Biology Reference
In-Depth Information
repaired using the homologous DNA engineered into the insertion vector as tem-
plate. Integration events can be isolated by screening for WT animals (unc-119
positive) that have lost the transgenic array (do not express the fluorescent reporters).
8. Advantages and Disadvantage
MosSCI is a potentially powerful approach that eliminates many of the problems
associated with generating transgenic arrays. The insertion of a single copy of the
construct circumvents the issues of germ line silencing and overexpression, leading to
more stable native gene expression. The technique is however relatively labor inten-
sive, requiring the generation of several potentially complex DNA constructs and the
use of animals containing specific Mos1 insertions ( Frokjaer-Jensen et al., 2008 ).
Screening for rare integration events also requires the use of a fluorescent microscope.
Additionally, this technique only allows for efficient insertion constructs of around 15
kb and up to 30% of the integrants will not be full length. This method is however
currently the only reliable method available for creating single copy integrants.
E. Use of Transgenic Arrays as Balancers
Under certain conditions, a transgenic array can be considered a specialized form
of duplication. In this context, they have been used to rescue and maintain specific
mutations. However, their rescuing ability has also been utilized as part of a system
for isolating suppressors of mutations that result in low-viability phenotypes ( Fay
and Han, 2000; Fay et al., 2002 ). In these screens, wild-type copies of the gene are
carried in the array, rescuing the detrimental phenotype of the mutated animals. An
advantage of using a transgenic array is that it can be engineered to meet the needs of
the screen, for example, Fay et al. created rescuing arrays containing wild-type
copies of thelin-35gene with a GFP reporter to facilitate ease of screening.
Transgenic lines made in this manner transmit the extrachromosomal array only
to a fraction of their progeny. The screen exploits this characteristic by screening for
viable animals that have lost the array after a mutagenesis treatment. Such animals
are likely to have acquired a second site suppressor mutation.
1. Advantages and Disadvantages
Transgenic arrays can be used to great effect for the rescue of specific single-gene
mutations. Transgenic arrays, unlike large rearrangements, can also be tailored to
specific requirements such as the inclusion of specific promoter elements or the use
of fluorescent marker for rapid identification. It is however difficult to rescue
mutations in genes that are required in the germ line due to silencing of the array.
The use of transgenes for mutagenesis screens can also create complexity when
screening for suppressor mutations as integration of the transgene sequence into the
genome or disruption of the fluorescent markers can lead to false positives that are
difficult to identify and eliminate.
Search WWH ::




Custom Search