Biology Reference
In-Depth Information
improved light emission properties of GFP-aequorin are still not completely
understood, it is thought that they likely relate to an improved stability of the
apoaequorin protein or to an increased quantum yield of the Ca
2
þ
-activated
photoprotein complex, or perhaps both.
The cloned GFP-apoaequorin gene has been engineered to target di
V
erent
subcellular compartments using similar strategies as those developed and described
already for aequorin (
Rizzuto et al., 1992
). This approach has also been extended
to include other spectral variants of these aequorin-based reporters, by replace-
ment of the GFP gene with the sequences encoding Venus (YFP), mRFP, and
more recently, mOrange (
Bakayan et al., 2009; Curie et al.,2007
;
Manjarr´s et al.,
2008
). The development of these bifunctional reporters together with new imaging
technologies (see
Section IV
) has considerably extended the number of applica-
tions possible with aequorin and in particular, has facilitated important advances
in multicompartment measurements of Ca
2
þ
concentrations and in noninvasive
whole animal Ca
2
þ
-imaging studies of the mammalian system.
A. Expression of Apoaequorin
1. Protocol 1: Preparation of Transgenic Zebrafish that Express Apoaequorin in a Tissue-Specific
Manner (e.g., in the skeletal musculature)
a. Materials
piP-HE (apoaequorin plasmid;
Inouye et al., 1989
)
a
p-SK plasmid (
a
-actin promoter;
Higashijima et al., 1997
)
pIRES2-EGFP plasmid (Clontech Laboratories, Inc., Mountain View, CA,
USA)
pCMVT
N
T vector (Promega Corp., Madison, WI, USA)
f-coelenterazine (C-6779; Molecular Probes, Invitrogen Corp., Eugene, OR,
USA)
Methyl cellulose (M0387; Sigma-Aldrich Corp., MO, USA)
b. Methods
i. Preparation of the p
-KS-aeq-IRES-EGFP plasmid. To prepare the p
a
-KS-
aeq-IRES-EGFP plasmid, use PCR to amplify the apoaequorin gene from the
piP-HE plasmid, with the following oligonucleotide primers: 5
0
-accagaattcatgacaag-
caaacaatactcagtcaagcttacatcagac-3
0
and 5
0
-accagtcgacttaggggacagctccaccgtagag-3
0
,
such that EcoR1 and Sal1, are added to the 5
0
and 3
0
ends of the apoaequorin
gene, respectively. The apoaequorin gene can then be cloned into the pIRES2-EGFP
plasmid using these restriction sites. Excise the aeq-IRES-EGFP fragment with
EcoR1 and Not1, and then clone it into the
a
p-SK plasmid to obtain an aeq-
IRES-EGFP fragment with an
a
-actin promoter (i.e.,
a
a-aeq-IRES-EGFP). In paral-
lel, amplify the SV40 late polyadenylation signal (pA) from the pCMVT
N
Tvector
using the following oligonucleotide primers: 5
0
-accagcggccgccagacatgataagatacattg-
3
0
and 5
0
-accagagctctctagaaccggttaccacatttgtagaggtttt-3
0
, adding Not1tothe5
0
end,