| OCR Text |
Show 7 these approaches (Bai et al., 2003; Borrell et al., 2005; Nakahira and Yuasa, 2005; Saito and Nakatsuji, 2001; Tabata and Nakajima, 2001). Following injection of DNA into the lateral ventricles of the embryonic brain, a transuterine electric field is applied to facilitate transfection of glia and neuron progenitors which eventually divide and migrate throughout the brain. The first objective of this thesis work was to design and build a toolset of plasmid constructs, carrying GCaMP variants to be used for calcium imaging of the rat brain following IUE. In Chapter 2, a characterization of these constructs is presented, demonstrating stable and functional expression of GCaMP in astrocytes and neurons in adult acute cortical slices or hippocampal organotypic slice cultures. Due to well-established methods for manipulating the mouse genome, it is the preferred model in which to express GECIs. Existing transgenic lines expressing GCaMPs under the control of Thy1 promoters have demonstrated that noninvasive genetic approaches exhibit good signal and viability of expressing cells (Chen et al., 2012; Dana et al., 2014). Cre/lox technology offers robust cell-type specific and reproducible expression as has been demonstrated with the Cre-dependent GCaMP3 reporter (Ai38) line (Zariwala et al., 2012). The second objective of this thesis work was to build an improved conditional GCaMP-expressing mouse line. In Chapter 3, I describe the Cre-dependent GCaMP5G-expressing reporter mouse line, termed PC::G5-tdT (Polr2a, CAG, GCaMP5G, tdTomato; Gee et al., 2014). The PC::G5-tdT line enjoys significant functional enhancements over previously generated GECI reporter mice including co-expression of tdTomato in order to facilitate the identification of quiescent cells. By targeting the construct to the intergenic region 3' of the ubiquitously expressed Polr2a gene, transcriptional integrity of the mouse genome was maintained. Robust calcium activity was observed in astrocytes, microglia and various neuronal subtypes, both in somas and fine |
