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

Abstract

as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue. ) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity. 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.

Background

as a reporter, in that they can be easily visualized without recourse to the vital substrates required to visualize β-gal in living tissue.

Results

) pattern. The resulting EYFP or ECFP expression patterns indicated that the reporter strains function as faithful monitors of Cre activity.

Conclusions

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.

Background

]].

] http://www.clontech.com/gfp/pdf/LivingColors.pdf.

]. The availability of different Cre reporter strains will be valuable, not only because of the advantages of different reporter proteins, but also because the efficiency of Cre-mediated excision may be dependent on the target locus.

Results and Discussion

], were injected into C57BL/6J blastocysts, and the resulting chimeric mice were bred to C57BL/6J females to pass the mutation through the germ line. Thus, the resulting mice were a mixture of strains 129X1/SvJ and C57BL/6J.

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.

].

transgene. They are visualized with a YFP filter set (A), with bright field illumination (B), or with a CFP filter set (C).

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embryo, showing a comparable pattern of β-gal staining.

).

expression in the same mid-hindbrain region.

). This is not surprising, given the higher quantum yield and extinction coefficient of EYFP as compared to ECFP, both of which result in higher fluorescence intensity.

Conclusions

expression, which cannot be easily detected in living tissue, the EYFP and ECFP reporter strains (together with the GFP reporter strains currently available) will be very useful for monitoring the expression of Cre in living tissues, or tracing the lineage of these cells and their descendants, in cultured embryos or organs. Furthermore, by using modified forms of Cre whose recombinase activity is inducible, one can use these reporter mice to perform detailed analysis on the lineage of cells at different time points during development.

Sources of Plasmids

].

The plasmids pEYFP-N1 and pECFP containing cDNA for EYFP and ECFP were purchased from Clontech Laboratories Inc.

Targeting Constructs

] was replaced by a linker (PacI, SwaI, AscI), so that it could be digested with PacI and AscI, and receive the bigT sequence.

and bpA sequence but leave behind the SA, the ends were Klenow filled and the plasmid self-ligated. The resulting plasmid was digested with SacI and PstI to remove the 5' MCS, which was replaced by a PacI linker. The resulting plasmid was called pPacSA.

sequences, but leave behind the bpA, and self-ligated. The resulting plasmid was digested with XbaI and ApaI to remove most of the 3' MCS (except for the terminal KpnI site), which was replaced with an AscI linker. The resulting plasmid was called pbpAAsc.

site by digesting the DNA with XhoI and KpnI and ligating in the MCS, which had been synthesized to have compatible cohesive ends. This plasmid was called pSAleotpA. The bpA sequence along with the 3' AscI site was excised from pbpAAsc by digesting it with SacI and KpnI, and inserted downstream of the MCS in pSAleotpA by digesting it with SacI and KpnI. The resulting plasmid was called pBigT, and its MCS contains sites for the restriction enzymes NheI, SalI, AccI, XhoI, ApaI, SacII, NotI, SacI and BclI.

genomic sequence compatible with the pBigT plasmid, pROSA26-1 was digested with XbaI, Klenow filled and a linker (PacI, SwaI, AscI) inserted. This plasmid was called pROSA26PA.

assembly, and inserted into pROSA26PA digested with PacI and AscI. This plasmid was subsequently linearized with KpnI and used for electroporation.

cDNA. This was inserted into BigT digested with XhoI, Klenow filled, and then digested with NotI. BigT was then digested with PacI and AscI to release the entire floxed neo-tpA and CFP assembly, and inserted into pROSA26PA digested with PacI and AscI. This plasmid was subsequently linearized with KpnI and used for electroporation.

An appreciable amount of recombination was observed while trying to grow up the final targeting vector, resulting in an aberrant plasmid of smaller molecular weight. Therefore, the mixture of theses two plasmids was digested with KpnI which linearized both plasmids, and the correct targeting vector was gel purified. It was subsequently confirmed to be the correct targeting vector by diagnostic PCR, restriction digests and sequencing (data not shown).

locus in ES cells

cells were electroporated with 10 μg of each targeting vector and grown without feeders under selection in 300 μg/ml G418 for seven days. 96 colonies were picked and 27 screened, for electroporations with each of the two constructs R26R-YFP and R26R-CFP. Genomic Southern blot hybridization was performed on DNA from ES cells digested with EcoRV. The 5' probe used detects a 11 kb wild type band and a 3.8 kb targeted band, due to the presence of an extra EcoRV site in the targeted allele.

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Construction of the Isl1-Cre mouse strain

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Mouse strains

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Detecting EYFP and ECFP expression

), unfixed embryos were photographed using a Nikon epifluorescence microscope fitted with Chroma filter sets for ECFP (cyan GFP Ex436/20 Dm455 Bar480/40) and EYFP (yellow GFP Ex500/20 Dm515 Bar535/30). Digital images were acquired using a Spot camera.

), embryos were fixed overnight in 4% paraformaldehyde at 4°C, washed 2x for 10 min. in PBS, then equilibrated in the following solutions until the embryos settled at the bottom (approx. 30 min): PBS, 5% sucrose in PBS, 10% sucrose in PBS, and 15% sucrose in PBS. They were then equilibrated in a 1:1 mixture of OCT (Tissue-Tek, Mile, Inc.) and 15% sucrose in PBS for >1 hour, and embedded in OCT over dry ice. Sections were cut at 8 - 12 μM, blow-dried for 30 min. at low heat, then stored at -80°C with desiccant in an air tight bag. Before being photographed, the slides were brought to room temperature, washed 3x in PBS, mounted in Vectashield (Vector Laboratories), covered with a cover glass and sealed with clear nail polish. Sections were photographed as described above.

Acknowledgements

We thank Alex Joyner and Wolfgang Wurst for the En-1/Cre mice, Philippe Soriano for ROSA26 genomic sequences, Roger Pederson for the JM-1 ES cell line, and Zaiqi Wu for excellent technical assistance. F.C. was supported by grants from the NIH. T.M.J. was supported by grants from the NIH and is an Investigator of the Howard Hughes Medical Institute.