CAAR transduction in cell line and measurement of expression levels
The validation of our CAAR T cell development requires a comprehensive series of steps. The process starts with the transfection of HEK293T cells with appropriate plasmids to produce the lentiviral vector. Following this, primary T-lymphocyte cell lines were expanded in culture. These expanded T-cells were then transduced with the lentiviral vectors to introduce the CAAR construct. The expression of CAAR has to be confirmed by staining the cells with specific antibodies. The functionality of the CAAR T cells is to be further evaluated through a range of assays, including assessments of cytotoxicity, proliferation, and cytokine production. The purpose of these evaluations was to demonstrate that the CAAR T cells effectively carried out their intended functions, supporting their potential for use in therapeutic applications.
CRISPR-dCAS9-VPR epigenetic induction of FOXP3
The goal of the experiments is to clone a pair of oligos into a lentiviral CRISPR vector for sgRNA expression. The lentiviral CRISPR plasmid has to be digested and dephosphorylated with BsmBI, followed by gel purification to isolate the desired vector fragment. Oligos have to be phosphorylated, annealed and ligated into the digested plasmid. After setting up the ligation reaction, a negative control also has to be prepared. The ligated product is then transformed into Stbl3 bacteria for propagation and further use.
T structure and H hairpins hybridization
After successfully designing and in silico testing both types of hairpins for the mechanism, it was necessary to validate their predicted function in the lab. This means assessing their hybridization of T-structures and subsequent release of the initiator, its hybridization to the HCR hairpins and the formation of the final four-part hybridisation chain reaction that releases the therapeutic molecules. After molecule behavior is verified via fluorescence and native gel electrophoresis it is important to determine the ideal quantity for their activation.