Difference between revisions of "Part:BBa K3335005"

Latest revision as of 18:14, 25 October 2020

We used it to express iRGD-Lamp2b and siRNA targeted at CD47

Tumor cells escape macrophage phagocytosis by overexpressing CD47.Therefore, we combined the fusion expression of iRGD and Lamp2b to characterize the exosome targeting.At the same time, siRNA targeting CD47 was produced, and mRNA expression was targeted to be reduced.This allows macrophages to avoid the effects of CD47 and attack tumor cells.

CD47 (Cluster of Differentiation 47) also known as integrin associated protein (IAP) is a transmembrane protein that in humans is encoded by the CD47 gene. CD47 belongs to the immunoglobulin superfamily and partners with membrane integrins and also binds the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). CD-47 acts as a don't eat me signal to macrophages of the immune system. Due to the ubiquitous expression of CD47, signaling differs according to cell type. Both activation and loss of CD47 can result in enhanced proliferation. And CD47 ligation leads to cell death in many normal and tumor cell lines via apoptosis or autophagy. Since then, CD47 has been found to be expressed on multiple human tumor types including acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, and other solid tumors. High levels of CD47 allows cancer cells to avoid phagocytosis despite having a higher level of calreticulin - the dominant pro-phagocytic signal.

CD47 siRNA overexpressed in HEK293T cell and exosome

First, we detected the correct expression of CD47 siRNA through RT-QPCR to ensure that sufficient targeted siRNA could be produced in cell. CMV-iRGD-siRC is this part. CMV-iRGD-siRP+C+K is a Composite part.

Figure.1 CD47 siRNA is overexpressed is HEK293T cell

Figure.2 CD47 siRNA is overexpressed in exosome

Figure.3 Absolute quantification of CD47 siRNA

At the same time, We detected siRNA in exosomes produced by HEK293T cells, confirmed that siRNA could be properly wrapped into exosomes, and prepared for its role (Figure.2).

According to the absolute quantification of CD47 siRNA, we found that the amount of siRNA in HEK293T cells is 5.710E-04 pM, the amount of siRNA in exosome is 2.544E-05 pM. The exosome siRNA is equivalent to 4.46% of the siRNA concentration in the cell.

Sequence and Features