Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 175

Unfortunately, the effect greatly reduces the mating efficiency of males, which can
make crosses more difficult. Some chromosomal integrants of rol-6D-marked trans-
genes show a much weaker Rol phenotype as heterozygotes, or they can be combined
with mutations in some genes (e.g., dpy-11), which can suppress the Rol effect.
Where a wild-type phenotype is desired from the transgenic animals, it is con-
venient to start with a strain carrying a recessive mutation and use rescue of the
mutation as the transgene marker ( Fig. 1B ). Markers commonly used are rescue of
pha-1, dpy-20,andunc-119.Lossofpha-1 is lethal, but the allele used is temper-
ature-sensitive (ts), so that animals are propagated at 15 C and selected for trans-
genics at 25 C( Granato et al., 1994 ). Loss of dpy-20 results in a viable dumpy
(Dpy) phenotype, but Dpy adults are more difficult to inject, so a ts allele is used
( Clark et al.,1995 ). Until such transgenes are integrated, maintenance requires
propagation at 25 C, which may make downstream genetics more challenging (e.g.,
if a transgene were to be crossed into another ts mutant background). unc-119
mutants do not form dauer larvae, an alternative larval stage that allows worms
to survive prolonged starvation. As a result, non-Unc-119 transgenics can be
identified from large populations since they are viable after starvation. For
MosSCI or microparticle bombardment, in which a very small fraction of animals
becomes transgenic, rescue of unc-119 has been the most frequently used marker,
although a number of other markers have been used successfully ( Praitis, 2006 ).
Use of the more compact C. briggsae homolog of unc-119 is convenient as it
facilitates cloning of the transgene and marker into the same plasmid. Inclusion
of the unc-119 marker into transgenic constructs has also been made simpler by a
recent modification of recombineering techniques ( Ferguson and Fisher, 2009;
Zhang et al.,2008 ).
As an alternative to using rescue of a mutation, transgenes can be marked by the
presence of a GFP reporter to myo-2 ( Okkema et al., 1993 ), elt-2 ( Fig. 3D )
( Fukushige et al., 1998 ), sur-5,orlet-858 ( Kelly et al., 1997; Yochem et al.,
1998 ). Access to a dissecting microscope equipped with a fluorescent lamp and
appropriate filters or a fluorescent worm sorter are necessary for identifying and
maintaining lines carrying extrachromosomal arrays. Other transgene markers
include antisense-unc-22, which imposes a twitching paralysis, and selection for
resistance to antibiotics ( Fire et al., 1991; Giordano-Santini et al., 2010; Semple
et al., 2010 ).
As a final consideration, expression of one gene on a single array may be pre-
cluded by the presence of the second gene. In such cases, it may be desirable to obtain
separate transgene reporters, and combine the two strains together. If this is done, the
researcher may wish to consider different strategies for marking the presence of
either transgene. For example, if both are rol-6D marked transgenes, it may be
difficult to identify double-transgenics or to even mate them together. In such cases,
rescue of unc-119 and rol-6D could be used to make separate transgenes, and then
the two strains can be combined by crossing rescued unc-119 transgenics to the
rol-6D strain that is homozygous for unc-119. The double transgenics will be Rol
non-Unc.