Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus - PubMed (original) (raw)

Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus

S Srinivas et al. BMC Dev Biol. 2001.

Abstract

Background: Several Cre reporter strains of mice have been described, in which a lacZ gene is turned on in cells expressing Cre recombinase, as well as their daughter cells, following Cre-mediated excision of a loxP-flanked transcriptional "stop" sequence. These mice are useful for cell lineage tracing experiments as well as for monitoring the expression of Cre transgenes. The green fluorescent protein (GFP) and variants such as EYFP and ECFP offer an advantage over lacZ as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize beta-gal in living tissue.

Results: In view of the general utility of targeting the ubiquitously expressed ROSA26 locus, we constructed a generic ROSA26 targeting vector. We then generated two reporter lines of mice by inserting EYFP or ECFP cDNAs into the ROSA26 locus, preceded by a loxP-flanked stop sequence. These strains were tested by crossing them with transgenic strains expressing Cre in a ubiquitous (beta-actin-Cre) or a cell-specific (Isl1-Cre and En1-Cre) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity.

Conclusions: In contrast to existing lacZ reporter lines, where lacZ expression cannot easily be detected in living tissue, the EYFP and ECFP reporter strains are useful for monitoring the expression of Cre and tracing the lineage of these cells and their descendants in cultured embryos or organs. The non-overlapping emission spectra of EYFP and ECFP make them ideal for double labeling studies in living tissues.

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Figures

Figure 1

Figure 1

Targeting of the ROSA26 locus. A, top to bottom: pBigT, a plasmid containing a loxP-flanked cassette with a PGK-neo selectable marker and a tpA transcriptional stop sequence, into which the EYFP or ECFP was cloned; pROSA26PA, containing genomic ROSA26 sequences for homologous recombination, and a diphtheria toxin gene (PGK-DTA) for negative selection in ES cells; the wild type ROSA26 locus, with the location of the probe indicated; the structure of the targeted locus; and the structure of the targeted locus after Cre-mediated excision of the _loxP_-flanked (PGK-neo, tpA) cassette. LoxP sites are indicated by solid arrowheads. B, Southern blot of DNA from seven ES cell lines, digested with EcoRV and hybridized with the probe indicated in A. The 11 kb band is the wild type band and the 3.8 kb band represents the targeted allele. Lines Y25 and C4 are wild type, while the remainder are heterozygous for the targeted allele.

Figure 2

Figure 2

Ubiquitous expression of EYFP or ECFP in R26R E8.5 embryos carrying a β-actin-Cre transgene. The two embryos, one carrying R26R-EYFP (right) and one carrying R26R-ECFP (left), were both heterozygous for the β-actin-Cre transgene. They are visualized with a YFP filter set (A), with bright field illumination (B), or with a CFP filter set (C).

Figure 3

Figure 3

Specific expression of EYFP in R26R-EYFP mice carrying Isl1-Cre. A, transverse section of an E14.5 R26R-EYFP/+; Isl1-Cre/+ embryo, revealing expression of EYFP in the motor neurons and dorsal root ganglia. The apparent expression in surface ectoderm is an artifact, as it was also seen in non-transgenic embryos (data not shown). Panel B, transverse section of E12.5 R26R-lacZ; Isl1-Cre embryo, showing a comparable pattern of β-gal staining.

Figure 4

Figure 4

Specific expression of EYFP at the mid-hindbrain junction in a R26R-EYFP E8.5 embryo carrying En1 Cki, an Engrailed-1 Cre knock-in allele. A, dark field illumination, showing anterior portion of embryo. B, YFP expression in the same embryo. The outline of the embryo is indicated by the dotted line. C, E8.5 embryo from a cross between_En1_ Cki and the R26R lacZ allele [4], resulting in lacZ expression in the same mid-hindbrain region.

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