Part:BBa_K3756013
RGD-Lamp2b-TSLP-siRNA-2
This DNA circuit is designed to express specific proteins and siRNA that specifically target TH2 cells and down-regulate the expression level of relevant target mRNA. It can express the fusion protein RGD-Lam2p, in which RGD is an epithelial-targeting peptide, and therefore can specifically target epithelial integrin. Lamp2b is an exosomal membrane protein. The fusion protein Rgd-lamp2b was thus able to target exosomes secreted by cells expressing the protein to epithelial cells. At the same time, the DNA circuit also expresses the siRNA of TSLP, which specifically degrades TSLP, an important factor involved in type 2 inflammation. Thus, cells expressing this DNA circuit could express siRNA of TSLP and specifically target TSLP in epithelial cells with the help of RGD-Lamp2b, thereby inhibiting its expression. Most importantly, the DNA circuit has attP and attB sites on both sides of the expression frame. In E. coli ZYCY10P3S2T expression system, Intergrase Φ31 can loop out the sequence between attP and attB to form minicircle DNA. The plasmid skeleton will be cleaved by I-SCEL enzyme. (Kay MA, et al. A Robust System for Production of Minicircle DNA Vectors. 2010) In our project, the DNA circuit will be prepared in E.coli ZYCY10P3S2T expression system to form Minicircle DNA, and then the latter will be used to verify its therapeutic effect on asthma mice by transfection of mouse lung adenoma cell line LA-4 and aerosol administration into mice. Details can be found in the project wiki.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1132
Illegal SpeI site found at 445
Illegal PstI site found at 829 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 445
Illegal PstI site found at 829 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1132
Illegal SpeI site found at 445
Illegal PstI site found at 829 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1132
Illegal SpeI site found at 445
Illegal PstI site found at 829 - 1000COMPATIBLE WITH RFC[1000]
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