Difference between revisions of "Part:BBa K3244025"

Latest revision as of 22:28, 21 October 2019

PD-1, NR4A and TOX siRNA cassettes for optimizing CAR-T cells for solid tumors

[http://2019.igem.org/Team:AFCM-Egypt# Team:AFCM-Egypt 2019] have added Part BBa_K3244024 (siRNA cassettes for optimizing CAR-T cells for solid tumors) has been composited into part BBa_K3244025 which consists of triple cassettes for U6, TOX and NR4A each consists PD-1 promoter, sense, hairpin loop, antisense and terminator. To form vector you will need RSV promoter, U6 promoter. bGh poly(A) for termination in mammalian cells. In our case we also added AmpR and AmpR promoter to compensate for the unavailability of chloramphenicol for research purposes. Functional characterization of BBa_K3244025: We performed gene Expression analysis of cultured T cell that was isolated from BC patients and engineered by using our first circuit by Real time quantitative PCR (qPCR) using Specific oligonucleotide sequences for NR4a and PD-1. A significant downregulation of PD1 and NR4a was observed in engineered patient-derived T-cells compared to un-engineered cells (p<0.01), however, higher levels were obtained in cells derived from the Schistosoma associated BC patient, although significance was not achieved (p<0.05). On the other hand, NR4a was decreased in engineered T cells (p<0.05). The gene expression was calculated according to un-engineered T cells (negative control)

For results of functional characterization please refer to our parts Page EGYPT-AFCM 2019 Parts Sequence and Features