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: amq@montanamolecular.com

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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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