Biology Reference
In-Depth Information
both temporal and spatial information regarding the Ca
2
þ
signals generated by the
musculature from these embryos up to 96 hpf. For imaging, we use two custom-
built Photon Imaging Microscope Systems (PIMS; Science Wares, Falmouth, MA,
USA), one based on an IPD-425 (Photek Ltd., Sussex, UK) and the other based on
a back-illuminated EMCCD (DU-897 iXon
EM
þ
camera) that was purchased from
Andor Technology (Belfast, Northern Ireland, UK) and then optimized by Science
Wares Inc. for detecting single photon events at the emission rates typical for
aequorin-based imaging (see
Section IV
for further details).
B. Expression of GFP-Apoaequorin
2. Protocol 2: Transient and Stable Transfection of Mammalian Cells with GFP-Apoaequorin
Using Plasmid DNA
Many of the expression vectors designed for gene delivery are commercially
available. They contain a number of units, including an immediate-early enhancer/
promoter sequence such as human cytomegalovirus (HCMV), a multiple cloning
region for insertion of the reporter gene, an antibiotic resistance gene (e.g., Ampi-
cillin) for selection of the vector in Escherichia coli, and an additional antibiotic
resistance gene for selection in mammalian cells (e.g., Neomycin G148). For
mammalian expression, a good starting point is to clone the reporter gene into a
vector containing an HCMV promoter (
Williams et al., 2005
). The constitutive
immediate-early HCMV promoter drives high levels of GFP-aequorin expression
in many mammalian cell lines following transient transfection.
Transfection reagents (e.g., cationic liposomes) enable recombinant DNA deliv-
ery into the nucleus of many immortalized cell types such as HEK293, HeLa,
COS7, CHO and NIH/3T3 cells, with high e
ciency. Following transient trans-
fection, stable clones can then be isolated using a combination of drug selection
(e.g., Neomycin (G418) resistance) and cell sorting using flow cytometry. On the
other hand, primary cells (e.g., cortical neurons) are usually transfected with very
low e
Y
ciency (i.e., less than 1-5%) using this method and better results can be
obtained by using recombinant viral vectors for gene delivery (
Rogers et al., 2005
).
Y
a. Materials
Transfection reagent (e.g., Fugene
Ò
6 reagent, Roche Applied Science; Lipofec-
tamine, Invitrogen; PolyFect, Qiagen).
Ultrapure plasmid DNA (1 mg/ml)—Plasmid DNA can be purified using a
plasmid purification kit (e.g., Qiagen
Ò
purification kits).
Optimem media (Invitrogen)
35-mm Petri dishes or 8-chamber slides (e.g., those available fromMatTek corp.
or Ibidi Gmbh). Any culture dish will do providing the bottom of the dish is
optimized for high-resolution microscopy on an inverted setup (e.g., dishes
prepared with a glass coverslip mounted underneath a hole cut in the bottom).
