Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 67

A. Transgenesis in C. elegans
The initial technique of transgenesis developed in C. elegans is based on DNA
microinjection in the syncitial part of the germ line ( Evans, 2006; Mello and
Fire, 1995; Mello et al., 1991; Stinchcomb et al., 1985 ). Injected DNA fragments
are randomly concatemerized in the germ line, resulting in the formation of large
arrays, which contain hundreds of copies of the injected DNA. These arrays
remain extrachromosomal and are replicated during cell division. This transgen-
esis technique is extremely simple and efficient, yet it has some intrinsic limita-
tions. First, genes contained in repetitive transgenic sequences are often over-
expressed in the soma, sometimes generating toxic effects and experimental
artifacts such as nonphysiological expression patterns. Second, repetitive
sequences present in extrachromosomal arrays are, most of the time, silenced
in the germ line ( Kelly et al., 1997 ). In addition, their presence triggers an RNA
silencing process known as cosuppression that induces the silencing of the
homologous endogenous locus ( Dernburg et al., 2000; Ketting and Plasterk,
2000; Robert et al., 2005 ). Cosuppression phenocopies loss-of-function alleles
of germ line-expressed genes, often resulting in animal sterility and preventing
the use of extrachromosomal arrays to study germ line-expressed genes in
C. elegans. Third, extrachromosomal arrays are not stably inherited during
mitosis and meiosis. As a consequence, transgenic animals are mosaic and
transgenic siblings can exhibit different phenotypes depending on how many
cells contain the extrachromosomal array. This problem can be solved by ran-
domly integrating the repetitive transgenes in the genome. Radiations or chemi-
cals are used to cause random double strand breaks (DSBs), thereby stimulating
DNA recombination between the transgene and the genome. The transgenic
population is subsequently screened for animals that segregate the transgenes
in a Mendelian fashion ( Evans, 2006 ). Fourth, structural rearrangements can
occur over time in the extrachromosomal array, hence causing variations of
transgene expression patterns and levels among different generations of the same
transgenic line.
A second technique of transgenesis was developed based on the bombardment
of the germ line with DNA-coated gold particles ( Evans, 2006; Green et al.,
2008; Praitis et al., 2001; Wilm et al.,1999 ). About 10% of the transformants
obtained with this ''biolistic'' transformation technique are unique low-copy
chromosomal insertions that can be readily identified by the use of a visible
counterselection marker ( Vazquez-Manrique et al.,2010 ). Biolistic has been used
to generate C. elegans strains stably expressing transgenes both in the germ line
(see for examples Cheeseman et al., 2004; Merritt et al., 2008; Sijen and
Plasterk, 2003 ) and the soma (see for examples Berset et al., 2005; Praitis
et al.,2005 ). Results obtained with such transgenes are thought to be more
physiologically relevant than the ones obtained with repetitive extrachromosomal
arrays ( Praitis et al.,2001 ).