Basic Research on Effectiveness of Vibratory Microinjection Method

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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.