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Fig. 2. Expression of ß-galactosidase in Osr2 heterozygous embryos.
(A) At E9.5, ß-galactosidase expression (blue) was detected specifically
in the mesonephric vesicles (arrow). (B) At E10.5, ß-galactosidase
activity was detected in the mesonephros (arrow), in the limb buds, in the
maxillary and mandibular processes, in the mesenchyme posterior to the eye and
in the mesenchyme adjacent to the first branchial cleft (arrowhead). (C)
Facial view of a stained E10.5 embryo showing ß-galactosidase expression
in the palatal primordia (arrowheads). (D,E) Frontal sections of E13.5 (D) and
E14.75 (E) heterozygous embryos showing ß-galactosidase expression in the
palatal mesenchyme, olfactory mesenchyme, tooth bud mesenchyme, and the
periocular mesenchyme and the eyelids. e, eye; fl, forelimb bud; hl, hindlimb
bud; mb, mandibular process; mx, maxillary process; oe, olfactory epithelia;
p, palatal shelf, t, tongue; tb, tooth bud.
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8 kb of genomic DNA. Boxes indicate
exons, with the protein-coding region marked in black. The positions of the
translation start (ATG) and stop (TAG) codons are also indicated. Restriction
sites are: B, BamHI; E, EcoRI; H, HindIII; P,
PstI; X, XbaI. The targeting vector used the 2.2 kb
XbaI-PstI fragment containing the intron 1/exon 2 junction
as the 5' arm and the 3.3 kb XbaI-HindIII fragment
3' to the Osr2-coding region as the 3' arm. A modified
bacterial lacZ gene and a neo expression cassette were
inserted in between the arms and a diphtheria toxin A (DTA)
expression cassette was cloned 3' to the 3' arm for negative
selection against random integration. Correct targeting results in the
lacZ gene and the neo cassette replacing most of the
Osr2 coding region, from the sixteenth codon of the open reading
frame to the XbaI site in the 3' untranslated region.
Arrowheads above the wild-type and mutant genomic schematics indicate the
positions of PCR primers used for genotyping. (B) Southern hybridization
analysis of tail DNA samples from a litter of F1 progeny of a chimeric male
generated with a targeted ES clone. Tail DNA samples were digested with
BamHI, separated by electrophoresis through a 1% agarose gel,
transferred onto a Zetaprobe nylon membrane (BioRad), and hybridized with
random prime-labeled probes made from the 600 bp
HindIII-EcoRI fragment isolated from the Osr2
genomic region 5' to the targeted region. The 14 kb BamHI
fragment corresponding to the wild-type allele was detected in all F1 progeny,
while the 7.7 kb mutant allele-specific fragment was detected only in
heterozygotes. (C) PCR analysis of tail DNA samples from a litter of newborn
F2 progeny. The fragments amplified from wild-type and mutant alleles are 490
bp and 460 bp, respectively. Homozygous mutants were born at the expected
Mendelian frequency (25%). m, DNA fragment size markers; +/+, wild type; +/-,
heterozygote; -/-, homozygote.
