Figure 1. Generation of the Hdh^ΔPRR allele.

(A) Schematic of the wild-type Hdh allele surrounding the first exon is shown (Hdh) along with the gene targeting construct (T) lacking the PRR, and the targeted locus following recombination (HdhΔP). The “~” in the targeting vector represents plasmid sequence, and the “II” indicates the restriction site used to linearize the targeting vector prior to ES cell electroporation. The location of the neomycin phosphotransferase cassette (pgkneo, box) flanked by loxP sites (black arrowheads) that was used for positive selection of the transfected ES cells is shown. The transcriptional orientations of the Hdh and pgkneo genes are indicated with arrows. The small gray arrows indicate the location of the forward and reverse oligonucleotide primers used for PCR genotyping. The sizes of the wild-type and targeted NcoI-digested genomic DNA fragments recognized by the 3´flanking probe (small black rectangle) are shown above (Hdh) and below (HdhΔP), respectively. Restriction enzyme sites are NotI (Not), NcoI (N), HindIII (H), XmnI (X) and KpnI (K). The schematic is not drawn to scale. (B) Exon-1 of the wild-type (Hdh) mouse htt gene was modified by gene targeting to generate a deletion of the sequence encoding the mouse PRR (Hdh^ΔP). The XmnI and KpnI restriction sites used for the modifications are shown. (C) DNA from tail biopsies was used to genotype progeny by PCR with primers that were designed to discriminate between the wild-type (Hdh) and modified (HdhΔP) alleles: Lane 1- Hdh^+/+; Lane 2 - Hdh^ΔPRR/ΔPRR; Lane 3 - Hdh^ΔPRR/+; M – 100bp DNA molecular weight marker. (D) Nucleotide and encoded amino acid sequence of the Hdh⁺ (top) and Hdh^ΔPRR (bottom) exon-1 XmnI–KpnI restriction fragment. Intron-1 sequence is presented in lower case, and the first and last codons of the PRR are indicated in larger font