Micromanipulation techniques such as ICSI (intracytoplasmic sperm injection) help realize the potential of the
mouse as a model organism in reproductive biomedicine and human genetics. Accordingly, ICSI is a resource
to preserve unique e.g. transgenic mouse genotypes in the research mouse facility. Common cryopreservation
methods cause reduction or loss of sperm motility, which is not required for ICSI. However ICSI by conventional
microcapillaries harms the delicate metaphase II (MII) mouse oocytes. To overcome this problem, Kimura and
Yanagimachi introduced piezo-assisted ICSI in 1995 [1], which led to dramatically higher success rates as
compared to the conventional procedure. In this Application Note the ICSI procedure is described using the new
device from Eppendorf for piezo-assisted manipulation, the PiezoXpert and the Prime Tech piezo system (PMM),
a piezo-drill device described elsewhere in combination with Eppendorf micromanipulators for mouse ICSI [2],
showing parameter settings and microinjection procedure in detail.
ICSI (intracytoplasmic sperm injection) is an important and commonly used assisted reproductive technology in
both, humans and animals. The Mouse is one of the most common model organisms of choice to study mammalian
fertilization. However, the ability to fertilize mouse eggs successfully by sperm injection has been hard to achieve due
to the fact that the metaphase II mouse oocytes are extremely sensitive and convential ICSI gives low survival rates
[1]. This problem can be solved with piezo-actuated micromanipulation where the capillary advances a very short
distance at a very high speed. This enables the capillary to penetrate the cell membrane with minimum distortion of
the cell and yields to high survival rates. The microinjection workstation required for this technique is very similar to
standard ICSI, but with the addition of a piezo-assisted unit attached to the capillary holder. In this Userguide, the
use of the Eppendorf PiezoXpert in combination with the Eppendorf TransferMan® NK 2 workstation is shown and
parameter settings as well as optimization of the piezo-actuated microinjection procedure itself are discussed.
Biopsy of equine embryos at the early/expanded blastocyst stage is a rather complex procedure, essentially due to the presence of a distinctive capsule in the perivitelline space that is extraordinarily resistant and difficult to penetrate using conventional micromanipulation methods. In addition, this embryonic capsule has been considered essential for the embryo survival and, until recently, many doubts persisted about whether it could be breached without impairing the viability of the biopsied embryo.
Recent studies, however, suggested that encapsulated equine blastocysts could be biopsied using a piezo-actuated micropipette that allows creating a small hole in the capsule to collect trophectoderm cells for genetic analysis without compromising their further viability. In this Userguide, the biopsy procedure of encapsulated equine embryos is described using the new device from Eppendorf for piezoassisted manipulation, the Eppendorf PiezoXpert, in combination with the Eppendorf TransferMan NK 2 micromanipulation workstation.
In this Application Note, the nuclear tranfer as a mean of cloning technique is described using the new device from Eppendorf for piezo-assisted manipulation, the Eppendorf PiezoXpert®, in combination with the Eppendorf Celltram vario.
