Leslie A. Lyons

A cat cloned by nuclear transplantation.

Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.

Cell biology: A cat cloned by nuclear transplantation

Sheep, mice, cattle, goats and pigs have all been cloned by transfer of a donor cell nucleus into an enucleated ovum, and now we add the successful cloning of a cat (Felis domesticus) to this list. However, this cloning technology may not be readily extendable to other mammalian species if our understanding of their reproductive processes is limited or if there are species-specific obstacles.

Comparative genomics: lessons from cats

The genomics era, spear headed by dazzling technological developments in human and mouse gene mapping, has additionally provoked extensive comparative gene mapping projects for domestic species of several vertebrate orders. As the human genome project promises a one dimensional string of 100,000 genes and sequences, comparative mapping will extend that inference to a second dimension representing index species of the 20 living mammalian orders and to a third dimension by phylogenetic description of the genomes of mammal ancestors. We review here the remarkable extent of genome homology conservation among mammals illustrated by technology applications in the feline genome project.

Comparative analysis of the domestic cat genome reveals genetic signatures underlying feline biology and domestication.

Significance We present highlights of the first complete domestic cat reference genome, to our knowledge. We provide evolutionary assessments of the feline protein-coding genome, population genetic discoveries surrounding domestication, and a resource of domestic cat genetic variants. These analyses span broadly, from carnivore adaptations for hunting behavior to comparative odorant and chemical detection abilities between cats and dogs. We describe how segregating genetic variation in pigmentation phenotypes has reached fixation within a single breed, and also highlight the genomic differences between domestic cats and wildcats. Specifically, the signatures of selection in the domestic cat genome are linked to genes associated with gene knockout models affecting memory, fear-conditioning behavior, and stimulus-reward learning, and potentially point to the processes by which cats became domesticated. Little is known about the genetic changes that distinguish domestic cat populations from their wild progenitors. Here we describe a high-quality domestic cat reference genome assembly and comparative inferences made with other cat breeds, wildcats, and other mammals. Based upon these comparisons, we identified positively selected genes enriched for genes involved in lipid metabolism that underpin adaptations to a hypercarnivorous diet. We also found positive selection signals within genes underlying sensory processes, especially those affecting vision and hearing in the carnivore lineage. We observed an evolutionary tradeoff between functional olfactory and vomeronasal receptor gene repertoires in the cat and dog genomes, with an expansion of the feline chemosensory system for detecting pheromones at the expense of odorant detection. Genomic regions harboring signatures of natural selection that distinguish domestic cats from their wild congeners are enriched in neural crest-related genes associated with behavior and reward in mouse models, as predicted by the domestication syndrome hypothesis. Our description of a previously unidentified allele for the gloving pigmentation pattern found in the Birman breed supports the hypothesis that cat breeds experienced strong selection on specific mutations drawn from random bred populations. Collectively, these findings provide insight into how the process of domestication altered the ancestral wildcat genome and build a resource for future disease mapping and phylogenomic studies across all members of the Felidae.

Feline Polycystic Kidney Disease Mutation Identified in PKD1

Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder in humans that causes the formation of fluid-filled renal cysts, often leading to renal failure. PKD1 mutations cause 85% of ADPKD. Feline PKD is autosomal dominant and has clinical presentations similar to humans. PKD affects approximately 38% of Persian cats worldwide, which is approximately 6% of cats, making it the most prominent inherited feline disease. Previous analyses have shown significant linkage between the PKD phenotype and microsatellite markers linked to the feline homolog for PKD1. In this report, the feline PKD1 gene was scanned for causative mutations and a C>A transversion was identified at c.10063 (human ref NM_000296) in exon 29, resulting in a stop mutation at position 3284, which suggests a loss of approximately 25% of the C-terminus of the protein. The same mutation has not been identified in humans, although similar regions of the protein are truncated. The C>A transversion has been identified in the heterozygous state in 48 affected cats examined, including 41 Persians, a Siamese, and several other breeds that have been known to outcross with Persians. In addition, the mutation is segregating concordantly in all available PKD families. No unaffected cats have been identified with the mutation. No homozygous cats have been identified, supporting the suggestion that the mutation is embryonic lethal. These data suggest that the stop mutation causes feline PKD, providing a test to identify cats that will develop PKD and demonstrating that the domestic cat is an ideal model for human PKD.

International equine gene mapping workshop report: a comprehensive linkage map constructed with data from new markers and by merging four mapping resources

A comprehensive male linkage map was generated by adding 359 new, informative microsatellites to the International Equine Gene Map half-sibling reference families and by combining genotype data from three independent mapping resources: a full sibling family created at the Animal Health Trust in Newmarket, United Kingdom, eight half-sibling families from Sweden and two half-sibling families from the University of California, Davis. Because the combined data were derived primarily from half-sibling families, only autosomal markers were analyzed. The map was constructed from a total of 766 markers distributed on the 31 equine chromosomes. It has a higher marker density than that of previously reported maps, with 626 markers linearly ordered and 140 other markers assigned to a chromosomal region. Fifty-nine markers (7%) failed to meet the criteria for statistical evidence of linkage and remain unassigned. The map spans 3,740 cM with an average distance of 6.3 cM between markers. Fifty-five percent of the intervals are ≤5 cM and only 3% ≧20 cM. The present map demonstrates the cohesiveness of the different data sets and provides a single resource for genome scan analyses and integration with the radiation hybrid map.

Pathogenesis of feline enteric coronavirus infection

Fifty-one specific pathogen-free (SPF) cats 10 weeks to 13 years of age were infected with a cat-to-cat fecal–oral passed strain of feline enteric coronavirus (FECV). Clinical signs ranged from unapparent to a mild and self-limiting diarrhea. Twenty-nine of these cats were FECV naïve before infection and followed sequentially for fecal virus shedding and antibody responses over a period of 8–48 months. Fecal shedding, as determined by real-time polymerase chain reaction (RT-PCR) from rectal swabs, appeared within a week and was significantly higher in kittens than older cats. FECV shedding remained at high levels for 2–10 months before eventually evolving into one of three excretion patterns. Eleven cats shed the virus persistently at varying levels over an observation period of 9–24 months. Eleven cats appeared to have periods of virus shedding interlaced with periods of non-shedding (intermittent or recurrent shedders), and seven cats ceased shedding after 5–19 months (average 12 months). There was no change in the patterns of virus shedding among cats that were excreting FECV at the time of a secondary challenge exposure. Four cats, which had ceased shedding, re-manifested a primary type infection when secondarily infected. Cats with higher feline coronavirus (FCoV) antibody titers were significantly more likely to shed virus, while cats with lower titers were significantly less likely to be shedding. Twenty-two kittens born to experimentally infected project queens began shedding virus spontaneously, but never before 9–10 weeks of age. Natural kittenhood infections appeared to be low grade and abortive. However, a characteristic primary type infection occurred following experimental infection with FECV at 12–15 weeks of age. Pregnancy, parturition and lactation had no influence on fecal shedding by queens. Methylprednisolone acetate treatment did not induce non-shedders to shed and shedders to increase shedding.

Nuclear transfer of sand cat cells into enucleated domestic cat oocytes is affected by cryopreservation of donor cells.

In the present study, we used the sand cat (Felis margarita) as a somatic cell donor to evaluate whether cryopreservation of donor cells alters viability and epigenetic events in donor cells and affects in vitro and in vivo developmental competence of derived embryos. In Experiment 1, flow cytometry analysis revealed that the percentage of necrosis and apoptosis in cells analyzed immediately after freezing/thawing (61 vs. 8.1%, respectively) was higher than that observed in frozen/thawed cells cultured for 18 h (6.9 vs. 3.3%, respectively) or 5 days (38 vs. 2.6%; respectively). The relative acetylation level of H3K9 was lower in frozen/thawed cells (5.4%) compared to that found in cultured cells (60.1%). In Experiment 2, embryos reconstructed with frozen/thawed cells had a lower cleavage rate (85%; day 2) than did embryos reconstructed with cultured cells (95%), while development to the blastocyst stage (day 8) was not affected by cell treatment (17.0% with frozen/thawed cells vs. 16.5% with cultured cells). In Experiment 3, pregnancy rates were similar between both cell treatments (32% with frozen/thawed cells vs. 30% with cultured cells), but the number of embryos that were implanted, and the number of fetuses that developed to term was lower for embryos reconstructed with frozen/thawed cells (1.2 and 0.3%, respectively) than those reconstructed with cultured cells (2.6 and 1.8%, respectively), while the number of fetuses reabsorbed by day 30 was higher (75%) for embryos reconstructed with frozen/thawed cells than those reconstructed with cultured cells (31%). A total of 11 kittens from cultured cells and three kittens from frozen/thawed cells were born between days 60 to 64 of gestation. Most kittens died within a few days after birth, although one kitten did survive for 2 months. In Experiment 4, POU5F1 mRNA expression was detected in 25% of blastocysts derived from frozen/thawed cells, whereas 88 and 87% of blastocysts derived from cultured cells and by in vitro fertilization, respectively, expressed POU5F1. We have shown that cell cryopreservation increased the incidence of necrosis and apoptosis and altered epigenetic events in donor cells. Consequently, the number of embryos that cleaved, implanted, and developed to term-gestation and POU5F1 expression in derived blastocysts indirectly was affected.

Albinism in the domestic cat (Felis catus) is associated with a tyrosinase (TYR) mutation.

Summary Albino phenotypes are documented in a variety of species including the domestic cat. As albino phenotypes in other species are associated with tyrosinase (TYR) mutations, TYR was proposed as a candidate gene for albinism in cats. An Oriental and Colourpoint Shorthair cat pedigree segregating for albinism was analysed for association with TYR by linkage and sequence analyses. Microsatellite FCA931, which is closely linked to TYR and TYR sequence variants were tested for segregation with the albinism phenotype. Sequence analysis of genomic DNA from wild-type and albino cats identified a cytosine deletion in TYR at position 975 in exon 2, which causes a frame shift resulting in a premature stop codon nine residues downstream from the mutation. The deletion mutation in TYR and an allele of FCA931 segregated concordantly with the albino phenotype. Taken together, our results suggest that the TYR gene corresponds to the colour locus in cats and its alleles, from dominant to recessive, are as follows: C (full colour) > cb (burmese) ≥ cs (siamese) > c (albino).

Detection of c-kit Mutations in Canine Mast Cell Tumors using Fluorescent Polyacrylamide Gel Electrophoresis

Mutations consisting of internal tandem duplications (ITDs) in exons 11 and 12 of the proto-oncogene c-kit are found in 30–50% of malignant canine mast cell tumors (MCTs). Traditionally, identification of such mutations in tumor specimens has been undertaken using standard polymerase chain reaction (PCR) and agarose gel electrophoresis. This procedure is limited to the detection of insertions and deletions larger than 9 base pairs in size. The purpose of this study was to compare the efficiency and accuracy of standard agarose gel electrophoresis with fluorescent polyacrylamide gel electrophoresis (PAGE) for the detection of ITDs in canine MCTs. The results of this study demonstrate that PAGE of labeled PCR products accurately predicts the size of the ITD in each tumor. In addition, other small insertions and deletions were not identified, suggesting that if they occur in canine MCTs, they do so infrequently. Because fluorescent and polyacrylamide formats are automated and have better resolution than agarose gels, fluorescent PAGE provides a more accurate, economical, and higher throughput method for the detection of c-kit mutations in canine MCTs.