SCID dogs: similar transplant potential but distinct intra-uterine growth defects and premature replicative senescence compared with SCID mice

Abstract

We have previously described DNA-dependent protein kinase (DNA-PKcs) mutations in horses and dogs that result in deficits in V(D)J recombination, DNA repair, and SCID. In this paper, we document substantial developmental growth defects in DNA-PKcs-deficient dogs that are not apparent in SCID mice. Fibroblast cell strains derived from either fetal or adult SCID dogs proliferate poorly in culture and undergo premature replicative senescence, somewhat reminiscent of cells derived from Ku-deficient mice. A limited number of animals have been immune reconstituted (by bone marrow transplantation) so that they can be maintained in a normal environment for long periods. Several of these animals have developed conditions associated with premature ageing at 2-3 years of age, roughly 20% of their expected lifespan. These conditions include intestinal malabsorption and primary neural cell neoplasia. These results suggest that DNA-PKcs deficiency is not tolerated equally in all species, perhaps providing insight into why DNA-PKcs deficiency has not been observed in humans. Finally, this study demonstrates the feasibility of maintaining SCID dogs for extended periods of time and documents their utility for bone marrow transplantation studies and as hosts for the propagation of xenografts. In sum, SCID dogs may present researchers with new possibilities for the development of animal models of human disease.

FIGURE 1

Immune reconstitution in SCID dogs by DLA-matched marrow transplantation. A, Histopathologic examination of a lymph node (LN) from an unaffected dog (right panel), a SCID dog 90 wk after DLA-matched bone marrow transplantation (middle panel), or an untreated SCID dog (left panel). B, Lymph node sections from an unaffected dog and a SCID dog 90 wk after DLA-matched bone marrow transplantation were stained with Abs to canine CD3 as described previously (8). C, Chimerism analysis of DNA prepared from various tissues obtained at necropsy (as indicated) 90 wk after DLA-matched bone marrow transplantation. The donor was heterozygous for the SCID allele; thus, the percentage of chimerism was established (shown below each lane) by phosphorimaging of amplification products from the two alleles, correcting for the different amplification efficiency as established by amplification of DNA from the heterozygous donor. D, Chimerism analysis of DNA prepared from PBMC taken at indicated times post-bone marrow transplantation. In this case, the marrow donor was homozygous for wild-type DNA-PKcs. Chimerism was analyzed by the PCR to detect presence of the MseI resistant (wild type) allele.

FIGURE 2

Intrauterine growth retardation in SCID puppies. A, Canine fetuses were harvested at 42 days of gestation. B, Genotyping was performed as described in Materials and Methods. Fetuses 2, 3, and 4 are homozygous for the SCID mutation.

FIGURE 3

Canine SCID fibroblasts replicate poorly and undergo premature replicative senescence. A, Fetal fibroblasts were derived from s.c. tissues of fetuses from two litters. Litter 1 included two normal and three SCID fetuses. Litter 2 contained three normal and three SCID fetuses. Cell growth curves were performed on each group of cell strains. Data is presented as average of fold increase in cell number for SCID and normal fibroblasts from each litter. Error bars represent SEM. B, Fetal fibroblasts were plated and stained with SA-β-galactosidase. Percentage of SA-β-galactosidase positive cells in fibroblast cell strains was derived from litter 1. Percentage is the average of two experiments performed after 4 and 5 wk of culturing the five cell strains. (FP1–5 denotes individual fetal puppies).

FIGURE 4

Defective telomere maintenance in SCID fibroblasts. DNA from both SCID and normal fetal and adult fibroblasts restricted with RsaI and HinFI was subjected to Southern hybridization using a probe specific for telomeric repeats. Average telomeric length in each sample was determined by measuring the mean mobility of hybridizing sequences.

FIGURE 5

Incidental observations in SCID dogs consistent with a premature ageing phenotype. A, H&E staining of a normal (left) and atrophic (right) pancreas from a SCID dog. B, H&E staining of spinal cord tumor consistent with primary meningiosarcoma.

FIGURE 6

Human tumor xenografts proliferate rapidly in SCID dogs. A, Growth of human tumor ovarian cell line, ES-2 injected s.c. at two sites in two SCID dogs and one normal dog. Tumors were measured and tumor volume in each dog was summed. B, Massive pulmonary metastasis of ES-2 cells is apparent at necropsy. C, Histopathologic examination of ES-2 pulmonary metastasis.