Biology Reference
In-Depth Information
adulthood (G
0
). Even after G
0
adults are obtained, damage caused by microinjec-
tion may result in early death or sterility in 30-50%.
Transformation does not take place in all germ-line cells in an injected
embryo. Usually, only a small fraction of the germ-line cells of a G
0
individual
produces transformed G
1
progeny. Thus, it is important to maximize the recov-
ery of G
1
progeny from each G
0
individual injected to increase the probability of
detecting progeny in which integration of
P
elements occurred. The size of the
introduced
P
element is another factor that may influence transformation suc-
cess; the larger the construct, the less frequent the insertion.
Detailed information on the life history and culture of
Drosophila
are avail-
able (
Roberts 1986, Ashburner 1989, Matthews 1994, Horn and Wimmer 2000
),
as are detailed protocols for transforming
Drosophila
with
P
vectors (
Spradling
1986, Karess 1987
). The protocols provide complete information on the appro-
priate equipment for microinjection, how to stage and dechorionate embryos,
align them on slides, desiccate them so that the eggs do not burst upon injec-
tion of the plasmid DNA, and inject them in the region that contains the pole
cells. Once eggs have been injected, they need to be held under conditions of
high relative humidity to prevent death by desiccation. Directions for preparing
the DNA for injection and for pulling the required very fine glass needles are
provided.
P
vectors have been engineered with different characteristics and functions
(for examples, see
Rubin and Spradling 1983, Karess and Rubin 1984, Cooley et al.
1988, Handler et al. 1993b, Horn and Wimmer 2000, Horn et al. 2000
) (
Figure 9.3
).
Generally, the vectors contain restriction sites for cloning, and usually contain one
or more selectable marker gene(s).
9.7.2 Characterizing Transformants
Identification of transformed flies is achieved in several ways. If a visible marker,
such as an eye color gene, is included in the vector then putatively transformed
D. melanogaster
can be determined visually.
Ideally, DNA from putatively transformed lines will be extracted and analyzed
by Southern blot to confirm the number of insertions in each line. If large num-
bers of fly lines need to be characterized, dot-blot analysis can be done.
In situ
hybridization of larval salivary-gland chromosomes will allow a determination to
be made of the number of insertions and their location(s). It is desirable to iden-
tify lines that carry only a single insertion if the timing and level of expression is
