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 efficiency of mCFP reporter gene can not even work. So we replace the mCFP reporter gene with the EGFP to test the cre-TM system. And because it is a really big part, 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.
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.
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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