Rapid genotyping of animals followed by establishing primary cultures of brain neurons.
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.
Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine; Department of Psychiatry, University of Iowa Carver College of Medicine.
Jin-Young
JY
Koh
Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine.
Sadahiro
S
Iwabuchi
EZ BioResearch LLC.
Zhengmin
Z
Huang
Department of Molecular Physiology & Biophysics, University of Iowa Carver College of Medicine; charles-harata@uiowa.edu.
N Charles
NC
Harata
D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D004195:Disease Models, Animal; D020821:Dystonic Disorders / Q000235:genetics; D060005:Genotyping Techniques / Q000379:methods*; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics; D009474:Neurons / Q000166:cytology*; D051381:Rats
C491548:Dyt1 protein, mouse; D018832:Molecular Chaperones