CN1078261A - Allogenic material is imported in a large number the method for viable cell - Google Patents
Allogenic material is imported in a large number the method for viable cell Download PDFInfo
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- CN1078261A CN1078261A CN 92103259 CN92103259A CN1078261A CN 1078261 A CN1078261 A CN 1078261A CN 92103259 CN92103259 CN 92103259 CN 92103259 A CN92103259 A CN 92103259A CN 1078261 A CN1078261 A CN 1078261A
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Abstract
The invention belongs to biological technical field.Soak cell with high osmotic buffer, make the cell interior portion water divide outflow, then cell is changed in the common nutrient solution that contains allogenic material, press low inside and high outside gradient to form Premeabilisation of cells, cause membrane perforation with physical method, allogenic material just enters cell in a large number.Can improve vegeto-animal hereditary property with present method, produce biologics and disease is treated.
Description
The field of the invention is a biotechnology.
Genetically engineered is the biological high-technology that the seventies grows up.This technology provides strong instrument for transforming biological character and rapid breeding.It comprises relatively independent two parts.One is isolated goal gene (as disease-resistant anti insect gene, saline-resisting and alkaline-resisting gene, drought resisting adversity gene or the like) with the molecular cloning means.Its two, goal gene is imported biomass cells, goal gene is expressed on cell or individual level.At present, the more existing effective meanss comparatively of clone's goal gene, and with external source goal gene transfered cell effectively, especially importing crop plant cells and fertilised non-human eggs then is engineered a great problem.
At present, import foreign gene toward vegetable cell and also do not have a kind of efficient ways.As soil Agrobacterium-Ti-plasmids carrier system, be the most deep plant conversion system of research, but it only is suitable for transforming dicotyledons, can not generally be suitable for monocotyledons.The virus vector method be with plant virus as carrier, foreign DNA is imported vegetable cell, its strictness is limited by the host range of plant virus.The microinjection rule utilizes microinjector directly foreign DNA to be injected in the cell, but its operation is very loaded down with trivial details, and technical qualification require high, is difficult to promote.Though PEG method, electric shocking method and liposome mediated-method increase the Plant Transformation opportunity of success, these methods are acceptor with the plant protoplast all, and workload is big, and success ratio is low.Because plant protoplast regeneration is the very work of difficulty, and many plant protoplasts are difficult to the regeneration plant so far.Though particle bombardment is an acceptor with the vegetable material that does not remove wall, the DNA amount that it imports acceptor is few, and actual transformation efficiency is not high.External existing human laser microbeam puncture vegetable cell attempt by the laser hole transformed plant cells, but they can only cause cell wall perforation, and can not guarantee that allogenic material enters in the cell in a large number, therefore produces little effect.
In the conversion of fertilised non-human eggs, the most frequently used is microinjection, and human PEG method, electric shocking method etc. are also arranged, and generally speaking efficient is not high.
We think that a kind of cell transformation method efficiently of creation is significant for accelerated gene engineering research process.New method for transformation must satisfy 2 requirements: 1. the damage that biomass cells is caused is little, does not influence the cells physiological activity.Allogenic material is entered in the cell in a large number,, goal gene is incorporated on the karyomit(e) to guarantee having enough dna moleculars to enter nucleus.
Principle of the present invention is: soak the cell certain hour with high osmotic buffer, make the cell interior portion water divide outflow, then cell is changed in the common nutrient solution that contains DNA, press low inside and high outside gradient to form Premeabilisation of cells.This moment, causing membrane perforation with physical method, under the effect that Premeabilisation of cells is pressed, pericellular material just enters rapidly in the cell by these micropores, and in a short period of time, cell perforation is closed naturally.Like this, the DNA in the extracellular environment has just entered perforated cell in large quantities.
The pre-treatment of recipient cell is the key of success of the present invention.The osmotic pressure of each kind of plant is generally between 0.25-0.65mol/L.Therefore, manually set up Premeabilisation of cells and press low inside and high outside gradient, just cell must be immersed than Premeabilisation of cells and press in the high damping fluid, so just can make the ICW outflow.We design high osmotic buffer concentration between 0.5-1.0mol/L in view of the above.According to different vegetable materials, prepare corresponding high sepage, the difference of pressing with high sepage concentration and Premeabilisation of cells is as the criterion above 0.05mol/L.
The preparation high osmotic buffer generally adopts the nontoxic glycitols molecule of pair cell, and as glucose, inositol, N.F,USP MANNITOL, sorbyl alcohol, sucrose etc., its general formula is: C
6H
12-14O
6Or C
12H
22O
11Damping fluid adopts Tris-hydrochloride buffer, phosphoric acid buffer etc., pH5.6-7.5.
The time that vegetable material soaks in high osmotic buffer is as the criterion tangible plasmolysis to occur, generally should surpass 20 minutes, and best soak time scope is 1-5 hour.
The cell that high osmotic buffer was soaked changes in the common nutrient solution that contains foreign DNA, has just set up Premeabilisation of cells and has pressed low inside and high outside gradient, at this moment should cause cell wall perforation with physical method as early as possible, and cell peripheral solution is entered in the cell rapidly.Cause the method for cell wall perforation to have: laser microbeam irradiation, metal particle, ultrasonic wave, high-voltage electric shock etc. with the particle gun acceleration.
With the example that is converted into of rice suspension cell, concrete operation method is as follows:
Rice suspension cell changed in the high osmotic buffer (10mM TrisCl, pH7.2,0.7M sorbyl alcohol) and soaked 3-4 hour after subculture 3-4 days.On super clean bench, the cell that high sepage was handled filters with 80 eye mesh screens, the small cell cluster of getting after 0.1ml filters changes in the 1.5ml Eppendorf pipe, wash once with common nutrient solution, add 5 μ g PBI, 121 plasmid DNA immediately, behind the mixing cell is put into the Rose cell and carry out the laser capture microdissection irradiation.Irradiating unit is the micro-instrument of Nd-YAG quadruple pulse.Output wavelength is 1060nm, 532nm, 355nm and 266nm, and pulsewidth is 10ns.Laser apparatus is when the 532nm wavelength, and the output energy is 10mJ.Laser beam is introduced a phase microscope through coupling, and in cell to be illuminated, spot diameter is 1.3 μ m with the 40X object lens focusing.During irradiation, make laser pulse roll into a ball enterprising line scanning irradiation at vegetable cell by moving stage all around, laser irradiation time is every sample 60 minutes, plays 4000 pulses approximately.Postradiation cell is cultivated according to a conventional method, screens, is detected and regenerates.
The advantage that the present invention has and range of application: it is that a cover is easy that osmotic pressure gradient adds the perforate membrane method for transformation, efficiently method for transformation. Because it can guarantee that allogenic material enters cell in a large number, it is minimum that the damage that recipient cell is suffered reduces to, and the cell after the conversion can be preserved its normal physiological status.
This method is theoretical as the basis with Premeabilisation of cells, therefore has plant cell and tissue, zooblast and the embryonated egg of keeping the Premeabilisation of cells compression functions, in addition microbial cell all available this method carry out the foreign gene importing. Theoretically, this method can import any gene that obtains through the molecular cloning means biological cell of any work. Therefore this method is applicable to many aspects: the 1. gene transferred crop cell that economical character is good, cultivate the New Crop Varieties that makes new advances. The gene that 2. will produce certain biologics imports the animal or plant cell, produces medicine by tissue culture method then. 3. the merit gene is imported fertilised non-human eggs, cultivate the animal new varieties. 4. when gene therapy, can with this method genes of interest be imported the human cell earlier, filter out behind the integrator cell again with in latter's patients with implantation body, just can reach therapeutic purposes.
Claims (6)
1, a kind of method that allogenic material is imported viable cell, it is characterized in that in high osmotic buffer, soaking cell more than 20 minutes, Best Times is 1--5 hour, then this cell is changed over to the common nutrient solution that contains allogenic material, use physical method to make cell wall perforation as early as possible, allogenic material just enters in the cell in a large number.
2, according to the described method of claim 1, it is characterized in that preparing high osmotic buffer and use the nontoxic glycitols molecule of pair cell, general formula is C
8H
12-14O
6Or C
12H
22O
11, adopt Tris-hydrochloride buffer or phosphoric acid buffer, pH5.6-7.5.
3, method according to claim 2 is characterized in that the glycitols molecule, preferably uses glucose, inositol, N.F,USP MANNITOL, sorbyl alcohol, sucrose etc.
4, method according to claim 1 is characterized in that the allogenic material that imports viable cell in advance mainly refers to separate the range gene that obtains by the molecular cloning means.
5, method according to claim 1 is characterized in that said physical method is the laser microbeam irradiation, with the metal particle that particle gun quickens, ultrasonic wave, high voltage electric striking.
6, method according to claim 5 is characterized in that laser microbeam irradiation output wavelength is 1060nm, 532nm, 355nm and 266nm, and pulsewidth 10ns, laser export energy when 532nm be 10mJ.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 92103259 CN1078261A (en) | 1992-05-07 | 1992-05-07 | Allogenic material is imported in a large number the method for viable cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 92103259 CN1078261A (en) | 1992-05-07 | 1992-05-07 | Allogenic material is imported in a large number the method for viable cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1078261A true CN1078261A (en) | 1993-11-10 |
Family
ID=4940100
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 92103259 Pending CN1078261A (en) | 1992-05-07 | 1992-05-07 | Allogenic material is imported in a large number the method for viable cell |
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| CN (1) | CN1078261A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420278A (en) * | 2015-12-09 | 2016-03-23 | 苏州大学 | Method for preparing cells carrying exogenous molecules in photoinduced perforating mode, base material for preparing cells and cells |
| CN109468309A (en) * | 2018-12-12 | 2019-03-15 | 怀化市共生农业系统工程研究所(普通合伙) | A kind of method that plasm Micro trauma outside imports living cells |
| WO2023001156A1 (en) * | 2021-07-19 | 2023-01-26 | Wuhan University | Compositions and methods for effective delivery of polynucleotides to cells |
-
1992
- 1992-05-07 CN CN 92103259 patent/CN1078261A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420278A (en) * | 2015-12-09 | 2016-03-23 | 苏州大学 | Method for preparing cells carrying exogenous molecules in photoinduced perforating mode, base material for preparing cells and cells |
| CN109468309A (en) * | 2018-12-12 | 2019-03-15 | 怀化市共生农业系统工程研究所(普通合伙) | A kind of method that plasm Micro trauma outside imports living cells |
| WO2023001156A1 (en) * | 2021-07-19 | 2023-01-26 | Wuhan University | Compositions and methods for effective delivery of polynucleotides to cells |
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