Gene engineering cell and application thereof in NK (Nature Killer) cell proliferation
A technology of genetic engineering and NK cells, which is applied in the field of in vitro preparation of NK cells in NK cell immunotherapy, can solve the problems of NK cell proliferation limitation, etc., and achieve the effect of long duration of expansion, simple amplification method and high purity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0034] Embodiment 1, the preparation method of genetic engineering cell 4-1BBL-mbIL-21-K562
[0035] 1. Purchase the 4-1BBL / PCR4 TOPO plasmid from Open Biosysems (Thermo Fisher Scientific, CO, USA), amplify it by PCR, and insert it into the Nhe I-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct 4-1BBL / pSBSO transposon.
[0036] 2. Co-transfect K562 cells with 4-1BBL / pSBSO transposon and transposase SB11, and sort by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).
[0037] 3. IL-21-Fc(CoOp)-pSBSO and transposase SB11 were co-transfected into 4-1BBL-K562 cells and sorted by flow cytometry to establish 4-1BBL-mbIL-21-K562 genetically engineered cells. Flow cytometry analysis confirmed that 4-1BBL-mbIL-21-K562 cell surface had obvious expression of 4-1BBL and IL-21 ( figure 2 ).
Embodiment 2
[0038] Embodiment 2, NK cell expansion method
[0039] 1. Purchase the 4-1BBL / PCR4 TOPO plasmid from Open Biosysems (Thermo Fisher Scientific, CO, USA), amplify it by PCR, and insert it into the Nhe I-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct 4-1BBL / pSBSO transposon.
[0040] 2. Co-transfect K562 cells with 4-1BBL / pSBSO transposon and transposase SB11, and sort by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).
[0041] 3. IL-21-Fc(CoOp)-pSBSO and transposase SB11 were co-transfected into 4-1BBL-K562 cells and sorted by flow cytometry to establish 4-1BBL-mbIL-21-K562 genetically engineered cells.
[0042] 4. Separate human peripheral blood mononuclear cells with lymphocyte separation medium.
[0043]5. The genetically engineered cells were irradiated with 100Gy radiation, or treated with 15ug / mL mitomycin for 4 hours; after that, the cells were washed 2~3 times with PBS.
[0044] 6. Pretreated genetic...
Embodiment 3
[0047] Example 3, NK cell expansion mechanism research
[0048] 1. Purchase the 4-1BBL / PCR4 TOPO plasmid from Open Biosysems (Thermo Fisher Scientific, CO, USA), amplify it by PCR, and insert it into the Nhe I-XhoI site of the GlySer-EGFP(CoOp)-pSBSO Sleeping Beauty expression vector to construct 4-1BBL / pSBSO transposon.
[0049] 2. Co-transfect K562 cells with 4-1BBL / pSBSO transposon and transposase SB11, and sort by flow cytometry to obtain K562 cells stably expressing 4-1BBL (4-1BBL-K562).
[0050] 3. IL-21-Fc(CoOp)-pSBSO and transposase SB11 were co-transfected into 4-1BBL-K562 cells and sorted by flow cytometry to establish 4-1BBL-mbIL-21-K562 genetically engineered cells.
[0051] 4. Separate human peripheral blood mononuclear cells with lymphocyte separation medium.
[0052] 5. The genetically engineered cells were irradiated with 100Gy radiation, or treated with 15μg / mL mitomycin for 4 hours; after that, the cells were washed 2~3 times with PBS.
[0053] 6. Pretreat...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com