Biology Reference
In-Depth Information
by the normal transposition method because it lacks 23 bp of one terminal repeat,
hence the designation wc for “wings clipped.” The inverted repeats are necessary
for transposition.
(B) Microinject both the vector and helper plasmids into embryos (G
0
) from an
appropriate host strain with
white
(
ww
) eyes. Embryos should be in the preblas-
toderm stage, when the cells in the embryo are still in a syncytium.
(C) Mate the G
0
individuals that survive to adulthood after injection with
males or females that are homozygous for
white
(
w/w
). If the wild-type gene
(w
+
) inserted into the chromosome of the injected embryo, then the progeny
(G
1
) will have red eyes but is heterozygous (w/w
+
).
(D) Mate G
1
red-eyed progeny (
w/w
+
) with wild-type (
w
+
/w
+
) flies to produce
the next generation (G
2
) with wild-type eyes. These flies may be transgenic.
(E) Select individual G
2
lines with wild-type (red) eyes.
(F) Identify possible transformants containing single insertions at unique sites
(single-insert lines) and verify insert structure.
(G) Analyze the properties of the transformed lines, including the level of
expression of the inserted DNA and the stability of the transformed line.
(H) Cross the most useful lines to balancer stocks to enable the lines to be
maintained in a stable condition.
Insertion of
P
-element vectors DNA into germ-line chromosomes is enhanced
if preblastoderm embryos are microinjected. At that stage, the cleavage nuclei
are in a
syncytium
(lacking nuclear membranes), and the
P
elements can more
easily be inserted into chromosomes of the
pole cells
that will give rise to the
ovaries and testes. Insertion of exogenous DNA into the chromosomes of
the germ line results in stable transformation. If only somatic cells obtain the
exogenous DNA, the flies cannot transmit the desired trait to their progeny.
Such adult G
0
flies may exhibit the trait, but they are only transiently trans-
formed. During embryogenesis and development of the larvae hatching from
the injected eggs (G
0
indicates the generation that is injected, G
1
indicates
their progeny, and so on), transcription and translation of the wild-type gene
(
w
+
) can produce sufficient xanthine dehydrogenase to produce a red eye color
in the adults. G
0
flies with a red eye color do not necessarily have the
white
+
gene inserted into the germ-line chromosomes because the injected DNA may
be transcribed and translated while in the cytoplasm, or the DNA may only be
inserted into the chromosomes of somatic cells (
=
transient transformation
).
Only a portion of the
P
vector should insert into the chromosome. The DNA
