Biology Reference
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markers, including a plasmid encoding the temperature-sensitive, dominant negative
selection marker twk-18(cn110). The plasmids are microinjected simultaneously in a
manner similar to that for conventional arrays, but the wait time following micro-
injection is about 10-14 days. Some 25-50 animals are needed for injection, and the
equipment is identical to that needed for normal extrachromosomal arrays (
Frokjaer-
Jensen et al., 2008
). Hence, laboratories that are already established for regular
microinjection will find it easier to work with Mos-directed insertions if their goal is
to obtain low copy number arrays.
Researchers wishing to try both microparticle bombardment and MosSCI may
consider constructing their transgene into a targeting vector for MosSCI, as the
resultant plasmid can then be used directly for bombardment, MosSCI or a conven-
tional multicopy array transgene, as all can be delivered to unc-119 mutants. It is
worth noting that as MosSCI insertions are targeted to predetermined locations,
researchers may wish to consider which location (and corresponding targeting
vector) they will use if there is a later need to combine transgenes into one strain.
There are currently two locations, on chromosomes II and IV (
Frokjaer-Jensen et al.,
2008
), though it is anticipated that additional targeting loci will become available
over time.
C. Integration of Extrachromosomal Arrays
Transgenes carried on extrachromosomal arrays can be integrated into a chromo-
somal location, which eliminates mitotic and meiotic loss of the array (
Evans, 2006
).
Spontaneous integration of extrachromosomal arrays has been observed by many
investigators, which may be more likely to be seen in large populations propagated
for many generations, especially if there is a selective advantage to the integrants.
Otherwise, spontaneous integration is rare enough that it is not convenient to expect
it to occur for any given transgene. Hence, most investigators use chemical mutagens
or ionizing radiation (gamma rays or ultraviolet light) to induce integration of an
array into a chromosome. This is usually done by mutagenizing a small starting
population of animals, establishing several hundred single F
1
animals, and testing F
2
progeny individually for 100% transmission of the transgene to subsequent genera-
tions (
Evans, 2006
). Coinjection of oligonucleotides can also stimulate integration
of arrays (
Mello et al., 1991
), and integration is observed if oocyte nuclei are directly
injected (
Fire, 1986
). However, neither of these approaches appears to be in wide
use. Once integrated, it is usually no longer necessary to follow a transgene by the
coinjection marker. This may simplify subsequent genetic manipulations and permit
combining multiple transgenes into a single strain.
For all integrated transgenic lines, strains should be backcrossed several times to
eliminate background changes to the genome introduced by the integration treat-
ment. It is also important to examine phenotypes and expression patterns in several
integrated lines to be assured that results are not dependent on the site of integration
or any linked background mutations.
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