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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Optogenetic and multiplexed gene editing in primary T-cells.

Lake, Daniel January 2023 (has links)
Current T-cell tracking techniques in vivo are limited. The ability to successfully target a gene in vivo in T-cells and track movement throughout its life cycle provides an exciting opportunity to elucidate the functions of genes. The aim of this study was to test an optogentically inducible Cre recombinase as well as a self-cleaving gRNA which can find and associate with Cas9 in vivo. Mouse T-cells which consecutively produce Cas9 (Cas 9, Jackson laboratory) were transduced and transplanted in immunodeficient mice (TCRb-/-, Jackson laboratory). The optogenetic component of the system is activated upon blue light stimulation and is introduced to the T-cell through a mouse stem cell virus (MSCV). The TCRb-/- mice underwent surgery which exposed their lymph nodes to blue light pulses from a fibre optic wire, this process is referred to as blue light surgery. BLU-VIPR T-cells which express self-cleaving gRNAs reduced the relative abundance of the target protein (Thy1.2), after blue light surgery in vivo. Furthermore, the optogenetic system showed minimal leakiness when used for gene targeting using gRNAs. This suggests that the gRNAs had associated with Cas9 and were able to successfully target the Thy1.2 gene. Results from the optogentically induced Cre recombinase showed that Cre was expressed in significant amounts without blue light stimulation, suggesting some background leakiness in the BLU-VIPR system.

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