Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 176

B. Delivery Methods
The C. elegans germ line is the target organ for microinjection. It contains a
syncytium of germ-line nuclei sharing a common cytoplasm ( Klass et al., 1976 ).
Researchers have two choices for delivery of transgenes to C. elegans, microinjec-
tion or microparticle bombardment ( Fig. 1C ).
Microinjection is typically the first technique tried, as it requires only a small
number of animals, and F 1 animals are scored within a few days after injection. An
inverted microscope setup with differential interference contrast (DIC) or similar
optics, a needle puller, and a micromanipulator are necessary. Pressurized nitrogen,
delivered through a regulator with a foot pedal controller, is usually used to force
injection mixtures through the needle. Alternatively, other lower-cost arrangements
are possible. Laboratories performing Drosophila microinjection may have similar
setups that can be used. A detailed protocol for C. elegans transformation using
microinjection can be found online from the WormMethods section of WormBook
and from other published sources ( Evans, 2006; Kadandale et al., 2009; Mello and
Fire, 1995 ). For laboratories that desire low copy number transgenes, for example, to
avoid toxicity or to achieve maternal expression, injection can be modified by the
inclusion of digested genomic yeast or nematode DNA ( Kelly et al., 1997 ).
Alternatively, bombardment or MosSCI, both of which yield low copy integrants,
can be used. When stable lines are required, extrachromosomal transgenic arrays can
be integrated using chemical mutagens or radiation, as described below.
Microparticle bombardment requires more time initially, as a large number of
starting unc-119 mutant animals are required, and there is usually a 10-14-day post-
treatment wait time before active screening for transformants begins. However, the
chief advantages of the technique are that both integrants and extrachromosomal
arrays are obtained in the same procedure, the technique relies on a selection that
yields only the most stable lines, and it requires little technical expertise. Access to a
Biolistic PDS-1000 Helium Microparticle Bombardment machine or other delivery
device and several consumables are needed for this procedure. Laboratories may
find access to such a machine if there is a nearby facility that performs plant cell
transformations. Detailed descriptions of the microparticle bombardment procedure
are available in WormBook or in other published sources ( Evans, 2006; Green et al.,
2008; Jackstadt et al., 1999; Praitis et al., 2001; Rieckher et al., 2009; Wilm et al.,
1999 ).
The delivery of transgenes for directed chromosomal insertion using Mos trans-
position ( Fig. 1C ) is really just a special case of direct microinjection into unc-119
mutant animals, as the desired transgenics do not require infinite passage of the
extrachromosomal transgene array. The injected plasmids serve primarily as the
chromosomal repair source (the ''targeting plasmid'') and to activate Mos transpo-
sition in the germ line. To distinguish bona fide chromosomal insertions from
transmission of an extrachromosomal array, several reporter plasmids are coinjected
simultaenously. In one version of the approach, chromosomal insertions are rescued
for the unc-119 phenotype but fail to express the other coinjected transformation