Transgenesis in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 160

protocols have been published, and hence in this chapter, we will review various
approaches to making C. elegans transgenics, discuss their applications, and con-
sider their relative advantages and disadvantages. Comments will also be made on
anticipated future developments and on the application of these methods to other
nematodes.
I. Introduction
Following the generation and characterization of chromosomal mutations
( Brenner, 1974 ), the ability to generate transgenic lines in C. elegans ( Fire, 1986;
Mello et al., 1991; Stinchcomb et al., 1985 ) opened the system for the rapid genetic
characterization of many diverse biological phenomena. The assembly of the
genome sequence ( C. elegans sequencing consortium, 1998 ) accelerated the rate
of gene identification because candidate genes could be identified by first correlat-
ing the genetic and physical maps and then transforming easily obtained subclones of
the genome into mutants to look for complementation rescue. The advent of green
fluorescent protein (GFP) as a reporter seemed destined for this system, as
C. elegans animals are essentially transparent at all life stages and exhibit little
autofluorescence ( Chalfie et al., 1994 ). The discovery of RNA-mediated interfer-
ence (RNAi) ( Fire et al., 1998 ) expanded further on this set of tools, and the vast
majority of work published in the C. elegans field uses a combination of all three
approaches: genetics, transgenes, and RNAi.
Early approaches for transgenesis in C. elegans involved microinjecting DNA into
either the hermaphrodite gonad or into unfertilized oocytes for the generation of
transgenic animals ( Fire, 1986; Mello et al., 1991 ). In contrast with other systems,
C. elegans embryos are not used for injection, because it is technically much more
challenging and less efficient than gonadal injection, which typically produces many
transformed F 1 animals per hermaphrodite. Unlike other systems in which trans-
genic DNA is generally integrated into chromosomal DNA in single copy ( Ringrose,
2009; Ziemienowicz, 2010 ), C. elegans transgenes obtained following microinjec-
tion assemble into multicopy extrachromosomal arrays that are transmitted to prog-
eny at 5-95% fidelity ( Mello and Fire, 1995 ). While an extrachromosomal transgene
is sufficient or even required for many purposes, arrays can be made to integrate
following treatment of a transgene strain with ionizing radiation or chemical muta-
genesis (see Evans, 2006 ).
The ease of producing transgenics in C. elegans, and the general reliability of
transgene-expression patterns, have permitted rapid characterization of gene expres-
sion and often function without the use of in situ hybridization or antibodies ( Fig. 1 ).
To a first approximation, genes in arrays are expressed similarly to endogenous
genes, although the relative expression may be increased due to a higher gene dosage
or reduced due to silencing of repetitive sequences ( Fire and Waterston, 1989; Kelly
et al., 1997; MacMorris et al., 1994; Okkema et al., 1993; Stinchcomb et al., 1985 ).
For many years it was observed that promoters normally active in the germ line fail to