Biomedical Engineering Reference
In-Depth Information
Table 4.9
Modes of transfection
Method
Properties
References
Microinjection
Transitive/sustained
expression, best used on
smaller populations,
in
vitro
Shen, Hirschhorn
et al.
1982; Dean 1997
Lipofection kits
Ideal for diffi cult to infect
cell types, good cell
viability, transduction
effi ciency
FuGENE6, Roche
Applied Science,
Lipofectamine 2000;
Invitrogen
Electroporation
Normally transitive, highly
effi cient, best with cell
cultures
Nature Methods (2006)
Retroviral - Lentovirus
For non-dividing cells
Naldini, Blomer
et al.
1996
Adenoviral - Adeno
associated virus
Transitive expression
Monahan and Samulski
2000
Retroviral -
Alphavirus
Ideal for nervous system
Ehrengruber, Hennou
et al.
2001
Retroviral - Pox virus
Short-term expression
Mastrangelo, Eisenlohr
et al.
2000
not yet been determined for the protein of interest, co-localization may also be
performed (Manders
et al.
, 1993). Common areas for non-specifi c localization
are nuclei and diffuse background noise.
4.4.4
In situ
assays
For applications requiring a detailed analysis of cell-ECM interactions, espe-
cially of living cells,
in situ
hybridization (ISH)-type assays are used. These
can range from commercially available kits, such as the luciferase-based
kits available from Promega and New England Biosciences, to specifi cally
developed
in situ
PCR protocols.
Luciferase
The luciferase assay contains a luminescent probe tagged with the naturally
occurring bioluminescent luciferase protein, which is derived from fi refl ies
(FLuc) and marine animals (GLuc). The luciferase assay is easy to complete
with commercially available kits and is useful for live-cell assays, especially
ATP measurements, as no lysis step is necessary. Additionally, luciferase
can be used to monitor biological processes, including cell differentiation,
both
in vitro
and
ex vivo
(Wurdinger
et al.
, 2008). Readings are taken via
luminometer, a device designed to measure luminescence, or by an optical
microscope. Due to the wide range of luciferase applications no protocol
