Team:UT-Tokyo/CTCD/Content

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Fig.3 the construct of our circuit

EGP-2 promoter

EGP-2 promoter is the promoter of Epithelial cell adhesion molecule (EpCAM) protein.EpCAM is a trans membrane glycoprotein which is expressed in epithelial cells and shows high level expression in various type of human epithelial carcinomas[2].Using this promoter, reporter genes can be expressed only in cancer cells. EGP-2 promoter is originally about 3500bp and it is not convenient to make a DNA construct. But, in the previous research, it is known that EGP-2 promoter can work if a part of its sequence is deleted[3]. In this project ,We cloned a part of EGP-2 promoter, and made it useful as a biobrick parts.

miRNA-142

MiRNA is a class of non-coding RNA.MiRNA binds the miRNA-recognition element in the 3' untranslated region of the target gene. MiRNA shows the tissue-specific expression pattern. In our project, we use miRNA-142, which is expressed only in hematopoietic cells[4].After miRNA-142 is transcribed, it is cleaved to miRNA-142-5p and miRNA-142-3p. Adding the recognition element of miRNA-142 in the 3' untranslated region of the reporter genes, leaky expression in non-carcinoma hematopoietic cells can be reduced.

Fig.4 When this construct is transfected to the epithelial cells like cancer cell, pEGP-2 promote the transcription of EGFP, and the mRNA is not degraded by miRNA because miR 142 is specific to hematopoietic cells.

Fig.5 When this construct is transfected to the hematopoietic cells, pEGP-2 does not work as a promoter, and mRNA is not transcribed. Even if the leak of the promoter makes few mRNA ,this mRNA is degraded by RNAi caused by miR 142.

We focused on a tissue-specific promoter and miRNA in order to detect CTCs.

As well as hematopoietic cells, many other tissues shows specific miRNA expression patterns[3]. Therefore, if by making a circuit that returns an output to a certain miRNA expression pattern, we may identify where a CTC comes from.

In addition to the detection of CTCs, combinations of tissue-specific promoter and miRNA can be applied to many treatments. For example, suicide genes can be expressed in cancer cells in a specific tissue.