Transfecting avian motor neurons and their axons using in ovo electroporation.

INTRODUCTIONIn ovo electroporation of half of the avian neural tube is a simple procedure in which one places the electrodes parallel to the neural tube, flanking the intended axial region of transfection. It is possible to modify this technique to target the ventral quadrant of the neural tube that contains motor neurons in the lateral motor column (LMC) and their axons by positioning the electrodes in an offset dorsal/ventral configuration, instead of the standard parallel position. If the electroporation is performed in the neural tube of stage 15 chick embryos, the medial portion of the LMC is targeted primarily; however, if neural tubes of stage 17 embryos are electroporated, the entire LMC will be transfected. This technique can be used to examine the behavior of motor axons as they project into the developing limb when genes are misexpressed, overexpressed, or knocked down via RNAi (using short hairpin RNA [shRNA]). The un-electroporated half of the neural tube serves as an internal control, or an enhanced green fluorescent protein (EGFP) reporter construct (pCAX) serves as a control for the electroporation and for EGFP expression. By electroporating a DNA construct that contains EGFP, or co-electroporating the DNA of interest with a GFP reporter construct, it is possible to verify the success of the electroporation in ovo.