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. 2001 Jul;21(14):4829–4836. doi: 10.1128/MCB.21.14.4829-4836.2001

FIG. 3.

FIG. 3

Retroviral insertion into the Neph1 locus. (A) Structure of the gene trap vector integration site in the Neph1 locus. The retroviral construct, which contains flanking viral long terminal repeats (LTRs), is inserted in the intron downstream of the ATG-containing Neph1 exon. Transcripts initiating at the Neph1 promoter (indicated by an arrow followed by broken and solid lines) encode a 4.5-kb message that utilizes the splice acceptor (SA) site upstream of the β-geo fusion gene. Transcripts initiating from the PGK promoter utilize the splice donor site in the inserted heterologous exon 1-splice donor (SD)-intron 1 cassette and splice into the downstream Neph1 exon (EX) to produce a roughly 8.7-kb RNA which is detectable only in ES cells and is absent in vivo (see text). The heterologous exon 1 is a noncoding exon that contains a natural in-frame stop codon (black diamond) that eliminates the likelihood of expression of a protein from the trapped Neph1 locus. A probe spanning the ATG-containing exons and downstream exons of Neph1 (black bars) was used to perform the Northern blot shown in Fig. 2 and 3C. The OST sequence is represented by a double line. The primers used to clone the Neph1–β-geo and the exon 1-Neph1 fusion transcript splice junctions are indicated by arrows and are expected to produce 354- and 485-bp bands, respectively; the sequence of the 5′ transcript is given in panel B. (B) Sequence of the hybrid transcript spanning the splice junction of the Neph1 ATG-containing exon and the β-geo-coding region. The ATG in the Neph1 coding region (boldface) is underlined, and the primers used for RT-PCR of this fragment are designated by broken arrows. (C) Northern blot of kidney RNA obtained from 1-day-old mice bearing either one copy (+/−) or two copies (−/−) of the trapped Neph1 locus. The blot, hybridized with a probe spanning the Neph1 ATG-containing and downstream exons (see panel A), shows RNA transcripts of 9 and 4.5 kb. (D) Ethidium bromide-stained gel showing RT-PCR products from the heterologous exon 1-Neph1 fusion transcripts. Lane 1 contains molecular-weight standards. The expected band size of 485 bp (see panel A) is clearly observed in the RNA samples from the ES cell line with the Neph1 mutation (lanes 2 and 3). This band is undetectable in kidney RNA from Neph1+/+ and Neph1+/− mice (lanes 4 and 6, respectively) and is negligible in kidney RNA from Neph1−/− mice (lane 5).