Development of efficient micro-scale gene transfer technique by combination of retrovirus vector and electroporation.

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The Goal of present study is to develop efficient and reversible microscale gene delivery technique as a tool for an analysis of molecular regulation of oro-facial tissue morphogenesis. For this porpose we carried out three lines of experiments. (1) Retroviral gene transfer is known as an efficient gene transfer technique. Cre-lox-P system is a way of controlled expression of introduced gene. Thus we first tried to combine these advantages in a single vector system. Lox-P sequences were introduced in 5 and 3 prime of the cloning site of an avian retrovirus vector RCAS(E). We then subcloned Green fluorescent protein gene(GFP) into the above vector (RCAS(E)-GFP). Cre recombinase was subcloned into RCAS(B) vector (RCAS(B)-Cre). More than 70% of RCAS(E)-GFP infected fibroblast were GFP positive within 1 week after infection. We then superinfected RCAS(B)-Cre into the same cells to turn off the expression of GFP.After 3 days, most of the doubleinfected cells became GFP negative. Thus these vectors turned out to be effective for efficient and controllable gene transfer. (2) Preparation of virus particle is cumbersome and needs a lot of experience. Therefore in the second experiment, we applied electroporation for microscale gene delivery of vector cDNAs instead of virus infection. We tested various conditions for electroporation and found that the pulses for 4 times at 20-60 V/cm for 50 msec was the most efficient. We also designed micro electrode for microscale gene delivery. (3) In the last experiment, we tested whether above technique is feasible to introduce foreign gene into developing mouse tooth germs. We injected Sonic hedgehog gene(Shh) into enamel knot and applied electroporation. We fixed the explants two days later and confirmed the expression of Shh specifically in enamel knot.