sternum, and lower jaw aplasia were also commonly
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20. We performed mouse skin grafts as in (
that we used transgenic or wild-type dorsal trunk
skin from B6CBF2 embryos that had been dissected
away from underlying muscle and grafted it onto 8-
to 12-week-old male
recipient mice. Dressings
were removed after 3 weeks. Each animal was pho-
21. Genomic sequences containing
from a bacterial artificial chromosome library ob-
tained from Research Genetics. Primers used to
screen this library from exon 2 [ACC GAG GGC TGG
GAC GAA GAT GGC and GCG AGC CAG CAT GCC
GTA CTT GCT G (
)] identified BAC 270A17, which
was digested with restriction enzymes and ligated
with vectorette linkers (
). Exon-intron boundaries
for the three exons were determined by sequencing
polymerase chain reaction (PCR) products amplified
using the universal vectorette primer and
primers selected from published sequences. Since
we were unable to obtain sequences from the exon
2–intron 3’ boundary, a primer from the 3
exon 2 was used. Primers used to amplify genomic
SHH were as follows: exon 1, CCG CCG CGC GCA
CTC G and AAG GAG CGG GTG AAA TCA CC; exon
2, TAA CGT GTC CGT CGG TGG G and TGC TTT
CAC CGA GCA GTG G; and exon 3, CCT CCT CCC
CGA GAC GC and GGC CCC CTC CCG CGC C.
Mutations were identified by single-strand confor-
mation polymorphism (SSCP) analysis of PCR prod-
ucts amplified from genomic DNA. The PCR prod-
ucts were sequenced on both strands directly from
the PCR-produced templates and after cloning into
Bluescript. Subsequent to our completion of this
work, another group has published intron sequenc-
es and primers useful for amplifying SHH exons from
genomic DNA (
22. DNA from one other BCC had a methionine to iso-
leucine change at position 115, but this change was
also present in DNA from the patient’s blood. This
unusually large BCC was diagnosed at age 40 in a
patient with no other phenotypic abnormalities sug-
gestive of BCNS.
23. K. F. Liem, G. Tremmi, H. Roelink, T. M. Jessell,