77and injected into either of the embryo’s pronuclei under a microscope. Pronuclear
microinjection remains the predominant method of delivering exogenous materials
into fertilized zygotes.
4.2 ES Cell Injection
The mouse ES cells derived from the inner cell mass of the blastocyst-stage embryos
are a pluripotent cell type with the potential to develop into all tissues of the body.
To manipulate the genomic DNA sequence, ES cells can be transfected with a tar-
geting vector carrying the desired mutations (e.g. knock-out, knock-in, or condi-
tional allele) as well as selectable markers flanked by homologous sequences. After
screening for mutations or selectable markers, correctly targeted ES cell clones are
selected for microinjection into embryos at either 8-cell or blastocyst stage [ 2 , 3 ].
Modified ES cells contribute to part of the host body and develop into chimeric
animals which are able to transmit the targeted allele to the offspring via the germ
line at a certain frequency. For injection into eight-cell embryos, two to eight ES
cells are injected into the perivitelline space between the zona pellucida and the
blastomeres using a beveled and sharp-tipped glass needle (15–20 μm in inner
diameter) [ 4 ]. If injecting ES cells into blastocysts, 10–15 ES cells are injected into
the blastocoel cavity. Injected embryos are subsequently transferred to pseudopreg-
nant females for continued development [ 2 ]. We prefer to inject ES cells into eight-
cell embryos rather than blastocyst embryos because it gives a higher rate of
high- percentage chimeric mice. From our experience, about 10% of the chimeric
mice we generated were fully ES cell-derived animals.
4.3 Cytoplasmic Microinjection
Foreign DNA, or other genome editing materials, can be delivered into the cyto-
plasm of fertilized zygotes using a similar technique to pronuclear microinjection,
except that the glass pipette does not penetrate the pronucleus. Another method is to
utilize a piezo-driven microinjection technique, by which the pipette holder is
equipped with a piezoelectric actuator that creates a quick mechanical pulse to
vibrate the pipette tip and pierce the zona pellucida and oocyte membrane (oolemma)
without a damaging effect. The exogenous editing materials loaded into the glass
pipette are able to pulsate into the cytoplasm when the glass pipette punctures the
oolema. The oolema then heals itself after the glass pipette is retrieved [ 5 ]. We typi-
cally use a blunt-end glass needle of 8 μm in inner diameter and obtain more than a
90% survival rate following the injection.
4 A Transgenic Core Facility’s Experience in Genome Editing Revolution