Target Discovery and Validation Reviews and Protocols

166 Houdebine
concentrations become toxic. In rabbits and pigs, the transgenesis yield is
increased but not linearly between 0, 5, and 7 to 8 ng/µL. These concentrations
correspond to approx 1000–5000 copies of the genes. When long DNA fragments
are used, the same amount of DNA as for short fragments must be
injected irrespective of the copy number. In ruminants, two reasons are convergent
to reduce microinjection efficiency: (1) reproduction is slow with a limited
number of available embryos, and (2) integration of foreign DNA occurs less
frequently than in other mammals.
DNA microinjection in cytoplasm is used in some nonmammalian species in
which pronuclei are not visible. Very high amount of DNA (1–20 million copies
in up to 20 nL) must be injected for success. For unknown reasons, this method
does not lead to an integration of foreign DNA with a significant yield in several
species, such as chicken, Xenopus, and medaka. The purity of DNA to be
microinjected must be as high as possible. Usually, fragments from plasmids or
bacterial artificial chromosome (BAC) vectors have to be isolated using agarose
gene electrophoresis. It is recommended to not add ethidium bromide to DNA.
The band of interest may be visualized in a separate and independent lane of the
gel, by using ethidium bromide. It is also preferable not to illuminate the gel with
UV light, which breaks DNA. Several kits including columns to isolate the DNA
fragments from the gel are available. They generally give satisfactory results.
When long DNA fragments have to be isolated from BAC, electrophoresis
must be followed by a digestion of agarose with agarase (7). The activity of
commercial agarase may be variable and leads to incomplete agarose digestion.
Batches of agarase must preferably be tested before use. The fraction must
be dialyzed on a filter to eliminate agarose digestion products and to add
polyamines (spermidine and spermine) to stabilize the long DNA fragments.
For long DNA fragments, but also for plasmid fragments, electroelution from
the agarose gels may be used. The eluted material is trapped in a filter and
finally eluted from the filter. Electrocution is simple, and it gives concentrated
and highly purified DNA fragments. The concentration of DNA must preferably
be evaluated by using both a spectrophotometer and an agarose gel electrophoresis.
Isolated plasmid fragments may be kept frozen for months before
microinjection. Long DNA fragments must be kept at 4°C and used within a
few weeks. Integrity of long DNA fragments must be checked after 2 or 3 wk
of storage. Any fraction that contains impurities must be eliminated and replaced
by a new preparation.
2.1.2. DNA Transfer by Using Electroporation
Electroporation of medaka embryos generated a large number of transgenic
animals (8). Many groups have not adopted this method, and its efficiency has
not been proved in other species.