and its particle diameter is 10～500nm; Described foreign gene is DNA or RNA, and described foreign gene is single-gene or polygene.
In the present invention, more preferably, the mass ratio of described magnetic Nano genophore and foreign gene is 2:1～1:2, most preferably 1:1.
The present invention also provides a kind of genetically modified organism to cultivate with the application of magnetic Nano genophore in transformed animal cell or vegetable cell, and wherein, described magnetic Nano genophore is Fe
3O
4Transformed animal cell or vegetable cell after/PEI particle, described magnetic Nano genophore link to each other with foreign gene, under the external source field drives of 0.1～0.6T.
Preferably, described magnetic Nano genophore is at Fe
3O
4/ PEI particle surface is formed with the particle of phosphor layer.
In the above-mentioned method that enters zooblast, preferred described zooblast is porcine fetus fibroblasts or spermatid, and preferred described foreign gene is the pEGFP-N1 plasmid DNA, and described method comprises the steps, the preparation nanometer Fe
3O
4/ PEI/pEGFP-N1 complex body: respectively with pEGFP-N1 plasmid DNA and carrier Fe
3O
4/ PEI particle is diluted in the nutrient solution, gently mixing; Transform: with nanometer Fe
3O
4/ PEI/pEGFP-N1 complex body joins in the zooblast of required conversion, places under the magnetic field to drive, and incubated at room 5～50min changes nutrient solution and continues to cultivate 10～50h behind conversion 2～20h; Detect: detect conversion situation and/or transformation efficiency.More preferably, in aforesaid method, the zooblast that need to transform in the described step of converting is for through the cultivation of spending the night, its cell attachment rate being 70～80% zooblast, and the magneticstrength in described magnetic field is 0.2～0.6T; Detect the conversion situation in the described detection step and realize by inverted fluorescence microscope or laser confocal microscope observation egfp expression, detect transformation efficiency and realize by flow cytometer, immunohistochemical methods detection or real-time quantitative PCR method.
In the above-mentioned method that enters vegetable cell, preferred described vegetable cell is in phytoplasm somatocyte or the plant pollen cell.
In the above-mentioned method that enters the phytoplasm somatocyte, preferred described phytoplasm somatocyte is tobacco protoplast cell or Arabidopis thaliana protoplasm somatocyte, preferred described foreign gene is the pCM1205-GFP plasmid DNA, and described method comprises the steps, the preparation nanometer Fe
3O
4/ PEI/pCM1205-GFP complex body: respectively with pCM1205-GFP plasmid DNA and carrier Fe
3O
4/ PEI particle is diluted in the nutrient solution, gently mixing; Transform: with nanometer Fe
3O
4/ PEI/pCM1205-GFP complex body joins in the required plant transformed protoplasm somatocyte, places under the magnetic field to drive, and incubated at room 5～50min continues to cultivate behind the conversion 24h; Detect: detect conversion situation and/or transformation efficiency.More preferably, in aforesaid method, need the plant transformed protogonocyte for density is adjusted to (1～5) * 10 with W5 solution in the described step of converting
5The pure protoplastis of cell/ml; Detect the conversion situation in the described detection step and realize by inverted fluorescence microscope or laser confocal microscope observation egfp expression, detect transformation efficiency and realize by flow cytometer, immunohistochemical methods detection or real-time quantitative PCR method.
In the above-mentioned method that enters plant pollen, preferred described plant pollen is cotton pollen, and preferred described foreign gene is Insect Resistant Cotton plasmid pGBIGhSNRK, and it contains conversion Bt anti insect gene and cotton development genes involved plant expression vector, described method comprises the steps, the preparation nanometer Fe
3O
4/ PEI/pGBIGhSNRK complex body: respectively with pGBIGhSNRK plasmid DNA and carrier Fe
3O
4/ PEI particle is diluted in the solution, gently mixing; Transform: cotton flower pesticide is soaked in nanometer Fe
3O
4In/PEI/pGBIGhSNRK the complex solution, place under the magnetic field to drive, pollen, artificial pollination are collected in airing behind 15～30min; Detect: detect conversion situation and/or transformation efficiency.More preferably, in aforesaid method, the cotton flower pesticide that needs in the described step of converting to transform is the fresh flower pesticide of not opening flower on the same day, and described artificial pollination acceptor material is to grow normal, as to castrate bagging isolation flower; Detection conversion situation realizes by the screening of kantlex in the described detection step, detects transformation efficiency and realizes by the PCR method.
Advantage of the present invention is: at first, preparation method of the present invention and method for transformation are simple to operate, are easy to grasp, and are convenient to promote.Secondly, method for transformation of the present invention is not limited by the animal and plant kind, has not required the recipient cell specificity, can be widely used in the animal and plant transgenic technology.Again, in method for transformation of the present invention, make gene delivery carrier with magnetic Nano gene particle, under the mediation in magnetic field, external source goal gene and transformant suspension culture are incorporated into cellular genome, have significantly improved the transformation efficiency of gene.At last, the present invention can control the copy number of the foreign gene that changes over to effectively by controlling the amount of the foreign gene of being combined with the magnetic Nano genophore.
Description of drawings
The nanometer Fe that a and b prepare for embodiment 1 among Fig. 1
3O
4/ PEI and its corresponding Fe
3O
4The sem photograph of/PEI/DNA mixture;
The nanometer Fe that a and b prepare for embodiment 1 among Fig. 2
3O
4The transmission electron microscope picture of the different amplification of/PEI;
The Fe of Fig. 3 for preparing among the embodiment 1
3O
4The transmission electron microscope picture of/PEI/DNA mixture;
The nanometer Fe that a and b prepare for embodiment 1 among Fig. 4
3O
4The AFM figure (height map and phasor) of/PEI;
A and b are that the magnetic Nano genophore transforms porcine fetus fibroblasts in embodiment 2 and the Comparative Examples 2 among Fig. 5, respectively the expression effect figure under the condition that magnetic field and no magnetic field are arranged.
Embodiment
Embodiment 1
The preparation of magnetic Nano genophore; (1) preparation nanometer Fe
3O
4Colloidal sol: accurately take by weighing 0.2molFeCl
3And 0.1molFeCl
2, join in the HCl solution of 1L0.1mol/L, fully stir after the dissolving.With the flow velocity of 10ml/min, slowly add the ammonia soln of 1.4L1.5mol/L in the above-mentioned solution, drip and finish the back and continue stirring reaction 30min.The HCl solution that slowly adds 0.1mol/L then in the reaction system, the pH value to 7.0 of regulation system, continuation stirring reaction 30min.Entire reaction course is at N
2Carry out under the protection.Take out reaction system, supernatant liquor is abandoned in centrifuging, reclaims precipitation, cleans repeatedly several times, removes residual ion, to detect less than Cl
-Till.To react the gained solid transfer to flask, add the 0.8L deionized water, fully stir, evenly disperse, add 0.2L30%H then
2O
2, being heated to 95 ℃, dispergation reaction 8h obtains nanometer Fe
3O
4Colloidal sol, its particle diameter is between 10～500nm.(2) preparation PEI-CH
2Cl
2Solution: at 10ml CH
2Cl
2Add 1g polyetherimide (PEI) in the solution, regulating temperature is 35 ℃, adds the 0.1g sodium lauryl sulphate, configuration PEI-CH
2Cl
2Solution.(3) compound: as to get nanometer Fe
3O
4Colloidal sol dropwise is added drop-wise to PEI-CH
2Cl
2In the solution, complex reaction 1h under 35 ℃ of degree makes nanometer Fe
3O
4Surface parcel one deck PEI film.(4) clean, drying: 10000rpm, 4 ℃ of centrifugal 20min abandon supernatant liquor; Add the 10ml deionized water, will be deposited in the nanometer Fe of centrifuge tube
3O
4/ PEI disperses again, and recentrifuge is abandoned supernatant liquor; Repeatable operation 3 times is cleaned nanometer Fe
3O
4Residual free CH on the/PEI surface
2Cl
2And PEI, centrifugal, reclaim throw out, 4 ℃ of following lyophilize 1h obtain the magnetic Nano Fe of PEI coating
3O
4Carrier particle (nanometer Fe
3O
4/ PEI), this carrier particle diameter between 20～520nm, magnetic Nano Fe wherein
3O
4Core diameter is between 10～500nm, and the PEI film thickness is between 5～20nm.
A and b are respectively the nanometer Fe that present embodiment prepares among Fig. 1
3O
4Fe in the sem photograph of/PEI and the present embodiment
3O
4/ PEI connects the Fe behind the DNA
3O
4The sem photograph of/PEI/DNA.As seen from the figure, nanometer Fe
3O
4/ PEI and Fe
3O
4/ PEI/DNA complex body is spherical in shape, is uniformly dispersed uniform particle diameter in dispersion liquid.The nanometer Fe that a and b prepare for embodiment 1 among Fig. 2
3O
4The transmission electron microscope picture of the different amplification of/PEI.Also can be seen the magnetic Nano genophore Fe for preparing by Fig. 2
3O
4/ PEI is globosity, and better dispersed, and Fig. 2 (b) can see magnetic Nano Fe
3O
4Particle is core, coated outside one deck PEI film.The Fe of Fig. 3 for preparing among the embodiment 1
3O
4The transmission electron microscope picture of/PEI/DNA mixture can be seen in nanometer Fe
3O
4Combine a large amount of netted DNA chains around the/PEI, the nanometer Fe of preparation is described
3O
4/ PEI and DNA have stronger binding ability.The nanometer Fe that a and b prepare for embodiment 1 among Fig. 4
3O
4The AFM figure (height map and phasor) of/PEI can see the nanometer Fe for preparing intuitively
3O
4The pattern of/PEI.
Comparative Examples 1
Prepare the magnetic Nano genophore by the method for announcing among the CN101748150A: get 5.6mmol FeCl
3And 2.8mmolFeCl
2Be dissolved in the 7ml de aerated water, regulate temperature in the frozen water at 2-4 ℃, add N
2Stir, dropwise the NaOH of Dropwise 5 M regulates pH to 11, reacts 30min in the ice-water bath; Regulate bath temperature to 80 ℃ then, heating 1h.After reaction finishes, 90000rpm, 4 ℃ of centrifugal 10min abandon supernatant liquor, and the collecting precipitation thing obtains nanometer Fe
3O
4Particle.This particle is resuspended with the 7ml deionized water, get the Fe of 200ul after resuspended then
3O
4Solution is added drop-wise to dropwise that (collocation method of phosphate buffered saline buffer PBS is as follows: take by weighing 8g NaCl, 0.2g KCl, 1.44g Na in the PBS solution of PEI25000 of 200ul0.075g/ml
2HPO
4With 0.24g KH
2PO
4, be dissolved in the 800ml deionized water, with the pH value to 7.4 of HCl regulator solution, adding distil water is settled to 1L at last), stirring reaction 3h under the room temperature places the dialysis tubing 4h that dialyses then, obtains magnetic Nano genophore Fe
3O
4/ PEI.
Table 1 has compared particle size data and the surperficial Zeta potential numerical value of gained magnetic Nano genophore in embodiment 1 and the Comparative Examples 1.
Table 1
As can be seen from Table 1, in Comparative Examples 1, the nanometer Fe that it is synthetic
3O
4There is reunion to a certain degree in particle, and particle diameter reaches micron order more greatly, after modifying PEI, disperses better and particle diameter reaches nano level, and after DNA was combined, DNA may be hidden between the branch of PEI at carrier, did not have bigger variation in conjunction with the front and back particle diameter; Compare the Fe of embodiment 1 gained with Comparative Examples
3O
4Particle has reached nano level, and is little by its magnetic Nano genophore particle diameter for preparing, thereby specific surface area is bigger, schemes Fe as can be known from SEM, TEM etc.
3O
4/ PEI size distribution is even, good dispersity.
In addition, about surperficial Zeta potential, in the Comparative Examples 1, nanometer Fe
3O
4After/PEI combined DNA, Zeta potential became greatly on the contrary, may be because the combination of DNA has played agglomeration to PEI, and it is assembled more closely, and is big to the surface potential contribution; Compare the nanometer Fe of embodiment 1 gained with Comparative Examples
3O
4The Zeta potential height of/PEI is easier to be combined with electronegative DNA, decreases in conjunction with the back Zeta potential, and the better in conjunction with effect of nano-carrier and DNA is described, is a kind of good gene delivery carrier.
Embodiment 2
Under the condition of externally-applied magnetic field, the magnetic Nano genophore transforms porcine fetus fibroblasts: with 1 * 10
5Individual porcine fetus fibroblasts is inoculated in 6 orifice plates in transforming the day before yesterday, places 37 ℃, 5%CO
2Incubator incubated overnight to cell attachment is bred to 70～80%.Carry out cell before the conversion and change liquid, use the DMEM nutrient solution of serum-free to cultivate.1 μ g pEGFP-N1 plasmid DNA is diluted in the 50 μ l nutrient solutions (the DMEM nutrient solution that does not contain foetal calf serum) mixing gently.Be 1 μ l/ μ g with 2 μ l(concentration) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier is diluted in the 50 μ l nutrient solutions, mixing gently, incubated at room 5min.Behind the 5min it is mixed with above-mentioned plasmid DNA, and mixing gently, incubated at room 20min obtains nanometer Fe
3O
4/ PEI/pEGFP-N1 complex body.Complex body is joined in each hole of culture plate that contains porcine fetus fibroblasts, place under the magnetic field of 0.3T and drive incubated at room 15min.Change the DMEM nutrient solution continuation cultivation that contains 10% foetal calf serum after transforming 5h, observe the expression of green fluorescent protein behind the 24h with inverted fluorescence microscope, detect transformation efficiency with flow cytometer.The weight ratio of carrier and plasmid is 2:1 in the present embodiment.Gained cell transformation efficient is 18.6 ± 1.8%.
Fig. 5 (a) is that the condition magnetic nano-gene carrier at externally-applied magnetic field transforms porcine fetus fibroblasts expression effect figure in the present embodiment.In Fig. 5 (a), under fluorescent microscope, to observe, under the exciting light of 470～490nm wavelength, cell sends green fluorescence, shows that the external source goal gene is directed to porcine fetus fibroblasts, and realizes expressing.
Comparative Examples 2
Under the condition that does not add magnetic field, the magnetic Nano genophore transforms porcine fetus fibroblasts: with 1 * 10
5Individual porcine fetus fibroblasts is inoculated in 6 orifice plates in transforming the day before yesterday, places 37 ℃, 5%CO
2Incubator incubated overnight to cell attachment is bred to 70～80%.Carry out cell before the conversion and change liquid, use the DMEM nutrient solution of serum-free to cultivate.1 μ g pEGFP-N1 plasmid DNA is diluted in the 50 μ l nutrient solutions (the DMEM nutrient solution that does not contain foetal calf serum) mixing gently.Be 1 μ l/ μ g with 2 μ l(concentration) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier is diluted in the 50 μ l nutrient solutions, mixing gently, incubated at room 5min.Behind the 5min it is mixed with above-mentioned plasmid DNA, and mixing gently, incubated at room 20min obtains nanometer Fe
3O
4/ PEI/pEGFP-N1 complex body.Complex body is joined in each hole of culture plate that contains porcine fetus fibroblasts, change the DMEM nutrient solution continuation cultivation that contains 10% foetal calf serum after transforming 5h, observe the expression of green fluorescent protein behind the 24h with inverted fluorescence microscope, detect transformation efficiency with flow cytometer.The weight ratio of carrier and plasmid is 2:1 in the present embodiment.
Fig. 5 (b) is for transforming porcine fetus fibroblasts expression effect figure at the condition magnetic nano-gene carrier that does not add magnetic field in this Comparative Examples.In Fig. 5 (b), under fluorescent microscope, observe, under the exciting light of 470～490nm wavelength, examine just and can recognize a small amount of and faint green fluorescence, illustrate that when not having magnetic field to exist, the transformation efficiency of porcine fetus fibroblasts is extremely low.
Comparative Examples 3
Under the condition that does not add magnetic field, utilize liposome lipo2000 to enter porcine fetus fibroblasts for carrier transforms the pEGFP-N1 plasmid: to transform and carry out bed board the day before yesterday, in 6 orifice plates, make cell concn reach 0.5～2 * 10 cell inoculation
5Cells/well, 37 ℃, 5%CO
2The incubator incubated overnight treated that cell density reached 90% and transforms when above in second day.0.8ug pEGFP-N1 plasmid DNA is dissolved in the 50ul DMEM serum free medium, 2ul lipo2000 is dissolved in the 50ul DMEM serum free medium, mixing, room temperature is placed 5min.They are mixed, obtain the lipo2000/pEGFP-N1 complex body, room temperature is placed 20min.The lipo2000/pEGFP-N1 complex body is joined in each hole of culture plate that contains porcine fetus fibroblasts, and 4-6 as a child changed and contained blood serum medium continuation cultivation.Observe the expression of green fluorescent protein behind the 24h with inverted fluorescence microscope, detect transformation efficiency with flow cytometer.Its transformation efficiency is 16.7 ± 2.1%.The carrier that uses in this Comparative Examples and the ratio of plasmid DNA are 2ul/0.8ug, and the ratio that namely is equivalent to carrier and plasmid DNA is 2.5ul/ug, and the ratio 2～3ul/ug of this and art-recognized optimum changing effect is consistent.
Embodiment 3
Under the condition of externally-applied magnetic field, the magnetic Nano genophore transforms the pig spermatid: the pig spermatid is diluted to 1 * 10
6Individual/ml.1 μ g pEGFP-N1 plasmid DNA is diluted in the 50 μ l nutrient solutions (the DMEM nutrient solution that contains 10% foetal calf serum), gently mixing.Be 1 μ l/ μ g with 2 μ l(concentration) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier is diluted in the 50 μ l nutrient solutions, mixing gently, incubated at room 5min; Then it is mixed with above-mentioned plasmid DNA, and mixing gently, incubated at room 20 minutes obtains nanometer Fe
3O
4/ PEI/pEGFP-N1 complex body.Complex body is joined in the 1ml spermatid solution, and the culture plate that rocks back and forth is evenly distributed it, places under the magnetic field of 0.3T to drive, hatch 30min after, put into 17 ℃ of thermostat containers and cultivate.Detect and real-time quantitative PCR method detection sperm transformation efficiency by immunohistochemical methods.The weight ratio of carrier and plasmid is 2:1 in the present embodiment.The gained transformation efficiency is 22.6 ± 4.5%.
Comparative Examples 4
Under the condition that does not add magnetic field, the magnetic Nano genophore transforms the pig spermatid: the pig spermatid is diluted to 1 * 10
6Individual/ml.1 μ g pEGFP-N1 plasmid DNA is diluted in the 50 μ l nutrient solutions (the DMEM nutrient solution that contains 10% foetal calf serum), gently mixing.Be 1 μ l/ μ g with 2 μ l(concentration) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier is diluted in the 50 μ l nutrient solutions, mixing gently, incubated at room 5min; Then it is mixed with above-mentioned plasmid DNA, and mixing gently, incubated at room 20 minutes obtains nanometer Fe
3O
4/ PEI/pEGFP-N1 complex body.Complex body is joined in the 1ml spermatid solution, and the culture plate that rocks back and forth is evenly distributed it, puts into 17 ℃ of thermostat containers and cultivates.Detect and real-time quantitative PCR method detection sperm transformation efficiency by immunohistochemical methods.The weight ratio of carrier and plasmid is 2:1 in the present embodiment.The result shows that do not adding under the magnetic field condition, transformation efficiency is extremely low.
Embodiment 4
The magnetic Nano genophore transforms porcine fetus fibroblasts: experimentation is basic identical with embodiment 2, only with among the embodiment 2 " 2 μ l(concentration are 1 μ l/ μ g) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier " change into use " 1 μ l(concentration is 1 μ l/ μ g) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier "; Be that the weight ratio of carrier and plasmid is 1:1 in the present embodiment.Gained cell transformation efficient is 32.6 ± 4.2%.
Embodiment 5
The magnetic Nano genophore transforms porcine fetus fibroblasts: experimentation is basic identical with embodiment 2, only with among the embodiment 2 " 2 μ l(concentration are 1 μ l/ μ g) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier " change into use " 0.5 μ l(concentration is 1 μ l/ μ g) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier "; Be that the weight ratio of carrier and plasmid is 1:2 in the present embodiment.Gained cell transformation efficient is 12.6 ± 2.4%.
Embodiment 6
The magnetic Nano genophore transforms the pig spermatid: experimentation is basic identical with embodiment 3, only with among the embodiment 3 " 2 μ l(concentration are 1 μ l/ μ g) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier " change into use " 1 μ l(concentration is 1 μ l/ μ g) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier "; Be that the weight ratio of carrier and plasmid is 1:1 in the present embodiment.The gained transformation efficiency is 42.1 ± 2.7%.
Embodiment 7
The magnetic Nano genophore transforms the pig spermatid: experimentation is basic identical with embodiment 3, only with among the embodiment 3 " 2 μ l(concentration are 1 μ l/ μ g) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier " change into use " 0.5 μ l(concentration is 1 μ l/ μ g) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier "; Be that the weight ratio of carrier and plasmid is 1:2 in the present embodiment.The gained transformation efficiency is 18.4 ± 3.6%.
Transformation efficiency in embodiment 2～7 and the Comparative Examples 2～4 is tabulated in table 2.
Table 2
As can be seen from Table 2, utilizing nanometer Fe
3O
4/ PEI carrier transforms in the porcine fetus fibroblasts process, externally-applied magnetic field be important must obligato condition, nanometer Fe
3O
4The carrier mediated porcine fetus fibroblasts transformation efficiency of/PEI is under the condition of field drives, and can realize being higher than in the prior art with the liposome is the transformation efficiency of the zooblast of carrier.In addition, as can be seen from the table, work as nanometer Fe
3O
4When the mass ratio of/PEI carrier and DNA was 1:1, under the condition of externally-applied magnetic field, it is maximum that the transformation efficiency of porcine fetus fibroblasts reaches.Simultaneously, utilize the nanometer Fe of preparation
3O
4/ PEI carrier can be successful the conversion of realization pig spermatid, lay a good foundation for further carrying out transforming in the animal body.
Embodiment 8
Magnetic Nano genophore transformation of tobacco protoplastis, step is as follows.
The preparation of tobacco protoplast.The tobacco aseptic seedling places the constant temperature illumination box, sets 28 ℃ of daytimes, humidity daytime 75%, illumination 10 hours; At 22 ℃ of nights, humidity 70% dark 14 hours, was cultivated 40～60 days; Take by weighing the above-mentioned cultured tobacco leaf of 1g, place centrifuge tube 5ml enzyme liquid system (1.5% cellulase, 0.3% macerozyme, 0.4mol/L N.F,USP MANNITOL, 0.02mol/L KCl, 0.02mol/L MES, 0.01mol/L CaCl2,0.1%BSA, pH5.8); 26.5 ℃, enzymolysis 3～4h under the dark condition.30min placed 26.5 ℃ of constant-temperature shaking culture case 50r/min oscillation treatment before enzymolysis finished, and discharged protoplastis.Then with protoplastis together with enzyme liquid through 240 order stainless steel sift net filtrations, filtrate is centrifugal 2min again.Remove supernatant, be precipitated as protoplastis.In precipitation, slowly add 2ml W5 solution (154mmol/L NaCl; 125mmol/L CaCl2; 5mmol/L KCl; 2mmol/L MES), gentle resuspended protoplastis.The centrifugal 1min of 100 * g abandons supernatant, repeats previous step and washs 1 time again, obtains pure protoplastis, and microscopically is observed its state, with W5 solution protoplastis density is adjusted to (1～5) * 10
5Cell/ml is stand-by.
Be 1 μ l/ μ g with 8 μ l(concentration) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier and 8 μ g pCM1205-GFP plasmid DNA (expressing green fluorescent protein), incubated at room 15～45min obtains Fe
3O
4/ PEI/pCM1205-GFP complex body; Get the tobacco protoplast that 1ml prepares, the centrifugal bottom that made protoplastis concentrate on centrifuge tube in 30 seconds of 300rmp/min adds the real Fe of 8 μ g
3O
4/ PEI/pCM1205-GFP complex body, at centrifuge tube bottom externally-applied magnetic field, effect 20min; Above-mentioned leaving standstill under the Arabidopis thaliana protoplastis room temperature of transduction cultivated 24h, observe the green fluorescent protein of expressing in the tobacco cell under fluorescent microscope, gained protoplasm somatocyte transformation efficiency is 53.1 ± 2.8%.
Embodiment 9
Magnetic Nano genophore arabidopsis thaliana transformation protoplastis: be 1 μ l/ μ g with 8 μ l(concentration) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier and 8 μ g pCM1205-GFP plasmid DNA, incubated at room 15～45min obtains Fe
3O
4/ PEI/pCM1205-GFP complex body; Arabidopis thaliana is 22 ℃ of temperature, humidity 80%, and illumination 10 hours, 19 ℃ of temperature, humidity 75%, dark culturing 14h, about 30～40 days rear blades of growth of seedling can be used; The protoplastis for preparing Arabidopis thaliana by the method for tobacco protoplast preparation among the embodiment 8; Get the Arabidopis thaliana protoplastis that 1ml prepares, the centrifugal bottom that made protoplastis concentrate on centrifuge tube in 30 seconds of 300rmp/min adds the real Fe of 8 μ g
3O
4/ PEI/pCM1205-GFP complex body, at centrifuge tube bottom externally-applied magnetic field, effect 20min; Cultivate 24h with leaving standstill under the above-mentioned Arabidopis thaliana protoplastis room temperature through transforming, observe the green fluorescent protein of expressing in the arabidopsis cell under fluorescent microscope, gained protoplasm somatocyte transformation efficiency is 66.7 ± 4.4%.
Embodiment 10
The magnetic Nano genophore mediates dual-gene converting cotton pollen: select normotrophic flower, castrate bagging and isolate, as the pollination material; Be 1 μ l/ μ g with 3 μ l(concentration) nanometer Fe for preparing among the embodiment 1
3O
4/ PEI carrier and 3 μ g plasmids (pGBIGhSNRK, it is Bt anti insect gene and cotton development genes involved integrative gene expression vector, the kalamycin resistance mark) add in the 100 μ l systems, and incubated at room 15～45min obtains Fe
3O
4/ PEI/pGBIGhSNRK complex body; With 1000 times of above-mentioned system dilutions, the flower pesticide of winning fresh not open flower soaks wherein, enters pollen cell at field drives magnetic nano-gene carrier-DNA mixture, handles 10～30min; Take out flower pesticide, blot, treat to collect pollen, artificial pollination after pollen sheds; Field sowing behind the seed maturity obtains positive plant through the kalamycin resistance screening; Win the positive plant blade, extract genomic dna and carry out the PCR detection, further determine positive plant, the statistics transformation efficiency is 10%(positive plant/conversion flower number).
Comparative Examples 5
Agrobacterium ovary injection: change plasmid pGBIGhSNRK over to LBA4404 with freeze-thaw method, the preparation engineering Agrobacterium.Picking engineering Agrobacterium bacterium colony is in YEB liquid nutrient medium shake-flask culture (200rmin
-1), be 10 up to the OD600 value for the 0.8(bacterial concentration
6～10
7CellmL
-1, dilution method is determined bacterial concentration); In the bacterium liquid that shakes, add Syringylethanone (30mgL
-1) place shaking table to cultivate 2h again, get 100 μ L bacterium liquid, 650 μ L sucrose solutions (5%), 250 μ L glycerine (60%) mix, 4 ℃ of preservations; 3d before the injection will contain the Agrobacterium activation of goal gene, and in 28 ℃ of incubators, growing diameter on the YEB solid medium is single bacterium colony of 1mm; Inject the same day, survey bacterium liquid OD value, the sucrose solution with 5% is diluted to 10
5Individual/mL, add Syringylethanone (0.1mmolL
-1), Silwet L77(0.05%) mixing; The morning 9:00-10:00, between 25～30 ℃ of the temperature, select in the field fruit branch and flower position preferably ovary as transforming object, with the ovary of plant middle part fruit branch for well; Stamen and corolla are peeled off, the right hand is held microsyringe, left hand is gently held up the ovary behind the excision petal, inject ovary 2/3 place with the y direction of microsyringe from column cap along ovary along style, vertically inject half bacterium liquid, extract 1/3 again, vertical second half bacterium liquid of injection, can not damage locule, inject field sowing behind the bacterium liquid 10 μ L seed maturities altogether. detect through kalamycin resistance screening and PCR, transgene efficiency is 1%.
The transformation efficiency of bt-cotton plasmid reaches 10% among the embodiment 10, and far above the transformation efficiency of Agrobacterium ovary injection 1% in the Comparative Examples 5, and its operation is more easy.
Claims (10)
1. a genetically modified organism is cultivated the preparation method who uses the magnetic Nano genophore, comprise the steps,
Steps A, the preparation nanometer Fe
3O
4Colloidal sol: be that 1～3:1 takes by weighing molysite and ferrous salt and adds in the dilute acid soln by the ratio of amount of substance, the back is stirred in dissolving makes the pH value of solution value be alkalescence to wherein adding weak caustic solution, continues to be stirred to reaction and finishes, with sour neutralization solution pH value to 6～8; Aforementioned preparation process is carried out under nitrogen or argon shield; With the reaction system solid-liquid separation, clean solid repeatedly to remove the negatively charged ion of bringing in the diluted acid; Add deionized water or ethanol in the gained solid, be stirred well to even dispersion, add H then
2O
2And be heated to 70～100 ℃ of reaction 2～24h, and obtain brown transparence solution, be nanometer Fe
3O
4Colloidal sol;
Step B, preparation PEI-CH
2Cl
2Solution: get PEI and sodium lauryl sulphate joins CH
2Cl
2In the solution, be made into PEI-CH
2Cl
2Solution;
Step C is compound: with above-mentioned nanometer Fe
3O
4Colloidal sol adds PEI-CH
2Cl
2In the solution, carry out complex reaction;
Step D cleans and drying: after the composite solution solid-liquid separation that step C obtains, add ethanol or deionized water wash in the solid phase to remove magnetic Nano Fe
3O
4The CH of/PEI remained on surface
2Cl
2And PEI, reclaim throw out and lyophilize, obtain the nanometer Fe of PEI coating
3O
4/ PEI particle is described magnetic Nano genophore.
2. method according to claim 1 is characterized in that, also is included among the described step D to clean the residual CH of nanoparticle surface
2Cl
2Behind PEI, with nanometer Fe
3O
4/ PEI particle is scattered in ethanol or the deionized water again, and to wherein adding fluorescent substance, fully the reaction back forms phosphor layer at particle surface, obtains the magnetic Nano genophore with fluorescent layer after the lyophilize.
3. method according to claim 2 is characterized in that, described fluorescent substance is to be selected from red fluorescent protein RFP, green fluorescent protein GFP, fluorochrome, the fluorescence quantum one or more.
4. the application of magnetic Nano genophore in zooblast or vegetable cell conversion for preparing according to any described method in the claim 1～3; Preferred described zooblast is porcine fetus fibroblasts or pig spermatid, and preferred described vegetable cell is tobacco protoplast cell, Arabidopis thaliana protoplasm somatocyte or cotton pollen.
5. application according to claim 4, it is characterized in that, transformed animal cell or vegetable cell after described magnetic Nano genophore links to each other with foreign gene, under the external source field drives of 0.1～0.6T, preferred described magnetic Nano genophore links to each other with foreign gene by electrostatic interaction or chemical coupling effect.
6. application according to claim 5 is characterized in that, contains fluorescence protein gene in the described foreign gene, can realize the expression of fluorescin.
7. according to claim 5 or 6 described application, it is characterized in that described magnetic Nano genophore has superparamagnetism, and its particle diameter is 10～500nm; Described foreign gene is DNA or RNA, and described foreign gene is single-gene or polygene.
8. according to claim 5 or 6 described application, it is characterized in that the mass ratio of described magnetic Nano genophore and foreign gene is 2:1～1:2, most preferably 1:1.
9. the application of magnetic Nano genophore in transformed animal cell or vegetable cell, wherein, described magnetic Nano genophore is Fe
3O
4Transformed animal cell or vegetable cell after/PEI particle, described magnetic Nano genophore link to each other with foreign gene, under the external source field drives of 0.1～0.6T; Preferred described zooblast is porcine fetus fibroblasts or spermatid, and preferred described vegetable cell is tobacco protoplast cell, Arabidopis thaliana protoplasm somatocyte or cotton pollen.
10. application according to claim 5 is characterized in that, described magnetic Nano genophore is at Fe
3O
4/ PEI particle surface is formed with the particle of phosphor layer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310153676.XA CN103233042B (en) | 2012-04-28 | 2013-04-27 | Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210129791 | 2012-04-28 | ||
| CN201210129791.9 | 2012-04-28 | ||
| CN201310153676.XA CN103233042B (en) | 2012-04-28 | 2013-04-27 | Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103233042A true CN103233042A (en) | 2013-08-07 |
| CN103233042B CN103233042B (en) | 2015-03-25 |
Family
ID=48881107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310153676.XA Active CN103233042B (en) | 2012-04-28 | 2013-04-27 | Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103233042B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652455A (en) * | 2019-02-19 | 2019-04-19 | 南京农业大学 | The Chinese cabbage high-efficiency genetic transforming method and its application that a kind of magnetic nano-carrier mediates |
| CN109797169A (en) * | 2019-03-06 | 2019-05-24 | 武汉轻工大学 | A kind of genophore and preparation method thereof |
| CN110541000A (en) * | 2019-09-10 | 2019-12-06 | 北京农业生物技术研究中心 | Method for obtaining magnetic nanoparticle-mediated antibacterial peptide chlorella |
| WO2020260104A1 (en) | 2019-06-27 | 2020-12-30 | Basf Plant Science Company Gmbh | Methods for transformation of fungal spores |
| CN112442476A (en) * | 2020-11-27 | 2021-03-05 | 江苏省农业科学院 | Method for preparing hydrangea protoplast and performing transient transformation |
| CN117025685A (en) * | 2023-07-07 | 2023-11-10 | 云南师范大学 | Magnetic nano-carrier mediated phytophthora infestans genetic transformation method and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748150A (en) * | 2010-01-28 | 2010-06-23 | 上海交通大学 | Method for preparing direct composite PEI magnetic gene vector |
-
2013
- 2013-04-27 CN CN201310153676.XA patent/CN103233042B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101748150A (en) * | 2010-01-28 | 2010-06-23 | 上海交通大学 | Method for preparing direct composite PEI magnetic gene vector |
Non-Patent Citations (2)
| Title |
|---|
| 李瑶等: "磁性纳米基因工程载体细胞转化方法研究", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 陈道桢等: "PEI-氧化铁磁性纳米颗粒作为基因载体的实验观察", 《第四军医大学学报》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652455A (en) * | 2019-02-19 | 2019-04-19 | 南京农业大学 | The Chinese cabbage high-efficiency genetic transforming method and its application that a kind of magnetic nano-carrier mediates |
| CN109797169A (en) * | 2019-03-06 | 2019-05-24 | 武汉轻工大学 | A kind of genophore and preparation method thereof |
| WO2020260104A1 (en) | 2019-06-27 | 2020-12-30 | Basf Plant Science Company Gmbh | Methods for transformation of fungal spores |
| CN114026238A (en) * | 2019-06-27 | 2022-02-08 | 巴斯夫植物科学有限公司 | Method for transforming fungal spores |
| CN110541000A (en) * | 2019-09-10 | 2019-12-06 | 北京农业生物技术研究中心 | Method for obtaining magnetic nanoparticle-mediated antibacterial peptide chlorella |
| CN112442476A (en) * | 2020-11-27 | 2021-03-05 | 江苏省农业科学院 | Method for preparing hydrangea protoplast and performing transient transformation |
| CN112442476B (en) * | 2020-11-27 | 2024-06-04 | 江苏省农业科学院 | Method for preparing and instantaneously transforming hydrangea protoplast |
| CN117025685A (en) * | 2023-07-07 | 2023-11-10 | 云南师范大学 | Magnetic nano-carrier mediated phytophthora infestans genetic transformation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103233042B (en) | 2015-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103233042B (en) | Preparation method and application of magnetic nano-gene vector for cultivating transgenic organism | |
| AU642889B2 (en) | Aerosol beam microinjector | |
| US7132289B2 (en) | Method for introducing foreign matters into living cells | |
| CN102925488B (en) | Magnetic nanocarrier-mediated plant transgenic method | |
| AU615463B2 (en) | Process for gene transfer in plants | |
| CN103080323A (en) | Linear DNA molecule delivery using pegylated quantum dots for stable transformation in plants | |
| CN109652455B (en) | Magnetic nano-carrier mediated efficient genetic transformation method for non-heading Chinese cabbages and application thereof | |
| CN113832035B (en) | Method for inducing aschersonia oligosporus to produce predatory organs by using extracellular vesicles secreted by caenorhabditis elegans | |
| CN104450779A (en) | Fast and efficient agrobacterium-mediated rice stem tip genetic transformation method | |
| CN103468638A (en) | Large-scale suspension cultivation method of 293 cells | |
| CN114621973B (en) | Method for preparing transgenic corn pollen and kit used by method | |
| CN107267549A (en) | A kind of method of the middle mesophyll protoplast of mountain China fir kind 406 separation, purifying and Efficient Conversion | |
| CN104152403B (en) | A kind of method that establishing goose embryonic epithelium cell line and the goose embryonic epithelium cell line of foundation | |
| Li et al. | High frequency generation of fertile transgenic rice plants after PEG-mediated protoplast transformation | |
| CN110499278A (en) | A kind of ginkgo leaf protoplast separation purification and the method for instantaneous conversion | |
| CN110331167B (en) | Corn genotype-independent DNA introduction method | |
| CN111849858A (en) | Preparation of moso bamboo protoplast and establishment of transient transformation system | |
| CN108949822A (en) | The breeding method of HRVVP7 transgenic safflower seed | |
| CN102102108A (en) | Method for cultivating efficient selected-marker-free transgenic crop by using double T-DNA+1 vectors | |
| Wang et al. | Establishment of a suspension cell system for transformation of Jatropha curcas using nanoparticles | |
| CN110541000B (en) | Method for obtaining magnetic nanoparticle-mediated antibacterial peptide chlorella | |
| CN102260704A (en) | Plant transgene method based on taking silicon dioxide nano particles as gene vector | |
| US6596540B2 (en) | Method for introduction of an exogenous genetic substance or a physiologically active compound | |
| Saunders et al. | Pollen electrotransformation in tobacco | |
| CN115287287B (en) | Method for analyzing characteristics and functions of novel alpha-bisabolol synthase (MrBAS) promoter in chamomile |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |