Basic NeuroscienceThe combination of limb-bud removal and in ovo electroporation techniques: A new powerful method to study gene function in motoneurons undergoing lesion-induced cell death

- A method combining induced motoneuron death and gene overexpression is described.
- The method is performed on chicken embryos developing in ovo.
- The electroporation rate of motoneurons is 40-50%.
- The survival rate one day after is 95% and decreases to 80% three days after.

BackgroundThe chicken embryo is an important model organism for developmental biology studies. At present, many techniques on this model have been set up, from surgical procedures to molecular biology methods, to answer capital questions of cell biology. The study of the genes involved in motoneurons (MNs) survival and cell death is critical for a better understanding of the molecular mechanisms leading to MNs degenerative diseases, such as amyothophic lateral sclerosis (ALS) and motor peripheral neuropathies.New methodHere, we describe the combination of a well known surgical procedure able to induce MNs cell death, the limb-bud removal (LBR), with a very popular method used in molecular biology to test gene function in living organisms, the in ovo electroporation (IOE). The aim of this work is to provide an effective method for the investigation of genes involved in MNs survival and cell death under lesion conditions.ResultsOur method allows the successful electroporation of the 40-50% of MNs on the side of LBR with a high survival rate early and late after procedure.Comparison with other methodsThis modified LBR technique combined with IOE allows a higher MN expression efficiency compared to an already published method.ConclusionsOur work opens the possibility of screening a multitude of genes involved in MNs survival or cell death in vivo with high reproducibility and efficiency on a flexible and inexpensive animal model. The LBR/IOE technique opens a new way for the optimization of subsequent studies on mammalian models of diseases affecting MNs survival.