Article Figures & Data
Figures
- Figure 1.
Generation of seven ataxin 3-120Q transgenic marmosets. A, Two types of constructs (CMV-ataxin 3-120Q-IRES-Venus and CMV-ataxin 3-120Q-2A-Venus) were introduced into marmoset embryos. B, Relative levels of transgene expression in PQD1 and three miscarried fetuses are shown. The relative gene expression level in PQD1 was set as 1. C, D, Genomic-PCR (top) and RT-PCR (bottom) analyses of each transgenic marmoset transduced with the CMV-ataxin 3-120Q-IRES-Venus construct (C) or the CMV-ataxin 3-120Q-2A-Venus construct (D). The results in seven transgenic marmosets (PQD1-7), a positive control (Plasmid), and negative controls (Wild-type and Water) are shown. E, Relative levels of transgene expression in ear fibroblast cells of each transgenic marmoset are shown. The relative gene expression level in PQD1 was set as 1.
- Figure 2.
Age-dependent changes in body weight and body weight gain. A, Age-dependent changes in the body weight of wild-type marmosets, symptomatic transgenic marmosets (PQD1, 2, and 6), and asymptomatic transgenic marmosets (PQD3, 4, 5, and 7). Arrows indicate the onset of neurologic symptoms of the three symptomatic transgenic marmosets. Averages ± 2 SD of body weight in wild-type marmosets (n = 4) are indicated as gray areas. B, Body weight gain of wild-type marmosets, symptomatic transgenic marmosets, and asymptomatic transgenic marmosets at 5–6 months of age. Each symbol represents an individual marmoset. Horizontal bars indicate the group average.
- Figure 3.
Transgenic marmosets show progressive neurologic symptoms. A, Age-dependent changes in 24-h spontaneous activity of a wild-type marmoset and PQD1 transgenic marmoset at 3 and 6 months. B, 24-h spontaneous activity of PQD2 at 3 months, PQD3 at 4 months, and PQD4–7 at 6 months of age. C, 24-h spontaneous activity levels of PQD1 at 5–6 months of age, PQD2 at 3 months, and PQD6 at 5–6 months of age, compared with those of wild-type marmosets and asymptomatic transgenic marmosets at 5–6 months of age. Each symbol represents an individual marmoset. Horizontal bars indicate the group average.
- Figure 4.
Age-dependent changes in grip strength. A, Age-dependent changes in grip strength of wild-type marmosets, symptomatic transgenic marmosets (PQD1, 2, and 6), and asymptomatic transgenic marmosets (PQD3, 4, 5, and 7). Averages ± 2 SD of grip strength in wild-type marmosets (n = 4) are indicated as gray areas. B, Grip strength of wild-type marmosets, symptomatic transgenic marmosets, and asymptomatic transgenic marmosets at 5–6 months of age. Each symbol represents an individual marmoset. Horizontal bars indicate the group average.
- Figure 5.
Expression of ataxin 3-120Q in transgenic marmosets PQD1 and 2. A, B, Genomic-PCR (top) and RT-PCR (bottom) analyses (A) and Western blot analysis (B) of the major organs and various brain regions of PQD1 and 2. A positive control (Plasmid) and negative controls (Wild-type and Water) were also included. Arrowheads indicate the position of the ataxin 3-120Q band.
- Figure 6.
Cerebellar atrophy and neurodegeneration accompanied by polyQ protein inclusions in the brains of symptomatic transgenic marmoset PQD1. A, B, Coronal MRI sections of the brain of PQD1 (A) and asymptomatic marmoset PQD5 (B). The arrow indicates the fourth ventricle. C, Quantitative analyses of brain MRIs of transgenic marmosets. The bar indicates the mean of PQD3–5 and 7. D, E, Anti-calbindin staining of the cerebellum of PQD1 (D) or a control wild-type marmoset (E). Arrowheads indicate degenerated Purkinje cells. F, G, Anti-GFAP staining of the cerebellum of PQD1 (F) or a control wild-type marmoset (G). H–K, Hematoxylin and eosin (HE; H), anti-1C2 (I and J), and anti-ubiquitin (K) staining of the spinal cord of PQD1. In H, the arrow indicates a degenerated anterior horn cell, and the arrowhead indicates an inclusion body. A magnified image of a 1C2-positive inclusion (I) is shown in J. L, M, Anti-GFAP staining of the spinal cord of PQD1 (L) and a control wild-type marmoset (M). N, HE staining of the cerebrum of PQD1. O, P, HE (O) and anti-1C2 (P) staining of the brainstem of PQD1. Scale bars: 100 µm in D–H and K; 200 µm in G; 50 µm in I, L, M, and O; 250 µm in N; and 10 µm in P.
- Figure 7.
Neurodegeneration and polyQ protein inclusions in the brain and spinal cord of symptomatic transgenic marmoset PQD2. A, B, Anti-calbindin (A) and GFAP (B) staining of the cerebellum of PQD2. C–H, HE (C and F), anti-1C2 (D and G), and anti-ubiquitin (E and H) staining of the brainstem (C–E) and spinal cord (F–H) of PQD2. I, Anti-GFAP staining of the spinal cord of PQD2. J, K, HE (J) and anti-ubiquitin (K) staining of the cerebrum of PQD2. These inclusions (arrowheads in K) were not immunoreactive for the 1C2 antibody. Scale bars: 100 µm in A, B, and F–H; 5 µm in C–E; 50 µm in I; 250 µm in J; and 10 µm in K.
- Figure 8.
Pathology of the peripheral nerves and muscles of PQD1 and 2. A, B, Toluidine blue staining of the peripheral nerves of the upper limbs of PQD1 (A) and PQD2 (B). Arrowheads indicate axonal degeneration. C–F, HE (C), modified Gomori trichrome (D), acid phosphatase activity (E), and NADH reductase activity (F) staining of the tibialis anterior muscle of PQD2. Marked fiber size variation and endomysial fibrosis were observed. Fibers with centrally placed large nuclei and intranuclear inclusions (arrows in C) or rimmed vacuoles (arrowheads in D) and acid phosphatase–positive fibers (asterisk in E) were also observed. The intermyofibrillar network was disorganized in many fibers (F). Dystrophic changes, group atrophy, and fiber-type grouping were not observed. G–J, Anti-1C2 (G and I) and anti-ubiquitin (H and J) staining of the quadriceps femoris muscle of PQD1 (G and H) and PQD2 (I and J). Scale bars: 100 μm in A and B; 20 μm in C–J.
- Figure 9.
Germline transmission of the ataxin 3-120Q transgenes. A, Genomic-PCR (top) and RT-PCR (bottom) analyses in second-generation transgenic offspring. The results in PQD1-1, 1-2, 1-3, and 1-4, a positive control (Plasmid), and negative controls (Wild-type and Water) are shown. B, Relative levels of transgene expression in ear fibroblast cells of each second-generation transgenic offspring are shown. The relative gene expression level in PQD1 was set as 1.
Tables
CMV-Ataxin3-120Q-IRES-Venus CMV-Ataxin3-120Q-2A-Venus Total Number of embryos transferred to surrogates 29 37 66 Number of surrogates 17 23 40 Number of pregnancies 7 7 14 Number of deliveries 3 2 5 Number of births (percentage of births per embryos transferred) 5 (17.2) 2 (5.4) 7 (10.6) Number of transgenic animals (percentage of transgenic animals per birth) 5 (100) 2 (100) 7 (100) A high titer of lentiviral vector was injected into a total of 66 embryos, followed by embryo transfer to 40 surrogate mothers. A total of 14 surrogates became pregnant, and five surrogates delivered seven offspring.
ID Sex CAG repeats Chromosomes with transgene integration Disease onset Disease progression PQD1 Male 120 1, 4, 17 4 months Moderate PQD2 Female 120 1, 17, 22 3 months Severe PQD3 Female ND PQD4 Female 120 PQD5 Female 120, 80 PQD6 Female 120 7, 22 4 months Mild PQD7 Male 120, 43 ND, not determined.
Although all marmosets except PQD3 carried the ataxin 3-120Q transgene, transgenes with other CAG repeat lengths were also found to be expressed in PQD5 and 7, respectively. PQD1, 2, and 6 had several integration sites and cellular mosaicism and developed neurologic symptoms of varying degrees at 3–4 months after birth.
PQD1 PQD2 13.5 months 5.5 months Degeneration Inclusion Degeneration Inclusion Cerebrum − − − + Cerebellum ++ − − − Brain stem − ± − + Spinal cord ++ ++ ++ ++ Peripheral nerves ++ N/E ++ N/E Skeletal muscles +++ ++ +++ ++ Two marmosets among the three symptomatic transgenic marmosets were subjected to immunohistochemical analysis. Note that cerebellar degeneration was observed in PQD1. N/E: not examined.
In this issue
