Sources of Plasmids
The components of the ROSA26 targeting vectors were a gift from Philippe Soriano. The adenovirus splice acceptor (SA) and bovine growth hormone polyadenylation sequence (bpA) were from plasmid pSAβgeo . The loxP flanked neo cassette, and trimer of the SV40 polyadenylation sequence (tpA), were from plasmid PGKneotpAlox2 . The ROSA26 genomic sequence and the diphtheria toxin (DTA) expression cassette were from plasmid pROSA26-1 , and the template for the external probe used to genotype ES cells was from plasmid pROSA26-5' .
The plasmids pEYFP-N1 and pECFP containing cDNA for EYFP and ECFP were purchased from Clontech Laboratories Inc.
The plasmid pBigT consists of the adenovirus splice acceptor sequence followed by a loxP site, neo expression cassette, strong transcriptional stop sequence (triple SV40 polyadenylation sequence), another loxP site in the same orientation as the first, a multiple cloning site (MCS), and the bovine growth hormone polyadenylation sequence. A PacI site was included 5' to the SA, and an AscI site 3' to the bpA. These two enzymes are rare eight base pair cutters and result in sticky ends upon digestion and can be used to excise the entire construct, for insertion into the plasmid with the ROSA26 genomic arms. The XbaI site used for insertion into the ROSA26 genomic locus  was replaced by a linker (PacI, SwaI, AscI), so that it could be digested with PacI and AscI, and receive the bigT sequence.
To make pBigT, plasmid pSAβgeo was digested with ClaI and XbaI to remove the βgeo 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.
Next, pSAβgeo was digested with SacI to remove the SA and βgeo 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.
The loxP-neo-tpA-loxP was excised from plasmid PGKneotpAlox2 by cutting with NotI, Klenow filling, and then cutting with KpnI. This was inserted downstream of the SA in pPacSA digested with SalI, Klenow filled, and then cut with KpnI. A MCS containing the restriction sites for XhoI, ApaI, SacII, NotI, SacI, EcoRV, and KpnI was synthesized and inserted downstream of the second loxP 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.
To make the ROSA26 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.
To make the R26R-YFP targeting construct, the EYFP cDNA was excised from pEYFP-N1 with ApaI and NotI and inserted into BigT digested with ApaI and NotI. BigT was then digested with PacI and AscI to release the entire floxed neo-tpA and EYFP assembly, and inserted into pROSA26PA digested with PacI and AscI. This plasmid was subsequently linearized with KpnI and used for electroporation.
To make the R26R-CFP targeting construct pECFP was digested with AgeI, Klenow filled then digested with NotI to excise the ECFP 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).
Targeting of the ROSA26locus in ES cells
JM-1 ES cells  were expanded by culturing on feeder cells in medium supplemented with LIF. Approximately 15 × 106 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.
Mice carrying the targeted allele were genotyped by the PCR as described .
Construction of the Isl1-Cre mouse strain
Isl1 genomic DNA had been previously isolated from a mouse 129/Sv genomic library (Stratagene) as described by Pfaff et al. . A PacI site had been introduced into an EcoRI site in the exon encoding the second LIM domain of Isl1. A cassette coating IRES Cre SV40 pA and pgk-neomycin was cloned into this PacI site to create a targeting construct with flanking 5' and 3' genomic DNA arms of 5 kb and 2 kb, respectively. ES cells were targeted and screened as described in Pfaff et al..
En-1/Cre mice (En1Cki allele) were a gift of Dr. Alexandra Joyner and Dr. Wolfgang Wurst . β-actin/Cre mice were a gift of Dr. Gail Martin . R26R-lacZ reporter mice were a gift of Dr. Philippe Soriano .
Detecting EYFP and ECFP expression
For whole mount photographs (Figures 2 and 4), 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.
For histological sections (Fig 3), 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.