Biology Reference
In-Depth Information
inserted should consist of the sequences contained within the inverted terminal
repeats of the
P
element. The plasmid DNA outside the inverted repeats should
not insert and should be lost during subsequent development.
The next generation of flies (G
1
) is the crucial generation to be screened
for transformation, because these flies should have red eyes
only
if the
white
+
gene did insert into the germ-line chromosomes. The presence of
one or more progeny with red eyes in the G
1
indicates
stable transformation
occurred. Once transformed fly lines are obtained, the lines should be stable
unless transposase is provided in some manner. If an experimenter wants to
induce movements of inserted DNA,
secondary transposition
can be induced
if transposase is introduced by injecting helper elements into a preblastoderm
embryo.
Individual G
1
flies may contain multiple insertions of the
P
element and the
P
element may have inserted into different sites in different G
1
flies. As a result,
colonies derived from single flies must be screened to identify colonies with a
single insert (
Figure 9.2
). To determine how many
P
elements inserted into the
chromosomes of each colony and their location, DNA from G
2
adult flies is pre-
pared from each isoline and evaluated by Southern-blot analysis. (See Chapter 5
for a description of Southern-blot analysis, which can document that the gene
inserted is in the chromosomes of the fly. Analysis by the PCR only indicates that
the target DNA is present; it could be present extrachromosomally. It does not
prove that the gene inserted into the chromosomes unless the surrounding DNA
is amplified by inverse PCR and shown to be
Drosophila
DNA.) In Southern-blot
analysis, DNA is cut with restriction endonucleases and probed with labeled
P
sequences to determine the number of insertions. Lines containing multiple
insertions should be discarded because these lines will be difficult to analyze. G
3
lines with single inserts are then crossed to
Drosophila
stocks containing appro-
priate
balancer chromosomes
. Balancer chromosomes prevent crossing over
between homologous chromosomes and thus help to maintain stable stocks.
The location of the transposon in each single-insert line can be determined by
in
situ
hybridization to salivary gland chromosomes.
Transformation rates vary, but may often be
<
5% of the embryos injected.
Furthermore, of those that are transformed, variability in expression of the
transgene can be extreme due to position effects. It usually is desirable to
obtain at least 10 single-insert lines containing a transgene of interest. This
may require microinjecting 600, or more, embryos because survival of embryos
after microinjection averages 30 to 70% and, of these, only 50-60% survive to
