An ovine transgenic Huntington's disease model

Abstract

Huntington's disease (HD) is an inherited autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat in the huntingtin (HTT) gene [Huntington's Disease Collaborative Research Group (1993) A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. The Huntington's Disease Collaborative Research Group. Cell, 72, 971-983]. Despite identification of the gene in 1993, the underlying life-long disease process and effective treatments to prevent or delay it remain elusive. In an effort to fast-track treatment strategies for HD into clinical trials, we have developed a new large-animal HD transgenic ovine model. Sheep, Ovis aries L., were selected because the developmental pattern of the ovine basal ganglia and cortex (the regions primarily affected in HD) is similar to the analogous regions of the human brain. Microinjection of a full-length human HTT cDNA containing 73 polyglutamine repeats under the control of the human promotor resulted in six transgenic founders varying in copy number of the transgene. Analysis of offspring (at 1 and 7 months of age) from one of the founders showed robust expression of the full-length human HTT protein in both CNS and non-CNS tissue. Further, preliminary immunohistochemical analysis demonstrated the organization of the caudate nucleus and putamen and revealed decreased expression of medium size spiny neuron marker DARPP-32 at 7 months of age. It is anticipated that this novel transgenic animal will represent a practical model for drug/clinical trials and surgical interventions especially aimed at delaying or preventing HD initiation. New sequence accession number for ovine HTT mRNA: FJ457100.

Figure 1.

HTT transgene and chromosomal location in HD founder transgenic animals (A) Schematic of the full-length human HTT cDNA ligation fragment used for transgenesis. The full-length cDNA includes exons 1–67 of the HTT gene ligated to a 5′ 1.1 kb fragment of the human HTT promotor region. The 3′-UTR is a genomic fragment of the bovine growth hormone (BGH) gene. (B) HD transgenic founder karyograms and predicted chromosome insertion sites. Location of the transgene is marked by yellow–green fluorescence. The chromosomal insertion site for G₀/1 was observed to be only at 1q43 without the necessity of making a karyogram (data not shown). G₀/3 is a very low-grade mosaic with no signals observed above background in any cells (data not shown). The insertion sites for the transgenes in karyograms of the other four animals are shown above.

Figure 2.

Molecular characterization of the HD founder transgenic animals. (A) Southern membrane hybridized with a radioactively labelled probe covering the promotor region (1.1 kb NruI/BssHII fragment). The 9.5 kb band is predicted to be the HindIII digested fragment corresponding to the wild-type ovine HD gene. About 4.9 and 3.6 kb bands are indicative of 5′–3′ and 5′–5′ transgene concatemerization, respectively. This is clearly illustrated in the multi-copy animals; G₀/2, G₀/4 and G₀/5. Real-time PCR transgene copy numbers are indicated below the Southern blot. The DNA was isolated from tail biopsy tissue. (B) Polyglutamine repeat region genomic PCR demonstrating the presence of the 73 unit expanded transgene repeat allele (expected size = 430 bp) and the ovine wild-type alleles (expected size = ∼220 bp; * marks those heterozygous for the wild-type ovine alleles). The positive control is the transgene ligation fragment (human cDNA'BGH plasmid). The transgene PCR product for the highly chimeric G₀/3 animal is not observed in this PCR reaction but the presence and size of the repeat region was confirmed by PCR analysis of sperm DNA (Fig. 2C). (C) PCR of the polyglutamine repeat region demonstrating stability of the CAG repeat in DNA isolated from sperm of the founder rams (G₀/1–4) and the G₂/5 transgenic rams. The positive control is genomic tail DNA from G₀/2 and the negative control is dH₂O. (D) RT–PCRs covering the BGH 3′ transcriptional tag of the founder transgenics. The preferentially amplified band is the unspliced product amplified from RNA isolated from tail biopsies. The positive control is the transgene ligation fragment (human cDNA'BGH plasmid) and the negative control is dH₂O. Reverse transcriptase minus controls were included for each animal and produced no amplification product, excluding the possibility of genomic contamination (data not shown).

Figure 3.

Expression in the G₀/5 pedigree. (A) Western blot immunoprobed with the antibody MAB2166 demonstrating the expression of the human HTT transgenic protein in the first sacrificed offspring of the G₀/5 line (1 month of age) as compared with the endogenous ovine HTT protein in eight different brain regions. The wild-type (WT) control animal is a true age matched sibling of the transgenic. Thal., Thalamus; G.P., Globus pallidus; C.N., Caudate nucleus; S.S.C., Somatosensory cortex; Cb., Cerebellum; Hipp., Hippocampus; S.N., Substantia nigra; V.C., Visual cortex. Gels were Coomassie stained to ensure even loading of samples (data not shown). (B) Pedigree of the G₀/5 line. Shaded boxes represent those animals in the pedigree that tested positive for the transgene in DNA extracted from tail biopsy tissue. SAC 1 and 7 months = ages at which animals were sacrificed for analysis; D.A.B., dead at birth; D.P.B., dead post birth (within 48 h); open diamond = unknown sex (aborted fetus).

Figure 4.

Wild-type ovine brain photographs depicting the gross anatomy of the basal ganglia. These photomicrographs demonstrate the gross anatomy of the basal ganglia in the ovine brain using various markers to characterize the striatal projection neurons. Immunolabelling through the caudate nucleus and rostral and caudal ends of the GP demonstrate comparable anatomy to the human basal ganglia with separate caudate nucleus (CN), putamen (Pt) and pallidal nuclei. Furthermore (D), (E) and (F) demonstrate that the GP is sub-divided further into an enkephalin rich GPe and SP rich GPi as observed in the human brain. (A), (D) and (G) show immunoreactivity for the neurotransmitter calbindin, a marker for neuronal cell populations which use calcium dependent signalling. Microscopic insets of the neuronal specific labelling can be seen in images (a), (d) and (g). (B), (E) and (H) show immunoreactivity for the neurotransmitter enkephalin, a marker of the GABAergic medium spiny neurons involved in the ‘indirect’ pathway projecting from the striatum to the GPe. Microscopic insets of this neurotransmitter labelling pattern can be seen in (b), (e) and (h). (C), (F) and (I) show immunoreactivity for the neurotransmitter SP, a marker of the GABAergic medium spiny neurons involved in the ‘direct’ pathway projecting from the striatum to the GPi and substantia nigra (not shown). Microscopic insets of this neurotransmitter labelling pattern are shown in (c), (f) and (i).

Figure 5.

Loss of CB1 and DARPP32 immunoreactivity in a 7-month-old G₁/5 animal as compared with control. (A) and (B) demonstrate macroscopically the loss in intensity of CB1 immunoreactivity in the GP (arrow head) of the transgenic animal (B) as compared with the control (A). In comparison, the cortex demonstrates a uniform staining intensity between the transgenic and control animals. Higher magnification illustrates the loss of immunoreactivity of fine synaptic labelling in the GP of the transgenic (b) as compared with control (a). (C) and (D) are macroscopic images of DARPP32 immunoreactivity in the control and transgenic animals, respectively. This clearly demonstrates the loss of immunolabelling intensity in the transgenic animal (arrow heads), which is observed in the absence of significant striatal cell loss. The loss of fine projection fibre labelling in the GP of the transgenic as compared with the denser labelling observed in the control is shown in (d) and (c), respectively. A photomicrograph of the putamen in the transgenic animal (d′) demonstrates loss of immunoreactivity in the absence of cell loss or dysmorphology. The corresponding control section is (c′).