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Fig. 3. Gene targeting at the Dusp6 locus. (A) Structure of
the linearized Dusp6 targeting vector and depiction of the wild-type
Dusp6 allele (Dusp6+), the correctly targeted
mutant allele in ES cells (Dusp6LACN) and the targeted
allele found in mice following expression of CRE in germlinetransmitting
chimeras (Dusp6L). Mouse genomic Dusp6 DNA is
depicted with solid thick lines; dotted lines indicate Dusp6 genomic
DNA not present in the targeting vector; open boxes indicate untranslated
regions; solid boxes indicate protein coding regions. The LacZ gene
(nls-lacZpA) is shown as a dark gray box; the Cre/Neo `suicide cassette' (ACN)
as a light gray box; the stop codon in the DUSP6 frame in exon 3 as an
asterisk. Flanking thymidine kinase genes (TK1 and TK2, transcriptional
orientation indicated by arrows) and the plasmid backbone are depicted as open
boxes. Recognition sites for NdeI are indicated by `N'; probes used
for Southern analysis by black bars. Numbered arrows indicate the identity,
position and directionality of primers used in PCR assays. (B) Southern
blot hybridization assay demonstrating correct targeting of Dusp6 in
ES cells. NdeI-digested DNA from the R1 ES cell line (R1), a cell
line with a random insertion of the targeting vector (Ran) and a targeted cell
line (Tar) was probed sequentially with 3' and Cre probes. Correctly
targeted cell lines had a novel 7.0 kb fragment that hybridized with both
probes. (C) PCR assay used to detect the stop-codon-containing
insertion in exon 3. DNAs isolated from correctly targeted ES cells (Tar), a
control random integrant (Ran) and wild-type cells (R1) were PCR-amplified
using primers 376 and 331. Targeted cell lines (TAR*) that produced
both the wild-type (187 bp) and the insertion amplicon (198 bp) were selected
for germline transmission. (D) PCR assay used to genotype offspring of
Dusp6+/L intercrosses. Tail DNAs were PCR-amplified with
primers 344, 309 and 315. The mutant allele yielded a 363 bp band and the
wild-type allele yielded a 499 bp band. (E) Northern blot hybridization
of mRNA isolated from E11.5 embryos of the indicated genotypes was probed with
a fragment of Dusp6 3' UTR, revealing a wild-type transcript of
3 kb in +/+ and +/-samples that was absent from the -/-sample. A minor
read-through transcript of 7.5 kb was evident in +/- and -/-samples, but
due to the targeting strategy, it is incapable of encoding functional DUSP6.
Rehybridization with a Gapdh probe is shown below.
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