Development of Vibratory Microinjection System for Instantaneous Cell Membrane Piercing in Cytoplasmic Microinjection into Fertilized Eggs

Abstract

To complete microinjection as quickly as possible, we have developed Vibratory Microinjection Systems (VMSs) that vibrate a micropipette in its longitudinal direction and can significantly reduce the time needed for pronuclear microinjection compared to ordinary (non-vibratory) microinjection. The longest breakdown of the time is the time required to pierce the cell membrane and the pronuclear membrane simultaneously. Because cytoplasmic microinjection, which pierces the cell membrane alone, is far more difficult and time-consuming than pronuclear microinjection, we next aimed to develop a VMS capable of penetrating the cell membrane instantly. In this new and latest version, two types of ultrasonic-wave vibrators were developed: the first for commercially available micropipettes (Femtotip) and the second for self-made micropipettes. The two vibrators differ only in their airtight structure, where the micropipettes connect to their respective vibrators: a female screw plus O-ring for the first vibrator (VMS6_1) and a silicone-rubber tube for the second (VMS6_2). The tube-type joint used in VMS6_2 only slightly damped or amplified vibrations from the vibrator to the micropipette tip, propagating them much more accurately than the screw-type joint in VMS6_1. In addition, VMS6_2 significantly shortened the time taken to pierce the cell membrane of a fertilized egg: an average of 1.52 s (N = 410) vs. 3.62 s (N = 65) in VMS6_1. The VMS6_2 group achieved a piercing time of zero in 86.1% of the allocated eggs, while only 10.8% of the VMS6_1 group did. In each vibrator, we also compared vibratory microinjection (VM; N = 475) and ordinary microinjection (OM; N = 457), which uses injection pressure in place of vibration. None of the eggs in the OM group achieved the zero-second piercing time. Compared to the OM, the VM group showed a significantly shorter piercing time, 1.80 vs. 10.69 s on average, and a significantly better survival rate, 90.3 vs. 81.8% on average. VMS6_2 not only improved on the already demonstrated superiority of VM to OM but also enabled instantaneous piercing of the cell membrane.