NK (Natural Killer) trophoblast cell for expressing cytokine composition as well as preparation method and application of NK trophoblast cell
A technology of trophoblast cells and cells, applied in the field of NK trophoblast cells and their preparation
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Embodiment 1
[0059] Embodiment 1 lentiviral vector construction
[0060] Whole gene synthesis tandem CD19 coding sequence and CD86 coding sequence KCD19CD86 (SEQ ID NO:6), IL-21-CD8α coding sequence KIL21CD8α (SEQ ID NO:7), CD64 coding sequence KCD64 (SEQ ID NO:8), CD137L Coding sequence (SEQ ID NO:9), and restriction site BamHI / EcoRI or BamHI / NotI are added at both ends;
[0061] Prepare the enzyme digestion reaction system shown in Table 1, digest the synthetic gene in a water bath at 30°C for 1 hour and a water bath at 37°C for 1.5 hours, and then connect it into the linearized lentiviral vector pCDH digested with the same restriction endonuclease ;
[0062] Table 1
[0063] components volume Ultra-pure water 16μL BamHI 2.5μL EcoRI / NotI 2.5μL 10×K Buffer 2.5μL 0.1%BSA 5μL dna 22μL (5μg)
[0064] The digested products were detected by agarose gel electrophoresis. The electrophoresis condition was 120V constant current electroph...
Embodiment 2
[0065] Example 2 Recombinant lentivirus packaging and titer detection
[0066] In this example, the calcium phosphate transfection method (Beyotime-Calcium Phosphate Cell Transfection Kit, product number C0508) was used to co-transfect 293T cells with the lentiviral expression vector and the packaging plasmid, and the lentivirus was packaged, and the centrifugal ultrafiltration method (Millipore-Centrifugal Filter Units Amicn (R) Ultra-15, product number UFC910096, 100kD) for collection and concentration of lentivirus, the steps are as follows:
[0067] (1) 24 hours before transfection, digest 293T cells in the logarithmic growth phase with 0.125% trypsin (the degree of cell confluence is 85-95%), and use complete medium DMEM containing 10% fetal bovine serum + 1mg / mL double antibody Adjust the cell density and passage, re-seeded in the d15 culture dish, 37 ℃, 5% CO 2 , cultivated under saturated humidity;
[0068] (2) After 24 hours, replace the cell culture medium with fre...
Embodiment 3
[0077] Construction and identification of embodiment 3 NK trophoblast cells
[0078] Inoculate 3mL of K562 cell suspension in a good growth state into a 60mm culture dish, add the packaged recombinant lentivirus to K562 cells according to the ratio of MOI=100:1, and add polybrene (8μg / mL) to carry out lentiviral Transfection, puromycin selection to obtain K562 expressing CD19, CD86, IL21, CD64 and CD137L, as NK trophoblast cells;
[0079] take 10 7 Add 1mL Trizol to NK trophoblast cells, blow and mix with a pipette tip to promote cell lysis, and place at room temperature for 5min; add 0.2mL chloroform, mix by inverting repeatedly for 15s, centrifuge at 12000g for 10min at 4°C; take the upper aqueous phase in a new Add 0.5mL isopropanol to the centrifuge tube, mix gently, place at room temperature for 10min, centrifuge at 12000g at 4°C for 10min; discard the supernatant, add 1mL of 75% alcohol to gently wash the precipitate, and centrifuge at 12000g at 4°C for 10min; Discard ...
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