377 Development of a vibratory microinjection system to increase the production rate of transgenic animals.

Abstract

Pronuclear injection is the standard method to deliver genes to zygotes, especially of medium- to large-size animals, but the production rate of transgenic animals is still low. To increase it, we developed a vibratory microinjection system (VMS) that vibrates a micropipette (Femtotip�; Eppendorf Co., Ltd., Tokyo, Japan) longitudinally to facilitate its penetration of the zona pellucida and nuclear membrane. The VMS can provide a wide range of frequencies and amplitudes of vibration, and is different from the piezo impact micromanipulator that uses an internal force resulting from a rapid deformation of piezoelectric elements. P Vibratory microinjection (VM) at a frequency of 5 kHz and an amplitude of 0.5 �m, which was not at the tip of micropipettet but at the vibrator's level, was compared with ordinary microinjection (OM). No fragmentation of the Venus gene encoding a variant of yellow fluorescent protein was confirmed after it had been exposed to this vibration for 1 h. A total of 910 eggs obtained from 36 BDF-1 mice were used. This experiment consisted of 15 sets of injections. In each set, the same number (d40) of eggs was allotted to the VM and OM groups. The eggs in each group were injected with the Venus gene using a single micropipette. The two groups were treated simultaneously with two sets of injection apparatuses. Embryonic development was assessed after 4-day culture. The following results were achieved: (1) VM made the micropipette penetrate zona pellucida with much less cellular deformation; the deformation rates were 28.7 � 7.2% for VM and 35.4 � 7.3% for OM (P < 0.0001). (2) Ninety-six of 455 embryos (21.1%) in the VM group developed to the blastocyst stage, whereas only 41 of 455 eggs (9.0%) in the OM group did (P < 0.0001). The numbers of Venus-expressed blastocysts were 29 for the VM group and 19 for the OM group (P = 0.14). However, the death rates in VM and OM groups were 39.1% and 32.7%, respectively (P = 0.045). This higher death rate in the VM group might have been due to transverse vibration at the tip of micropipette because it was bent approximately 20�. (3) Two of 15 micropipettes in the VM group pulled out a part of the nuclear DNA and/or cytoplasm before finishing the given number of injections while 9 of 15 micropipettes in the OM group did (P = 0.0063). We conclude that the VMS achieved zygote injection with significantly less cellular deformation and also resulted in significantly better embryonic development, suggesting anticipated improvement in the production of transgenic animals. The VMS can also cut the cost of micropipettes and save the trouble of frequent replacement of micropipettes.