Part:BBa_K1440000:Experience
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===Applications of BBa_K1440000===
This part originates from BBa_K812032. This part is used to test the cre-TM system and pTRE system. It consists of loxp sites and pTRE promoter. both pTRE and cre-TM parts have not been used before in iGEM competition. So we introduce these two parts in this new part. In this part, although we use mCFP as the reporter gene, we find that the mCFP reporter gene can not even work. So we replace the mCFP reporter gene with the EGFP to test the cre-TM system. We also replace the mCFP with OSKM (four transcription factors) binding with T2A linkers to prove that using the pTRE promoter we found ourselves, we can successfully make fibroblast reprogram to iPSc.However, owning to this part 2 with OSKM together is more than 5kb, we try several times still fail to link itself to PSB1C3 together.
We will only show the result with EGFP and OSKM. Firstly, we show the data about Cre-TM system, we cotransfect the Cre_ERT2 (BBa_K1440001) plasmid and this part (mCFP replaced with EGFP).We choose HEK293 cells to do transfection. We plant the cells in a 24-wells dish. Before transfection, we make sure the density of the cell is about 50-70%. We use lipofectamine 2000 as the transfection reagent. After 24hr, we dose each dish with certain concentration of tamoxifen. And at 36hr, we get the result.
We analyze the data by cell counting with the help of blood cell counting chamber. We use "GFP-positive cells numbers/total cell numbers" to indicate the transfection efficiency（= 0.327） in "Control-GFP" cell plate. We use the same method to count the tamoxifen (0umol/ml, 1umol/ml, 2umol/ml, 5umol/ml, 10umol/ml ) induced GFP expression.(See figure below)
Then we construct the concept "REU"(Relative expression unit) that ((Cell density of GFP-positive cells by tamoxifen inducing/ total cells)/(Cell density of GFP-positive cells of control-GFP/ total control-GFP cells) to indicate the efficiency of tamoxifen system. The data show that Conc. tamoxifen 5umol/ml is the best condition for this system to work, and the inducing efficiency is more than 60%. Besides, the leakage of this system is only 0.9%, which indicates that it is a quite controllable system.
Then we use this part to test somatic cell reprogramming. We replace the mCFP gene with the OSKM four transcription factor binding with T2A linker. We make this part transfer to a kind of plasmid "plenti-OSKM" which can be used to package lenti virus. Then we use the lenti virus to stably transfect this part (mCFP replaced with OSKM) into MEF cells(mouse embroynic fibroblast). After 12 hours, we dose the cell plate with 1ug/ml dox. After 15 days cell culture, we get the result.
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