Utilizing Gene Electroporation and Role of FGFs in Mandibular Development

Objectives: During organogenesis, fibroblast growth factor (FGF) signaling plays an important role in early pattern formation and tissue differentiation. In craniofacial development, differentiation of various tissues and organs involves FGF signaling, such as craniofacial skeletogenesis. However, the roles of FGFs in mandibular morphogenesis are still unclear. We first optimized electroporation condition in combination with microinjection for gene transfer using expression vectors and examined the function of FGF8 and 10 in mandibular development. Materials and methods: We arranged various sizes of mammalian expression vector (3.8 to 5.5 Kb) for expression of enhanced green fluorescent protein (EGFP). We transferred expression vectors carrying EGFP, Fgf8 or Fgf10 gene by electroporation to embryonic day 12 rat mandibular explants and cultivated by modified Trowell method. Approximately 10μg/μl of the vectors were microinjected into the explants and electroporation was applied under various magnitudes of voltage. After optimization, we examined the influence of ectopic expression of Fgf8 and Fgf10 on the development of Meckel's cartilage in the mandibular explant. Expression of EGFP, Fgf8 and Fgf10 in mandibular explants was observed 24 hours after gene transfer and the morphology of the Meckel's cartilage was examined. Results: Vector size was significantly inversely proportional to the transfection efficiency, when analyzed by regression analysis (P<0.05). Histologically, mandibular explants transferred mock vector and loaded less than 20V of electrical pulses maintained symmetrical structure. Meckel's cartilage was spirally deformed by ectopic expression of Fgf10, while thickness increased by Fgf8, when vectors were electroporated at 20V. Length of Meckel's cartilage was significantly longer than that of the control, when Fgf10 was ectopically expressed (student t-test, P<0.05). Conclusion: Electroporation successfully induced the ectopic expression of EGFP, Fgf8 and Fgf10 in mandibular organ culture. The expression of FGFs affected the size and shape of the Meckel's cartilage.