Journal: Toxins

Article Title: The Bacterial Enzyme Cas13 Interferes with Neurite Outgrowth from Cultured Cortical Neurons

doi: 10.3390/toxins13040262

Figure Lengend Snippet: Neuron-specific Cas13 expression. ( A ) A schematic view of the neuron-specific Cas13 expression construct; ( B ) Distribution of LwaCas13a-GFP or GFP expression observed after two days in cultured mouse cortical neurons. LwaCas13a-GFP remains mostly restricted to the cell soma. Scale bar is 20 μm.

Article Snippet: Primary mouse cortical neuron cultures were prepared from neonatal mice according to the optimized protocol for primary mouse hippocampal and cortical neurons from Lonza.

Techniques: Expressing, Construct, Cell Culture

Journal: Neuron

Article Title: Plug and Play Protein Modification using Homology-independent Universal Genome Engineering

doi: 10.1016/j.neuron.2019.05.047

Figure Lengend Snippet: (A) Schematic of HiUGE KI application for C- or N-term protein labeling in vitro. Primary hippocampal cells from Cas9 mice were transduced with a combination of GS-gRNA and HiUGE donor AAVs and immunostained to detect epitope or smFP-HA labeling, with representative images displayed in panels B-S. (B-M) Examples of C-term HA-epitope KI to diverse targets (mouse Tubb3, Map2, Mecp2, Actr2, Clta, Nrcam, Ank3, Sptbn4, Scn2a, Gfap, Pdha1, and Dcx), showing the expected expression patterns of the translated proteins respectively. (N-P) C-term smFP-HA KI to mouse Insyn1, Insyn2, and Arhgap32, which encode the inhibitory postsynaptic density (iPSD) proteomic candidates. Colocalization of the HA-immunoreactivity with the juxtaposed inhibitory presynaptic marker vesicular GABA transporter (VGAT) immunosignal is shown in the insets. (Q-S) N-term Myc-epitope KI to mouse Actb, Lmnb1, and Nefm, showing the expected expression patterns of the translated proteins respectively. Scale bar is indicated in each panel, or within insets (2μm). GFP fluorescence of the Cas9-2A-GFP and nuclei labeling with DAPI are also shown. Arrowheads represent the subcellular features associated with the targeted genes, such as the dendritic spines, AIS, mitochondria, distal end of neurites, inhibitory synapses, and neurofilaments.

Article Snippet: Primary mouse hippocampal and cortical neuron / glia culture Primary neuron / glia cultures were derived from Gt(ROSA)26Sor tm1(CAG-cas9 * ,-EGFP)Fezh or C57BL/6J (Jackson Laboratory) neonatal pups (P0-P2).

Techniques: Labeling, In Vitro, Transduction, Expressing, Marker, Fluorescence