Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 174

expression ( Duret, 2000; Okkema et al., 1993 ). Hence, some labs may consider that
particular manipulations, such as modification of protein coding regions, might be
best achieved by direct synthesis, considering the time and/or number of manipula-
tions that would otherwise be necessary.
Expression of genes in the germ line or early embryo can be less straightforward
than expression in other tissues or stages of development. The repetitive nature of
conventional transgenes results in germ-line silencing ( Kelly et al., 1997 ).
Conventional extrachromosomal arrays are compatible with maternal (germ line)
expression for only a small subset of GFP reporters ( Fire et al., 2006 ). There are
several approaches for achieving expression of maternal transgenes. One is to use
conventional arrays, but to coinject genomic DNA that has been digested with a
restriction enzyme that leaves blunt ends ( Kelly et al., 1997 ). This approach appears
to achieve expression of maternal transgenes because the arrays are made complex
and less prone to silencing. In some instances, maternal expression can be achieved
by using a promoter and 3 0 UTR that seem to be compatible with expression from a
multicopy array, such as from glh-2 ( Bessereau et al., 2001 ). More reliable
approaches for germ-line or maternal expression use microparticle bombardment
or MosSCI ( Fig. 1B ), both of which deliver fewer copies of the transgene, which
makes them less prone to silencing. Both of these techniques require special con-
sideration for the plasmids that carry the transgenes, as described below.
IV. Obtaining Transgenic Animals
A. Considerations for Marking of Transgenics
It is usual practice to mark the presence of a transgene by a convenient marker that
can be scored visually in larvae or adults, to facilitate identification following
transgene delivery, and when transformants are obtained, during crosses or screening
for integrants ( Figs. 1A,B , Table IV ). Transgenes that confer a readily detectable
change in phenotype from nontransgenic animals (e.g., rescue of a visible mutation
or very bright GFP reporter), may not need a coinjection marker unless a positive
control for the injection process is desired. During the process that gives rise to
conventional transgene arrays, recombination among the injected plasmids (if pres-
ent at high enough relative concentrations) will almost always ensure that multiple,
separate plasmids become incorporated into the same array. For microparticle bom-
bardment, the marker is often but not always included in the same plasmid as the
transgene, because the low copy number of the resulting insertions makes it less
likely that both will become integrated. For Mos-directed chromosomal insertion,
the marker and transgene must both be included in between the flanking homology
segments.
Simple transgenes in a wild-type background can be marked with the plasmid
pRF4, which encodes the su1006 allele of the rol-6 gene, also called rol-6D ( Fig. 1A )
( Mello et al., 1991 ). pRF4 induces an obvious right-handed Roller (Rol) phenotype.