clontechnique note for cre (science).pages. - addgene · the cre stoplight plasmid switches color...
TRANSCRIPT
Cre Stoplight with Living Colors® is a faster, brighter
reporter for Cre recombinase.
Drago A Guggiana-Nilo1, Anne Marie Quinn 2§ ,Thomas E. Hughes 1
1Department of Cell Biology and Neuroscience, Montana State University,
Bozeman MT 59715
2 Montana Molecular, LLC,
Bozeman MT 59715
§Corresponding author
Email address:
Anne Marie Quinn: [email protected]
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Introduction
Cre recombinase is a powerful and widely used tool for manipulating the
genome. The enzyme targets areas of the chromosome that are flanked by
specific loxP recognition sites and can either delete genes, or release new
gene expression in a transgenic animal. Cre can introduce mutations that can
be specifically activated (the inducible knockout), or targeted to specific cells.
Many new genetic strains of mice have become available that either express
Cre recombinase in particular cells and tissues, or that harbor the loxP sites
that Cre recognizes (1).
The Cre enzyme is a 38kD recombinase that recognizes a 34 bp sequence,
the loxP site, in the target DNA. By a series of staggered cuts, recombination
occurs between the two loxP sites and the enzyme releases the DNA. When
the loxP sites are positioned in opposite orientations, the intervening DNA is
inverted, and when the sites are oriented head to tail, the DNA in between is
excised. Cre is powerful because the reaction it catalyzes is robust, it does
not require additional proteins, it can manipulate large regions of the genome,
and it can be targeted to specific cell types (2). The ability to detect exactly
which cells have been manipulated by Cre, in any experimental setting, is
essential to verifying and interpreting Cre-mediated events.
Using color as a reporter
The Cre Stoplight plasmid switches color to report Cre activity by conditionally
expressing two different fluorescent proteins (3). The original reporter uses
the red fluorescent protein DsRed (4) followed by a transcription terminator.
These two elements are flanked by loxP and loxH sites, which are followed by
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eGFP (enhanced Green Fluorescent Protein). Cells transiently transfected
with only the original Cre Stoplight produce red fluorescence. However, if Cre
is also expressed, DsRed is excised, along with the transcription terminator,
and eGFP is expressed. The loxP and variant loxH sites make this reaction
irreversible (5). The original Cre Stoplight has been widely used since it was
published (6-8) and we recently reengineered the plasmid to use Living
Colors® fluorescent proteins.
Cre Stoplight with Living Colors® is brighter, faster and more useful.
We improved the original Cre Stoplight by 1) using optimized fluorescent
proteins that mature quickly and produce strong fluorescence, 2) swapping
the arrangement of the fluorescent proteins, 3) removing superfluous
elements of the plasmid. The original Cre Stoplight expresses a tetrameric
DsRed (9) in the absence of Cre. The arrangement of red as the default and
green as the Cre indicator limits the utility of the original reporter in the context
of cell lines and animal strains that already contain a green fluorescent protein
(10). To address this, we inverted the order of the fluorescent proteins such
that green fluorescence would be expressed in cells without Cre, and red
would indicate functional Cre. After testing a series of fluorescent proteins,
we identified Zsgreen1 (4) and Mcherry (11) to be the brightest combinations
in this expression context. The original Cre Stoplight also contained a variety
of arbitrary tags, cloning sites, and vector sequence that had accumulated
during the history of the plasmid. We removed all of these extra elements to
create a more compact and efficient reporter system.
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Conclusions
A new and improved version of the Cre Stoplight reporter is now available that
is brighter, faster and more compatible with current techniques. Cre Stoplight
with Living Colors® employs ZsGreen1 and mCherry to yield bright
fluorescence 24 hours post transfection. This new version of the original
reporter detects Cre activity both qualitatively and quantitatively in living cells
when co-expressed with the Cre enzyme.
References
1. J. Livet, T. A. Weissman, H. Kang, R. W. Draft, et al., Nature 450, 56-62
(2007).
2. A. Nagy, Genesis 26, 99-109 (2000).
3. Y. S. Yang, T. E. Hughes, Biotechniques 31, 1036, 1038, 1040-1 (2001).
4. M. V. Matz, A. F. Fradkov, Y. A. Labas, A. P. Savitsky, et al., Nat Biotechnol
17, 969-73 (1999).
5. F. Buchholz, A. F. Stewart, Nat Biotechnol 19, 1047-52 (2001).
6. G. Briones, D. Hofreuter, J. E. Galán, Infect Immun 74, 1084-90 (2006).
7. R. G. Harris, E. L. Herzog, E. M. Bruscia, J. E. Grove, et al., Science 305,
90-3 (2004).
8. H. Li, X. Zhou, D. R. Davis, D. Xu, C. D. Sigmund, Am J Physiol Renal
Physiol 294, F1481-6 (2008).
9. G. S. Baird, D. A. Zacharias, R. Y. Tsien, Proc Natl Acad Sci U S A 97,
11984-9 (2000).
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10. T. Yoshimizu, N. Sugiyama, M. De Felice, Y. I. Yeom, et al., Development,
Growth & Differentiation 41, 675-84 (1999).
11. N. C. Shaner, R. E. Campbell, P. A. Steinbach, B. N. G. Giepmans, et al.,
Nature biotechnology 22, 1567-72 (2004).
SV40 poly A
loxp site
T7 transcription terminationloxH
BGH poly A
mCherryZsGreen1CMV promoter
A B
C D
100 pg of pBS18524 hrs A.T.
1 ug of pBS18548 hrs A.T.
1 ug of pBS18524 hrs A.T.
Control - No pBS18524 hrs A.T.
Figure 1. Cre Stoplight 2.4 with Living Colors® is a brighter option to the
original Cre Stoplight. Above: DNA map of the improved Cre Stoplight.
Below: testing Cre Stoplight 2.4. 500 ng of the Cre Stoplight 2.4 were co-
transfected into HEK 293 cells with varying amounts of the Cre expression
vector, and imaged at different times after transfection. A: Control experiment,
no Cre expression vector added, 24 hours after transfection; B: 100 pg of Cre
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expression vector added, 24 hours after transfection; C: 1 µg of Cre
expression vector added, 24 hours after transfection; D: 1 µg of Cre
expression vector added, 48 hours after transfection.
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