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Fig. 1. Tsix was truncated in the Xist gene by the
introduction of a multiple polyadenylation sequence. (A) The
genomic structure of the TsixpA allele is shown below the
overall structure of the Xist/Tsix loci. The second intron
of the human 
-globin gene, which harbors an mpA cassette in an
antisense orientation with respect to Xist transcription, was
introduced at the XhoI site (107228-107233 in GenBank Acc. No.
AJ421479). SD, splicing donor; SA, splicing acceptor. (B) Targeting
scheme for generating the TsixpA allele. Positions of the
recognition sites of the relevant restriction enzymes and the probes used in C
and D are shown. B, BamHI; E, EcoRI; H, HindIII; P,
PvuII; X, XhoI. (C) Homologous recombination in ES
cells was confirmed by Southern blotting. Probes and restriction enzymes used
are shown below the blot. Appropriate recombination in the 5' region was
confirmed using a 5'probe, Xist5-5, located outside of the fragment used
for the short arm of the targeting vector. Since the recombination event in
the 3' region could not be properly addressed with a single restriction
enzyme, it was confirmed by two steps. Although use of probe XB verified the
recombination event by the appearance of the expected 1.7 kb band upon
BamHI digestion, the presence of a 5.6 kb band detected using probe
3' inner upon EcoRI digestion, which was also seen in parental
R1 ES cells, verified that there was no other rearrangement in the fragment
used for the long arm of the targeting vector. (D) Excision of the
puromycin-resistance cassette by cre recombinase was confirmed by Southern
blotting using tail DNA. PvuII digestion and subsequent hybridization
with XB as a probe allowed differentiation of the wild-type,
TsixpA2lox and TsixpA alleles.
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