Biology Reference
In-Depth Information
i. Transgenic mice expressing GFP-apoaequorin reporters. GFP-aequorin has
also been expressed in a number of mammalian cell lines, as well as in organisms
such as Plasmodium bergei (
Billker et al., 2004
), Drosophila melanogaster (
Martin
et al., 2007
), and in mice (
Rogers et al., 2007
). Transgenic mice can provide a
source of cells, tissues, or organs for studies ex vivo or for studies in vivo (
Rogers
et al., 2007
). In the case of targeted reporters, these are especially useful because
they provide information regarding the localization of any probe-derived signals,
which would otherwise be di
Y
cult because of the low to moderate resolution
a
orded by bioluminescence imaging. In addition, an inducible or conditional
expression system could be introduced into the vector (e.g., Cre/Lox or Tet
inducible elements), to ensure the absence of a phenotype in the event that expres-
sing the reporter ubiquitously from the early stages of development is found to be
detrimental. A conditional expression system also enables expression to be acti-
vated in a specific cell population or at di
V
erent stages of development. Indeed, the
UAS-Gal4 system enables the specific expression of GFP-aequorin in neuronal
subsets of the fly brain, allowing specific neuroanatomical mapping of Ca
2
รพ
signaling pathways (
Martin et al., 2007
).
Transgenic mice can be generated via one of two methods: (1) using a 'classical'
transgenesis approach, where the transgene is randomly integrated into the
genome (
Constantini and Lacy, 1981
), or (2) by homologous recombination,
which enables directed integration of the transgene (e.g., knock-in of the HPRT
locus;
Rogers et al., 2007
). Similarly to pronuclear injection, lentiviral vectors can
be used to deliver the transgene into the fertilized mouse egg (
Ikawa et al., 2003
).
However, these methods can result in random and multiple integrations of the
transgene. In contrast, homologous recombination targets transgene insertion in a
single-copy to a known site in the genome ensuring a more predictable expression
pattern and phenotype based on the known integration site (
Bronson et al., 1996
).
Transgenic mice conditionally expressing mitochondrially targeted GFP-apoae-
quorin have already been generated using this method (
Rogers et al., 2007
).
Targeted insertion of the transgene was made 5' to the X-linked hypoxanthine
phosphoribosyltransferase (HPRT) locus (X-chromosome).
V
4. Protocol 4: Preparation of Acute Brain Slices from Transgenic Mice Expressing
Mitochondrially Targeted GFP-Aequorin
a. Materials
Vibratome (e.g., Model VT-1000, Leica)
Oxygen (95% O
2
/5% CO
2
)
Glass petri dish (3-5 cm diameter)
ACSF, pH 7.4 (124 mM NaCl, 3 mM KCl, 1.3 mM MgCl
2
, 25 mM NaHCO
3
,
1.25 mM NaHPO
4
and 10 mM glucose)
Superglue, Blades, Low temperature melting Agar
Specimen chamber for live imaging (e.g., RC-20 chamber from Harvard
apparatus)
