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associated with defects inXPD. As reported previously (de Boer et a!., 1998), a null
allele for XPD resulted in a very early embryonic lethality, probably as early as the
two-cell stage as anticipated for a gene essential for basal transcription (de Boer et
al., 1998). This paper describes the generation and characterization of a mouse
model for TTD, by mimicking a point mutation in the mouse germline, as found in
theXPD gene ofpatientTIDlBEL.
RESULTS
Gene-eDNA fusion targeting of the TTDIBEL mutation, XPD R722w
To generate a mouse model for trichothiodystrophy, we wished to mImIC a
causative point mutation, associated with the classical photosensitive form of TTD.
The arginine 722 to tryptophan single amino acid substitution (henceforth
designated XPDRl]]II) was selected, which is found in patient TIDlBEL
(Broughton et a!., 1994) and several other TID individuals (Takayama et a!., 1996,
and M. Stefanini personal communication). In view of the absolutely essential basal
transcription function of the XPD gene (de Boer et a!., 1998) introduction of a point
mutation is a very delicate operation: the precise protein conformation is likely to be
of cmcial importance as the protein is only partially inactivated by the mutation and
still has to fit into the 9-subunit TFIIH complex. To avoid that mouse/human
differences in the region of the mutation could cause complications we decided to
"humanize" the part of the XPD gene in which the R722 amino acid residue resides.
Furthermore, the insertion of a dominant selectable marker should not influence the
expression of the gene. This reasoning led us to design a novel gene targeting
strategy that we term "gene-cDNA fusion targeting". It involves a single step
targeting protocol in which part of the coding region of the gene of interest is
replaced by the corresponding part of the eDNA, that is cloned in frame and
contains the desired point mutation. After gene targeting in embryonic stem (ES)
cells the resulting allele will express a mutated, chimeric transcript. The targeting
construct used, the probe outside of the targeting construct for screening
homologous recombinants and the primers for amplification of XPD R7 :!:!1V mRNA
are outlined in Figure lAo To generate a XPDRl]]W allele, the most 3' 194 bp of the
human XPD eDNA including the C2166T mutation and stop codon, was cloned in
frame in exon 22 of the mouse XPD gene (see Figure la). A human p-globin
cassette including part of exon 2, intron 2, exon 3, 3'UTR and polyadenylation
signal was cloned behind the stop codon to serve as 3' UTR. The neomycin
resistance gene as dominant selectable marker was cloned downstream of the
polyadenylation signal of the globin cassette so that it would not interfere with
XPD R7 :!:!W transcription. The HSV-tk gene was used for selection against random
integration. Following electroporation and double-drug selection of ES cell clones,
A mouse model for TID 69