pSMB_miTuner Plasmid HD2 (BGH(rc)/shRNA10(rc)/sv40(rc)/CMV/Luc2_sv40/CMV/Kozag_hRluc_BGH)
miTuner plasmid used for binding site characterization and expression tuning (BB-2, RFC12).
scroll down for characterization results
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 1
Illegal BamHI site found at 3411
Illegal XhoI site found at 3153
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1537
Illegal NgoMIV site found at 2881
Illegal NgoMIV site found at 2902
Illegal NgoMIV site found at 3174
Illegal AgeI site found at 2605
- 1000Illegal BsaI.rc site found at 4590
Illegal SapI.rc site found at 2787
Promoter efficiency of miRNA kit was checked in HEK T-Rex cells. Therefor 50ng of each construct with different promoter set-ups (table 1) were transfected into HEK 293 T-REx cells in 96-well plate format using FuGENE transfection reagent. As every construct is expressing firefly luciferase (luc2) and renilla luciferase (hRluc) at the same time the setup is unaffected by transfection efficiency and cell number variations. Each sample was transfected and measured by Dual luciferase assay in 8 replicates. As by this time no shRNA has been cloned into plasmid NO knock-down of luc2 is expected and the different expression efficiencies allow for characterization of the different promoters combinations. BBa_K337032 leads to a relative luciferase unit (RLU) of luc2 to hRluc expression of 6 RLU. BBa_K337035 and BBa_K337035 are showing a comparable expression of 12 - 13 RLU, which is in line with the knowledge that both luciferases are driven by the CMV promoter. Hek 293 T-Rex cells stably express the Tet repressor thus allows us to observe very efficient repression of Firefly luciferse expression if a CMV-TetO2 promoter is driving luc2 (BBa_K337038 and BBa_K337046). BBa_K337040 transfection into Hek T-Rex cells results in an expression of 15 RLU. BBa_K337042 and BBa_K337044 are constructed in a way that luc2 is driven by the CMV promoter and hRluc is driven by the RSV promoter and show a comparable expression of 17-20 RLU. This leads to the conclusion that the CMV promoter shows comparable expression to the RSV promoter in Hek T-Rex cell lines.
|part||promoter driving luc2 (Firefly)||promoter driving (Renilla)||promoter driving shRNA expression|
Characterization of BBa_K337035 and BBa_K337036 in different cell lines
If transfecting BBa_K337035 and BBa_K337036 are transfected into different cell lines it is obvious that Hek293T cells are the easiest to transfect with both constructs an expression of 17-22 RLU is to be measured. Hek T-Rex cells are showing and expression level of 12 RLU of both constructs. Hela cells are also showing constant expression levels of 8 RLU with both constructs. A rather low expression of 2RLU is to be seen by transfecting the 2 constructs in Huh7 cells. This might be due to low transfection efficiency of this cell line in general. All together it is to say that BBa_K337035 and BBa_K337036 show comparable expression.