Biology Reference
In-Depth Information
A. Phenotypic Characterizations
To address whether and how mutations in a gene alter the development of a
neuron, the first step is to introduce a reporter into homozygous mutants through
standard genetic crosses. The next step is to compare the expression patterns of the
reporter in mutant versus wild-type animals. A rule of thumb in this kind of analysis
is to maintain identical culture conditions and blinding genotypes when possible. If
defects are observed, it is best to test a second transgenic marker or use other
methods to confirm the phenotype. For example, if one wants to observe whether
a gene affects axon guidance, one can cross a panel of neuronal morphology markers
into mutants (
Table I
) to generate homozygous mutants carrying that marker. If
abnormalities are observed, one also needs to confirm that the phenotypes are due to
mutation of the gene by transgenic rescue experiments. Further studies include
expressing the gene under specific neuronal promoters to determine whether the
gene functions cell-autonomously, as well as dissecting the domain requirement of
the protein.
B. Identifying Mutants in Forward Genetic Screens
Studying of the nervous system in C. elegans is facilitated by the ease of carrying
out forward genetic screens. Such screens are the most effective and valuable way of
discovering new genes and pathways. Before beginning, it is advised to read the
article for general guidelines on designing a screen by
Jorgensen and Mango (2002)
.
A forward genetic screen combines standard mutagenesis in a range of mutant
selection schemes as summarized below.
(1) Mutagenesis: Two major types of mutagens are chemical mutagens including (1)
ethyl methanesulfonate (EMS); (2) nitrosoguanidine (NTG); (3) diethyl sulfate;
(4) N-Nitroso-N-ethylurea; (5) formaldehyde; (6) acetaldehyde; (7) diepoxyoc-
tane; (8) diepoxybutane, and radiation mutagens including X-rays,
g
-rays, UV
light, and ionizing particles (
Anderson, 1995
). Among all these mutagens, EMS
is the most potent and widely used. Because over 90% of mutations are G/C to A/
T transitions, EMS is an excellent mutagen for generating nonsense or missense
amino acid coding alternates. Another commonly used mutagen is UV irradia-
tion, which frequently generates a large proportion of gene rearrangements
(deletions or insertions) and most of these mutants are likely null alleles.
(2) Genetic screen: The mostly commonly used procedure is manual visual inspec-
tion. Automated screening using worm sorters has also become feasible.
(a) Manual screen: For most screens, researchers prefer nonclonal F2 genetic
screens. Briefly, mutagenized P0 worms are plated one worm per plate in
seeded large plates and cultured for six days. The F2 generation is examined
for phenotypes. Such a scheme allows for screening a large number of
mutagenized genomes in a relatively short time, but lethal or sterile mutants
are easily missed. In a standard clonal screen, after mutagenesis 20 to 30 late
Search WWH ::
Custom Search