Generation of the targeting vector, ES cells, and chimeras.

A 2.5-kb PCR fragment of Aqp2 gene as the 3′ arm was amplified using primers WZ1499/1500 with mouse tail DNA as template. The fragment was cloned into pcDNA 3.1 (+) at PciI and BstZ171 to create p692. A 2.9-kb fragment containing ER^T2CreER^T2 was released from pCAG-ERT2CreERT2 and inserted into p692 at EcoRI and NotI to generate p693. The 5′ arm was created through overlapping PCR. The first, a 4.1-kb fragment of the Aqp2 gene, was amplified from mouse genomic DNA with primers WZ1494/1502. The second, a 1.6-kb fragment, was synthesized with primers WZ1495/1496 using pCAG-ERT2CreERT2 as the template. The final 5.7-kb 5′ arm, obtained via PCR using primers WZ1496/1502 with the two fragments as the template, was then cloned into p693 at EcoRI and AgeI to generate the final target vector p694. The authenticity of the whole insert sequence in the final target was confirmed by sequencing.

After linearization with KpnI, which trimmed the 5′ and 3′ arms to 1.8 kb and 2.3 kb, respectively, the targeting vector was electroporated into C57BL/6N JM8.N4 embryonic stem (ES) cells. The C57BL/6N background of the ES cells offers a unique advantage for identification of chimeras (see below). A total of 384 G418-resistant clones were picked, duplicated, and expanded, and total DNA was extracted for Southern analyses. Probes to screen for homologous recombination in Southern analyses were selected that were outside the region of homology used for the arms of the targeting vector. Probes were generated using PCR. The 3′ probe (893 bp) was amplified using primers TMF571/572. The 5′ probe (474 bp) was amplified using primers TMF241/242. DNA fragments were labeled using [α-³²P]dCTP and hybridized and washed to digested ES cell genomic DNA using standard methods. To detect recombination at the 3′ arm, DNA was digested with BamHI, with a 9.8-kb hybridizing band indicating a wild-type (WT) and a 12.5-kb band allele denoting targeted allele. For the 5′ arm, DNA was digested with EcoRV with band sizes of 9.7 kb (WT) and 8.6 kb (targeted) (Fig. 1A). Chromosome counting was carried out to verify the genome integrity of six correctly targeted ES clones. Two of these clones with >80% euploid cells were injected into C57BL/6J blastocysts to produce chimeras. Since the ES cells and the blastocysts are homozygous for the WT and mutant nicotinamide nucleotide transhydrogenase gene (Nnt^+/+ and Nnt^−/−), respectively (19), PCR analyses were done to identify the chimeras and to evaluate the relative contribution of the ES cells. The expected products are 576 bp for the WT allele and 743 bp for the mutant allele.

Generation of Aqp2^ECE/+ mice. Aqp, aquaporin; ECE, cassette expressing estrogen receptor (ER)^T2CreER^T2. A: schematic illustration of targeting strategy for the Aqp2^ECE knock-in allele. B and C: Southern blotting analyses of embryonic stem (ES) cell clones confirming the targeted Aqp2^ECE knock-in allele. D: PCR-based genotyping of chimeras showing male 5022 with almost 100% contribution of ES cells, as evidenced by the presence of the ES cell-derived WT allele coupled with barely detectable host blastocyst mutant allele of nicotinamide nucleotide transhydrogenase gene (Nnt). E: PCR-based genotyping showing that male 5022 had a high rate in germline transmission of Aqp2^ECE allele to offspring.