An Investigation of in Ovo Electroporation and High Titer Viral Injection Gene Transfer Methods in the Study of the Dorsal-Ventral Patterning of the Forebrain of Chick
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Signaling centers in the developing vertebrate brain are responsible for determination of dorsal or ventral characteristics of neural tube cells. These characteristics will eventually determine the final functions of the neuronal cell. Bone morphological proteins (Bmp) expressed in the roof plate, act as dorsalizing signals in the neural tube, patterning cells to dorsal fate. Antagonistically, centers such as the notochord and floor plate express sonic hedgehog (Shh), a powerful ventral patterning protein signal that competes with Bmp activity. A method to study these signals is to antagonize them and visualize effects in neural tissue by in situ hybridization. Dominant-negative Bmp Receptor 1 B, a mutant inhibitor of Bmp activity, can be transferred into neural tissue in order to see these effects. Two methods that have found success in gene transfer are in ovo electroporation and viral injection. Electroporation allows genes contained in plasmid vectors to be directed to specific points in tissue. The expression of the novel gene by the embryo is quick, but short term. Conversely, viral injection allows a gene contained in a retroviral vector to be spread by infection throughout the neural cavity. The expression of a gene takes longer to begin, but is permanent. In this study, we explored the signaling involved in dorsal-ventral patterning by utilizing these two methods. We found that electroporation of the human Efx plasmid with a ligated alkaline phosphatase gene, gave semi-satisfactory results. The electroporations were inconsistent in success, giving weak and imprecise direction of protein expression. We did not succeed in electroporating a retroviral based vector, RCAS, into the neural tissue, most likely due to the vector's large size along with poor electrodes. A noggin/pXex construct was made to replace the RCAS as another possible vector for electroporation. Dominant-negative BMPR-IB viral stocks concentrated 6-10x and injected into day two neural tube gave a high concentration of infection in growing cells. Further, dorsal tissue based choroid plexus formation was slightly deterred by the BMPR-IB; however, not the extent expected. It was concluded that viral injections must occur at an earlier age to see greater effects. Both methods can act as efficient methods of forebrain study in the chick.