Biology Reference
In-Depth Information
and Age1, Xba1, and Sac1tothe3
0
end of the SV40 late polyadenylation signal. The
SV40 late polyadenylation signal can then be cloned into the pBluescriptII-KS
þ
plasmid with Not1andSac1, after which the
a-aeq-IRES-EGFP fragment can also
be cloned into this plasmid using Xho1andNot1. The enhanced green fluorescent
protein (EGFP) marker gene is regulated by the IRES-sequence for the subsequent
identification of transgenic fish. Use of the IRES-sequence enables the translation of
both apoaequorin and the EGFP marker from a single mRNA; thus, the expression
level and distribution of EGFP reflects the expression level and distribution of
apoaequorin (
Fahrenkrug et al., 1999; Wang et al.,2000
).
ii. Generation of transgenic zebrafish that express apoaequorin in the skeletal
muscles. Linearize the p
a
-KS-aeq-IRES-EGFP plasmid with Xba1 and then
microinject
100-200 pg) into the center of the blastodisc of the
zebrafish embryos at the one-cell stage. The microinjection pipettes and pressure
injection system used are described in detail in
Webb et al.
1 nl (i.e.,
(1997)
. The injected
28.5
C and screened for the expression of
EGFP after 24 hpf. Embryos (F
0
) that express EGFP should be raised to adult-
hood for further transgenic germ line screening. This involves: (1) The F
0
fish being
crossed with the wild-type fish to get the F
1
generation. (2) If some of the F
1
embryos express EGFP, then this indicates that one of their parents was transgenic
(i.e., it was heterozygous). The F
1
embryos that express EGFP can then be raised
to adulthood and intercrossed with one another to produce the F
2
generation.
(3) In the F
2
generation, 50% of the o
embryos should then be maintained at
spring should be heterozygous, 25% should
be homozygous, and 25% should be wild-type. (4) The homozygous F
2
transgenic
fish may then be identified by crossing the EGFP-expressing fish with wild-type
fish; if all of the F
3
o
V
spring express EGFP, then their transgenic parent was a
homozygote; if 50% of the F
3
embryos express EGFP, then their transgenic parent
was heterozygote. The homozygous F
2
fish can then be intercrossed with one
another to obtain stable transgenic lines.
iii.
V
Dechorionate the
a
-actin-apoaequorin
transgenic embryos when they are at the eight-cell stage (we dechorionate embryos
manually with watchmaker's forceps) and incubate them in a custom-designed
injection/imaging chamber (described in
Webb et al., 1997
) with 20
m
g/ml f-coe-
lenterazine in 30% Danieau's solution to reconstitute the active aequorin. Prepare
the f-coelenterazine as a stock solution of 2 mg/ml in methanol and dilute it in 30%
Danieau's solution just prior to use. In this transgenic F
2
fish line, the EGFP and
thus the apoaequorin, are expressed in the musculature at low levels at
I n v i v o reconstitution of aequorin
.
12 hpf
(i.e., the
6-somite stage) and the level of expression increases in an approximately
linear manner up to
24 hpf EGFP and thus apoaequorin
were expressed throughout the entire musculature, that is, in both the slow and fast
muscles. Thus, this line of muscle-specific apoaequorin-expressing transgenic
zebrafish can be used to visualize and characterize the Ca
2
þ
signals generated in
the trunk musculature during its formation and function. For imaging, these later-
stage embryos may be immobilized with 3% methyl cellulose. We have collected
24 hpf. In addition, at
