Basic Research on Effectiveness of Vibratory Microinjection Method

为了提高转基因动物的产量，我们开发了振动显微注射系统（VMS）。该系统旨在通过在插入细胞时纵向振动微移液管来促进基因转移。对频率小于18 kHz的VMS进行评估。对不同频率下的振动显微注射（VM）与普通（非振动）显微注射（OM）进行了比较。BDF-1小鼠受精卵平均分配给VM组和OM组。每组的鸡蛋被分成若干组，每组30个鸡蛋。每组的30个卵子用一个微移液管注入金星基因。(1)与OM相比，VM使微移液器穿透透明带，细胞变形率显著降低，降低了约20%。(2)注射后存活的卵培养4天后，VM组的囊胚发育率比OM组高2 ~ 3倍，但死亡率略高。(3)“拔出”事件，即微移管在完成给定注射次数之前拔出一部分核DNA的事件，VM组的发生频率明显低于OM组。拔管事件通常需要更换新的微移管。研制了频率上限为100kHz的超声VMS （UVMS）。我们对UVMS进行了安全性评价，证实超声振动没有引起细胞损伤：细胞内压力的极端变化、空化或DNA的断裂。综上所述，VMS实现了受精卵注射，细胞变形明显减少，胚胎发育也明显改善，这表明转基因动物的生产得到了改善。VMS还可以降低微移液器的成本，省去频繁更换微移液器的麻烦。

To increase the production rate of transgenic animals, we developed Vibratory Microinjection System (VMS). This system was aimed at facilitating gene transfer by vibrating a micropipette longitudinally during insertion into cells.1.The VMS providing any frequency less than 18 kHz was evaluated. The vibratory microinjection (VM) at several frequencies was compared with the ordinary (non-vibratory) microinjection (OM). Fertilized eggs obtained from BDF-1 mice were equally allotted to VM and OM groups. The eggs in each group were divided into some sets of 30 eggs. The 30 eggs in each set were injected with Venus genes using a single micropipette.(1)Compared with OM, VM enabled micropipettes to penetrate zona pellucida with significantly less cellular deformation rates, reducing them by about 20%. (2)After four-day culture of eggs having survived the injection, the VM group showed 2 to 3 times higher rates of development to the blastocyst stage than the OM group did, although it did slightly higher death rates (NS). (3)‘Pulling-out' event, i.e., an event that a micropipette pulled out a part of nuclear DNA before finishing the given number of injections, took place significantly less frequently in VM group than in the OM group. The pulling-out event usually necessitates replacement of the micropipette with a new one.2.We developed ultrasonic VMS (UVMS) with upper limit of frequency of 100kHz. We conducted safety evaluation of UVMS, and confirmed no cellular damage caused by ultrasonic vibration : extreme change in intracellular pressure, cavitation or fragmentation of DNA.In conclusion, VMS achieved zygote injection with significantly less cellular deformation and also resulted in significantly better embryonic development, suggesting improvement in production of transgenic animals. VMS can also cut the cost of micropipettes and save the trouble of frequent replacement of micropipettes.