In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.
In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells. The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.
Rice
Heather
H
Center for Neurologic Diseases, Harvard Medical School.
Suth
Seiyam
S
Cavanaugh
William
W
Bai
Jilin
J
Young-Pearse
Tracy L
TL
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D004625:Embryo, Nonmammalian; D005260:Female; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008297:Male; D016501:Neurites / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D010957:Plasmids / Q000032:analysis / Q000235:genetics; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
D049452:Green Fluorescent Proteins; D004247:DNA