Biology Reference
In-Depth Information
Dominant transgenic markers are useful for following an array during crosses to
other strains. An example is the rescue of the rol-6(su106) mutation (
Mello et al.,
1991
), which causes the worms to roll around its long axis. However, the morpho-
logical phenotype can interfere with observations of phenotypes caused by the gene
of interest. Another popular dominant marker is suf-5::GFP which is benign mor-
phologically (
Gu et al., 1998
). Transgenic animals carrying the GFP marked array
can be followed using a fluorescent dissecting microscope.
D. Methods for Creating Transgenic Animals
1. Microinjection
The standard approach to making transgenic strains is to coinject two or more
DNAs into the distal gonad syncytium of an animal. One of the DNAs carries the
DNA construct to be studied (the transgene) and the other a plasmid that contains a
marker to select for successful transformation. Both linear and circular DNAs can
form into arrays. Injected DNAs undergo homologous recombination and in this way
become assembled into an array (
Mello et al., 1991
). Using injected DNAs that share
sequence homology can facilitate efficient homologous recombination, although
nonhomologous recombination has also been observed (
Mello and Fire, 1995
). It
is possible to introduce several DNA constructs, although independent verification
of each component should be performed (usually this can be done by PCR fromDNA
extracted from transgenic animals) to ascertain that the recovered array contains all
coinjected components.
Transgenic animals produced by injection typically have large extrachromosomal
arrays that contain many copies of the coinjected DNAs (
Mello et al., 1991
). A
fraction of these repetitive arrays become heritably stable and some of the first-
generation progeny (F1) will transmit the array through subsequent generations.
Once the strain is established, transmission is often reproducible with regard to
heritability or in the case of somatic promoters, expression. However, some degree
of mitotic instability will occur and these are incompletely inherited. If the arrays are
integrated into a chromosome, usually by treatment with radiation, it is possible to
create a strain that transmits the array in a Mendelian manner (
Mello et al., 1991
).
2. Advantages and Disadvantages
Transgenic constructs do not always reproduce the expression patterns of the
endogenous genes. Genes expressed in the germ line are problematic. Transgenes
in repetitive arrays are strongly silenced in germ cell nuclei (
Kelly et al., 1997
). It is
however possible to circumvent this issue by using mutant animals that are deficient
for genes that elicit germ line silencing (
Kelly and Fire, 1998
). Additionally, it is
difficult to predict and control the level of expression among different arrays, and
expression can be variable among siblings of a single strain even when using
integrated arrays showing expression variability (
Mello and Fire, 1995
); this can
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