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CURRENT PROTOCOLS IN MOLECULAR BIOLOGY


CHAPTER 9. INTRODUCTION OF DNA INTO MAMMALIAN CELLS
SECTION II USES OF FUSION GENES IN MAMMALIAN TRANSFECTION
UNIT 9.6 Overview of Genetic Reporter Systems
TABLE(S)

Table 9.6.1 Genetic Reporter Systems


Reporter gene
In vitro assay
In vivo assay

Strengths

Weaknesses

Chloramphenicol Chromatography,
acetyltransferase differential
(CAT)
extraction,
fluorescence, or
immunoassay

None commonly
available

Minimal endogenous
activity in mammalian
cells; stable protein;
various assay formats
available

Firefly luciferase Bioluminescence


(luminometer or
scintillation
counter)

Bioluminescence
assay in live cells
with luciferin
esters as
substrate

Nonisotopic; good
sensitivity and broad
linear range (4 orders
of magnitude); minimal
endogenous activity in
mammalian cells;
relatively inexpensive

Assays are time5-10 x 107


consuming and
laborious; most
formats require
expensive radioactive
substrate; relatively
low sensitivity and
narrow linear range
(2 orders of
magnitude);
fluorescent assays
offer better
sensitivity, but require
a fluorometer for
quantitation; short
half-life of CAT
mRNA
Short half-life of
1-2.5 x 105
protein; assay
requires a
luminometer or
scintillation counter;
conventional assay
lacks reproducibility

b-galactosidase

Colorimetric,
Histochemical
Nonisotopic; various
fluorescence,
staining with X-gal assay formats
chemiluminescence substrate;
available for different
(luminometer or
bioluminescence applications;
scintillation
assay in live cells chemiluminescent
counter)
with fluorescein assay is very
di-b-Dsensitive, and has an
galactopyranoside extremely broad linear
range (5-6 orders of
(FDG)
magnitude); well
suited as internal
control for normalizing
other reporters
Secreted alkaline Colorimetric,
None commonly Nonisotopic; secreted
phosphatase
bioluminescence, available
reporter protein;
(SEAP)
or
various assay formats
chemiluminescence
available;
(luminometer or
chemiluminescent
scintillation
assay is very
counter)
sensitive, and has a
broad linear range (4
orders of magnitude);
useful for high
throughput assays
performed in 96-well
microtiter plates
Human growth Radioimmunoassay None commonly Secreted reporter
hormone (hGH)
available
protein; direct
detection of protein
levels; simple assay,
easy to perform; highthroughput assay
performed in 96-well
microtiter plates;
useful as internal
control for normalizing
other reporters (e.g.,
SEAP)
Histochemical
Nonisotopic; various
b-glucuronidase Colorimetric,
fluorescence, or
staining with Xassay formats
(GUS)
chemiluminescence Gluc substrate
available for different
(luminometer or
applications; GUS
scintillation
protein is stable;
http://www.geguchadze.com/PDF/protocols/CPonline/Doc/16933-16933.html

Limit of
detection
(molecules)a

Cost/assay Available reporter


(U.S.
vectorsc
dollars)b
2.00-7.00

pSV0CAT and
pSV2CAT (Gorman et
al., 1982); pA10CAT2
(Laiminis et al., 1984);
pCAT-Basic, pCATEnhancer, pCATPromoter, and pCATControl (Promega)

0.10-0.25

pT8lluc, pSluc2,
pXP1/pXP2, and
pOLUC (Nordeen,
1988)
pMAMneo-LUC
(Clontech) pGL2Basic, pGL2Enhancer, pGL2Promoter, and pGL2Control (Promega)
pbgal-Basic, pbgalEnhancer, pbgalPromoter, and pbgalControl (Clontech)
pSV-b-gal (Promega)

Many cell types have 104-105


0.01-0.02
high endogenous b- (chemiluminescence
galactosidase
assay)
activity; assays
require fluorometer or
luminometer

May not be suitable 104-105


0.01-0.04
for cells expressing (chemiluminescence
low levels of
assay)
placental-type
alkaline phosphatase
(lung, testes, and
cervix); may not be
suitable if
experimental design
affects secretory
capacity of target
cells

pSEAP-Basic, pSEAPEnhancer, pSEAPPromoter, and pSEAPControl; (Clontech)


pBC12/RSV/SEAP
and pBC12/HIV/SEAP
(Berger et al., 1988)

Radioactive assay
3 x 108
requiring 125I-labeled
antibody; no signal
amplification as with
activity assays;
relatively low
sensitivity and narrow
linear range;
expensive

1.00-2.00

pXGH, pOGH, and


pTKGH (Selden et al.,
1986)

Assays with best


sensitivity require
fluorometer or
luminometer
(scintillation counter

N/A

pBI101, pBI101.2,
pBI101.3, pBI121,
pBI221, and
pGUSN358-S
(Clontech)

N/A

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counter)

Green
fluorescent
protein (GFP)

a,b,c

Fluorescence (UV
light box or
fluorescence
imaging device)

chemiluminescent
can substitute)
assay is very
sensitive, and has an
extremely broad linear
range (6 orders of
magnitude);
predominant reporter
used in plant genetic
research
Fluorescence (UV
light box,
fluorescence
microscopy, or
FACS analysis)

Reporter of gene
Signal intensity may N/A
expression and protein be too weak for some
localization in live
applications; this
cells; fluorescence
limitation is largely
occurs in a speciesovercome by brighter
independent fashion; GFP mutants such as
fluorescence is an
EGFP
intrinsic property of the
protein and does not
require additional gene
products, substrates,
or cofactors;
fluorescent signal is
highly resistant to
photobleaching; no
apparent toxic effects
of GFP expression in
bacteria or eukaryotes

Essentially
free, no
additional
reagents
necessary

pEGFP, pEGFP1,
pEGFP-N1,2,3, and
pEGFP-C1,2,3
(Clontech)

Much of this information was taken from Alam and Cook, 1990. The lists of available reporter vectors may not be complete. N/A: information not available.

From Current Protocols in Molecular Biology Online


Copyright 2003 John Wiley & Sons, Inc. All rights reserved.

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