CN107022571A - A kind of method of transfected Jurkat cells - Google Patents
A kind of method of transfected Jurkat cells Download PDFInfo
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- CN107022571A CN107022571A CN201710352851.6A CN201710352851A CN107022571A CN 107022571 A CN107022571 A CN 107022571A CN 201710352851 A CN201710352851 A CN 201710352851A CN 107022571 A CN107022571 A CN 107022571A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Abstract
The present invention discloses a kind of method of transfected Jurkat cells, and step includes：0.1mg/mL poly D lysines are added into 24 porocyte culture plates, 4 degree stand overnight, sucking liquid, drying of being uncapped in super-clean bench, are washed with PBS；It is inoculated with Jurkat cell, incubator and cultivates to above-mentioned culture plate, next day removes supernatant, is washed with PBS 3 times, Jurkat cell switchs to " adherent " state；The conventional steps transfected according to attached cell transfect pLL3.7 plasmids, and fluorescence microscopy Microscopic observation GFP expression quantity, determines transfection efficiency after transfecting 24 hours.The present invention is carried out to Jurkat cell after " adherent " processing, can be increased the touch opportunity of liposome complex and cell, be effectively improved transfection efficiency (more than 90%).The adherent processing procedure of the present invention does not influence cell viability, can be to the safe transfection exogenous plasmid of Jurkat cell.
Description
Technical field
The present invention relates to biological technical field, a kind of method of transfected Jurkat cells is particularly belonged to.
Background technology
Foreign gene transfer is that foreign gene is transferred to recipient cell by a kind of method with physics, chemistry or biology
It is interior, and it is allowed to realize the technology of gene magnification and expression in intracellular, it is widely used to modern life science research and clinical doctor
Learn in gene therapy.Conventional foreign gene transition strategy mainly uses calcium phosphate precipitation, DEAE- glucose method and liposome
Infection protocol etc., especially lipofection, which are received, to be widely popularized.However, liposome method is only applicable to attached cell, to outstanding
Floating cell is difficult to obtain gratifying transfection efficiency.Current suspension cell transfection mainly uses electroporation technology, not only needs
Specific electroporation, and to cellular damage it is larger, be difficult to ensure that cell viability.Slow virus and adenovirus mediated rotaring dyeing technology
Although solving the problem of transfection efficiency is low to a certain extent, but operating process is cumbersome, security risk big, applied to gene
Treatment easily causes serious immune response.Therefore, how to ensure that cell viability turns into while high-efficiency transfection suspension cell
Using suspension cell as a urgent problem to be solved of investigation of materials target gene specific function.
Jurkat cell derives from the peripheral blood of acute T- lymphocytic leukemias patient, belongs to suspension cell, is research
One of most widely used cell line in acute T- lymphocytic leukemias pathogenesis and medicine.Existing transfection side
Method, such as liposome transfection, calcium turn and electric shock transfection method transfected Jurkat cells generally existing transfection efficiency is low, cell viability
Poor the problem of, this just greatly limit application of the Jurkat cell in leukaemia Mechanism Study and medicament research and development.Therefore, find
Efficient and safe transfection method, is using pathogenesis of the Jurkat cell as investigation of materials T lymphocytic leukemias and targeting
Treat the technical barrier of leukaemia.
Lysine is a kind of positively charged amino acid, and the poly-D-lysine that molecular weight is more than 70KD can be used for promoting carefully
Born of the same parents' adherent growth.More poly- D-Lys and poly-l-lysine may be used to promote the adherent of cell.Li Molin etc. exists
1mg/mL poly-l-lysine processing Tissue Culture Plate is used in method disclosed in 10 days October in 2012 and suspension is added
The transfection efficiency of cytolipin plasmids.But, the method is not suitable for Jurkat cell.Reason is that poly-l-lysine can
By cell dissociation and to absorb, certain cytotoxicity can be produced by taking in excessive poly-l-lysine, and the concentration used is very
Greatly (1mg/mL), the waste of reagent is caused.
The content of the invention
It is an object of the invention to provide a kind of methods of high, the safe transfected Jurkat cells of transfection efficiency.
A kind of method for transfected Jurkat cells that the present invention is provided, comprises the following steps：
A. the PBS that 150~500 μ L contain the how poly- D-Lys of 0.1mg/mL is added into 24 orifice plates, to the another of 24 orifice plates
An outer hole adds 150~500 μ L PBS as a control group, and 4 degree of refrigerators are stood overnight；The how poly- D-Lys molecular weight is
150~300KD；
B. supernatant is sopped up, PBS is washed 3~5 times, by 0.5~1.5 × 106Individual Jurkat cell is resuspended in 500 μ L and contains 10%
NBCS and without antibiotic RPMI1640 culture mediums, in 37 degree, 5%CO2 incubators culture 10~16 hours,
Then not adherent cell is sopped up, is washed with PBS 3 times, 200~500 micro- Microscopic observation Jurkat cells of μ L PBS are added adherent
Situation calculates relative adherent rate；
C. when relative adherent rate is more than 3, according to the transfection method of attached cell, transfected with 3~9 μ L liposome 2000
1~3 μ g pLL3.7 plasmids containing GFP expressed sequences, 4~6 hours after transfection, replacing contains 10% NBCS
RPMI1640 culture mediums, fluorescence microscopy Microscopic observation GFP expression after 24 hours calculates transfection efficiency.
With 0.1mg/mL how poly- D-Lys and 1.5 × 106Individual Jurkat cell is acted on 38~46 hours, and mtt assay is surveyed
Cell viability, does not make significant difference.
Compared with prior art, beneficial effects of the present invention：
1) present invention is coated with 24 porocyte culture plates for 150~300KD how poly- D-Lys with molecular weight and is followed by advance
Jurkat cell is planted, the Jurkat cell of suspension is become into " adherent " cell, recycles liposome 2000 to be transfected, overcomes
The problem of Jurkat cell hardly possible transfection, transfection efficiency is up to more than 90%.
2) in addition, in 42 hours that transfection process is undergone, how poly- the D-Lys (0.1mg/L) of low concentration are right
The vigor of Jurkat cell does not make significant difference.Therefore, Jurkat is entered the invention provides a kind of transmission DNA of energy high effect nontoxic
The method of cell.The method can provide technical support to study the pathogenesis and targeted therapy of T lymphocytic leukemias.
Brief description of the drawings
Fig. 1 case study on implementation 1 of the present invention is with respect to adherent rate statistical result
Fig. 2 transfection efficiency statistical results of case study on implementation 1 of the present invention
Fig. 3 cell viability testing results of case study on implementation 1 of the present invention
Embodiment
A kind of method of the transfected Jurkat cells of embodiment 1, comprises the following steps：
A. the PBS that 150 μ L contain the how poly- D-Lys of 0.1mg/mL is added into 24 orifice plates, to other the one of 24 orifice plates
Hole adds 150 μ L PBS as a control group, and 4 degree of refrigerators are stood overnight.
B. supernatant is sopped up, PBS is washed 3 times, by 1.5 × 106Individual Jurkat cell is resuspended in the new born bovine that 500 μ L contain 10%
Serum and the RPMI1640 culture mediums without antibiotic, are cultivated 12 hours in 37 degree, 5%CO2 incubators.Then sop up and do not paste
The cell of wall, is washed 3 times with PBS, is washed every time 5 minutes, adds the micro- adherent situations of Microscopic observation Jurkat cell of 300 μ L PBS
(Fig. 1).8 visuals field are randomly selected, the cell number in each visual field, every group of cell number divided by the cell number of control group is recorded, obtains
Go out relative adherent rate.
C. when relative adherent rate is more than 3, according to the transfection method of attached cell, contained with liposome 2000 (3 μ L) transfection
There are the pLL3.7 plasmids (1 μ g) of GFP expressed sequences, 6 hours after transfection, change the RPMI1640 containing 10% NBCS
Culture medium, fluorescence microscopy Microscopic observation GFP expression after 24 hours calculates transfection efficiency (Fig. 2).Transfection efficiency calculating side
Method is that cell transfecting randomly selects 10 visuals field after 24 hours, taken pictures respectively under blue light and visible ray after each visual field is selected,
The cell number A1, A2, A3 ... ... of fluoresced green in each visual field, A10 and TCS B1, B2 are counted,
B3 ... ..., B10, with A1/B1 ... ..., A10/B10 average value ± SD, which does statistical analysis and does figure, to be transfected
Efficiency.
With the PBS and 1.5 × 10 of 0.1mg/mL how poly- D-Lys6Individual Jurkat cell is acted on 42 hours, mtt assay
Survey cell viability (Fig. 3).PBS is control group, i.e., with isometric PBS coated cell culture plates.Drawn by t-test detections
The how poly- D-Lys of conclusion 0.1mg/mL do not make significant difference to Jurkat cell vigor.
Claims (2)
1. a kind of method of transfected Jurkat cells, it is characterised in that comprise the following steps：
A. the PBS that 150~500 μ L contain the how poly- D-Lys of 0.1mg/mL is added into 24 orifice plates, to other the one of 24 orifice plates
Hole adds 150~500 μ L PBS as a control group, and 4 degree of refrigerators are stood overnight；
B. supernatant is sopped up, PBS is washed 3~5 times, by 0.5~1.5 × 106Individual Jurkat cell be resuspended in 500 μ L contain 10% it is new
Raw cow's serum and the RPMI1640 culture mediums without antibiotic, culture 10~16 hours in 37 degree, 5%CO2 incubators, then
Not adherent cell is sopped up, is washed with PBS 3 times, the micro- adherent situations of Microscopic observation Jurkat cell of 200~500 μ L PBS are added
Calculate relative adherent rate；
C. when relative adherent rate is more than 3, according to the transfection method of attached cell, 1~3 is transfected with 3~9 μ L liposome 2000
The μ g pLL3.7 plasmids containing GFP expressed sequences, 4~6 hours after transfection, replacing contains 10% NBCS
RPMI1640 culture mediums, fluorescence microscopy Microscopic observation GFP expression after 24 hours calculates transfection efficiency.
2. a kind of method of transfected Jurkat cells as claimed in claim 1, it is characterised in that the how poly- D-Lys point
Son amount is 150~300KD.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157739A (en) * | 2019-05-14 | 2019-08-23 | 天津市康婷生物工程集团有限公司 | A kind of method that can improve lymthoma Daudi cell transfecting marker representation amount proportion in cell mixing |
| CN110157740A (en) * | 2019-05-14 | 2019-08-23 | 天津市康婷生物工程集团有限公司 | A kind of method that can improve Lymphoma Raji Cells transfection markers object expression quantity proportion in cell mixing |
| WO2021031716A1 (en) | 2019-08-22 | 2021-02-25 | 浙江道尔生物科技有限公司 | Anti-pd-l1 single domain antibodies |
| WO2022174781A1 (en) | 2021-02-22 | 2022-08-25 | 浙江道尔生物科技有限公司 | Multi-domain fusion protein and use thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157739A (en) * | 2019-05-14 | 2019-08-23 | 天津市康婷生物工程集团有限公司 | A kind of method that can improve lymthoma Daudi cell transfecting marker representation amount proportion in cell mixing |
| CN110157740A (en) * | 2019-05-14 | 2019-08-23 | 天津市康婷生物工程集团有限公司 | A kind of method that can improve Lymphoma Raji Cells transfection markers object expression quantity proportion in cell mixing |
| WO2021031716A1 (en) | 2019-08-22 | 2021-02-25 | 浙江道尔生物科技有限公司 | Anti-pd-l1 single domain antibodies |
| WO2022174781A1 (en) | 2021-02-22 | 2022-08-25 | 浙江道尔生物科技有限公司 | Multi-domain fusion protein and use thereof |
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Application publication date: 20170808 |