Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 111, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/53783
Keywords
Neuroscience; Issue 111; Lentivirus; Retrovirus; Viral Packaging; High Titer; in vivo injection; Intracerebroventricular injection; CaPO4 Transfection
Categories
Funding
- NINH [R01MH097949]
- Autism Speaks Pilot Grant [7359]
- Norris Cotton Cancer Center Optical Imaging Shared Instrumentation Grant [P30CA023108]
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Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.
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