Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

Abstract

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences. Video LinkThe video component of this article can be found at

Localization of the signal of dystonia-associated protein torsinA near the Golgi apparatus in cultured central neurons

“…Thus the torsinA proteins are not overexpressed in the ΔE-torsinA knock-in mice, and in this respect, they closely resemble fibroblasts of human patients with DYT1 dystonia (Goodchild and Dauer, 2004;Goodchild et al, 2005;Hewett et al, 2008). We examined torsinA localization in primary cultures of central neurons obtained from the cerebral cortex, striatum and hippocampus, prepared as reported previously (Kakazu et al, 2012a;Kakazu et al, 2012b;Iwabuchi et al, 2013a;Iwabuchi et al, 2013b;Koh et al, 2013;Koh et al, 2015). The use of such cultures enabled us to examine the neurons of homozygous mice, which normally do not survive to adulthood (Dang et al, 2005;Goodchild et al, 2005;Tanabe et al, 2012).…”

“…Neurons from individual newborn mouse pups were cultured separately by a method described previously (Koh et al, 2013;Koh et al, 2015). In brief, the cerebral cortex, striatum and hippocampus were dissected at postnatal days 0-1.…”

“…The cells were cultured in a humidified incubator at 37°C, with 5% CO 2. Rat hippocampal neurons were isolated and cultured using the same method (Koh et al, 2015), and plated at a density of 6,000 cells per well. The cultured neurons were analyzed after 14-19 days in vitro (DIV).…”

Transcriptome profiles in brains of mice heterozygous for a DYT1 dystonia-associated mutation in the endogenous Tor1a gene

“…Every effort was made to minimize suffering of the animals. On postnatal days 0-1, pups of both sexes of the ΔE-torsinA knock-in model of DYT1 dystonia (Goodchild et al, 2005) were genotyped according to a fastgenotyping procedure (within ~5 hours, EZ Fast Tissue/Tail PCR Genotyping Kit, EZ BioResearch LLC, St, Louis, MO, USA) (Koh et al, 2015). The pups were identified long-term by tattooing the paw pads (NEO-9 Neonate Rodent Tattooing System, Animal Identification and Marking Systems Inc, Hornell, NY, USA) (Koh et al, 2015).…”

“…These sizes of the brain regions were large enough that RNA from a single brain was sufficient for a single profiling experiment. To ensure consistency between samples, all dissections were carried out by the same, experienced neuroscientist who has cultured primary neurons from these regions (Koh et al, 2015). After meninges were carefully removed from the surface, the brain regions were transferred to individual, dry, ice-cold RNase-free tubes.…”