Novel Methods used by Takeuchi

     Traditional methods of introducing DNA into a system (in this case a chick embryo) relied on the Replication-Competent Avian Sarcoma (RCAS) retrovirus system, which carried with it, a 16 hour time delay between injection of the virus and expression of the forgein DNA; a time-lag that is, consequently, unacceptable. The reason being the fact that limb-identity takes place at such an early stage in development, such that after a 16 hour lag, the identification process could be complete, resulting in prevention of conversion of the limb field due to misexpression of the T-Box genes.

     The time-delay problem was overcome by a novel system in which the RCAS retrovirus plasmids, containing the T-Box gene of choice are electroporated directly into the prospective limb-field; thus only a two hour time delay was observed. The ectopic Tbx5/Tbx4 genes were introduced to the prospective limb fields at stage 7-9.

     To visualize the introduced DNA, a fusion was made between the retroviral T-Box gene complex and an Enhanced Green Fluorescent Protein gene such that RCASBP-Tbx5-EGFP and RCASBP-Tbx4-EGFP are the complexes being electroporated into the limb fields of the chick embryos.

     In these misexpression studies, it was determined that misexpression of irrelevant genes, such as Alkaline Phosphatase from the viral plasmid, and Green Fluorescent Protein, had no consistent morphological changes in the limb buds. Thus, the mal-effects seen in limb development were due solely to misexpression of Tbx5 and Tbx4, as the nonspecific effects were minimal.