Part:BBa_K2446051
surface-EGFP
his part is the surface-expressed EGFP (surEGFP), Surface-expressed EGFP (surEGFP) is built by fusing CD8α signal peptid and EGFP to the transmembrane region of CD86 (NM_175862) from N terminal to C terminal. CD8α peptide guides surEGFP to be located to cell membrane. transmembrane region of CD86 supports surEGFP to fix on membrane.
surEGFP is the surface antigen of our SynNotch-anti-EGFPBBa_K2446055, and also the core part of the[http://2017.igem.org/Team:Fudan/Demonstrate surface antigen density heterogeneity model] Aantigen density has been considered as an obstacle to improve cellular immunotherapy for tumor [1] , rather than an opportunity to employ diverse and/or combined treatment. To demonstrate the feasibility of utilizing antigen density heterogeneity, we first established a cell model that is able to stably express different antigen densities on cell surface. We chose surEGFP as representative antigen due to the convenience of detecting, quantifying and observing.
Figure 1: Sketch map shows surface-expressed EGFP density heterogeneity model.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experiments
Confocal
We cotransfect HEK293T with target plasmids, two virus packing plasmids (CMV-VSVG & PCL) in lipofectine 2000 to pack retrovirus. After 48h of expression we filer the medium and get the virus solution and then add the solution to the medium of wide-type Hela cells. After 48h of acculturation the cells are ready to be screened. We use flow cytometer and screen out cells highly expressing GFP signal with FITC channel. After several days of culture expansion we use confocal microscope to capture cell state.
Figure 2 :Image shows that the endoplasmic reticulum (ER) membrane has strong fluorescence, while the plasma membrane has relative weak signal. It consists with the process that surEGFP is expressed, translocated and matured inside ER lumen with its transmembrane embed in the ER membrane, and later surEGFP is transported and exposed to culture media when fluorescence declines due to oxidation in the extracellular environment
Three level expression
We also build cell strain of K562 expressing surGFP using the same way as that of Hela cells. After culture expansion we use flow cytometer and screen out K562 expressing different level of surGFP to simulate tumor heterogeneity. Western result indicates that our screening is effective.
Figure 3 :Different gates according to different level of GFP signal enables flow cytometer to screen out K562 heterogeneous in expression of surGFP. The result also shows three sharp peaks indicating little crossover among these group of cells. Western result giving different level of GFP signal with little difference in GAPDH, indicating effectiveness in screening.
References
[1] K. Newick, S. O'Brien, E. Moon, S. M. Albelda, CAR T Cell Therapy for Solid Tumors. Annual Review of Medicine 68, 139--152 (2017).| None |