Difference between revisions of "Part:BBa K1440000:Experience"
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'''===Applications of BBa_K1440000=== | '''===Applications of BBa_K1440000=== | ||
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This part originates from BBa_K812032 and BBa_K747096. This part is used to test the cre-TM system and pTRE system. It consists of loxp sites and pTRE promoter. In this part, although we use mCFP as the reporter gene, we find that the efficiency of mCFP is not quite good, so we replace the mCFP reporter gene with the EGFP to test the cre-TM system. And because it is a big part, so we also replace the mCFP with OSKM (four transcription factors) binding with T2A linkers, aiming to prove that using the pTRE promoter we found ourselves, we can successfully make fibroblast reprogram to iPSc. | This part originates from BBa_K812032 and BBa_K747096. This part is used to test the cre-TM system and pTRE system. It consists of loxp sites and pTRE promoter. In this part, although we use mCFP as the reporter gene, we find that the efficiency of mCFP is not quite good, so we replace the mCFP reporter gene with the EGFP to test the cre-TM system. And because it is a big part, so we also replace the mCFP with OSKM (four transcription factors) binding with T2A linkers, aiming 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. | + | |
| + | 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. | ||
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| + | [[File:BBa_K1440000_with_CreERT2_TM_test.png.png]] | ||
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Revision as of 07:47, 23 October 2014
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===Applications of BBa_K1440000===
This part originates from BBa_K812032 and BBa_K747096. This part is used to test the cre-TM system and pTRE system. It consists of loxp sites and pTRE promoter. In this part, although we use mCFP as the reporter gene, we find that the efficiency of mCFP is not quite good, so we replace the mCFP reporter gene with the EGFP to test the cre-TM system. And because it is a big part, so we also replace the mCFP with OSKM (four transcription factors) binding with T2A linkers, aiming 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.
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