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

本研究的目的是建立一种高效、可逆的微尺度基因导入技术，用于分析口腔颌面部组织形态发生的分子调控。为此目的，我们进行了三个方面的实验。(1)逆转录病毒基因转移是一种高效的基因转移技术。Cre-lox-P系统是一种外源基因控制表达的方法。因此，我们首先尝试将这些优点联合收割机结合在单一载体系统中。在禽逆转录病毒载体RCAS（E）克隆位点的5和3 ′端引入Lox-P序列。然后我们将绿色荧光蛋白基因（GFP）亚克隆到上述载体（RCAS（E）-GFP）中。将Cre重组酶亚克隆到RCAS（B）载体（RCAS（B）-Cre）中。在感染后1周内，超过70%的RCAS（E）-GFP感染的成纤维细胞呈GFP阳性。然后将RCAS（B）-Cre转染到同一细胞中，关闭GFP的表达，3天后，大部分细胞变为GFP阴性。因此，这些载体被证明是有效的和可控的基因转移。(2)病毒颗粒的制备是繁琐的，需要大量的经验。因此，在第二个实验中，我们应用电穿孔代替病毒感染来进行载体cDNA的微量基因递送。我们测试了电穿孔的各种条件，发现以20-60 V/cm脉冲4次，持续50 msec是最有效的。我们还设计了用于微量基因传递的微电极。(3)在最后一个实验中，我们验证了上述技术将外源基因导入发育中的小鼠牙胚的可行性。我们将Sonic hedgehog基因（Shh）注入牙釉质结中并应用电穿孔技术。两天后固定外植体，证实Shh在釉质结中特异性表达。

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.