Skeletal Manifestations of Heritable Disproportionate Dwarfism in Cats as Determined by Radiography and Magnetic Resonance Imaging.

OBJECTIVE: The aim of this study was to characterize the radiographic alignment of thoracic and pelvic limbs and evaluate for intervertebral disc disease in cats with feline disproportionate dwarfism (FDD). STUDY DESIGN: Observational cross-sectional study. Radiographic joint orientation angles were measured in 10 thoracic and pelvic limbs from 5 FDD cats and compared with those angles measured in 24 thoracic limbs and 100 pelvic limbs from skeletally normal cats. Magnetic resonance imaging of the spine was performed in 2 FDD cats for the evaluation of pathology of the intervertebral discs or vertebrae. RESULTS: All limbs from FDD cats possessed deformities. FDD humeri demonstrated procurvatum proximally, and recurvatum distally in the sagittal plane, but showed no difference in the frontal plane. FDD radii possessed excessive recurvatum proximally, and procurvatum distally in the sagittal plane, and varus proximally and valgus distally in the frontal plane. Whereas no torsion was discernible in the humeri, all radii had external torsion. In the frontal plane, FDD femurs exhibited varus both proximally and distally whereas the tibia possessed proximal valgus and distal varus. No torsion in the pelvic limbs was observed. No spinal pathology was detected in the FDD cats included in the original study. CONCLUSION: Feline disproportionate dwarfism results in significant appendicular deformity in all limbs. The incidence of intervertebral disc degeneration in FDD cats is inconclusive.

Mutational analyses of novel rat models with targeted modifications in inflammatory bowel disease susceptibility genes.

Mutations and single base pair polymorphisms in various genes have been associated with increased susceptibility to inflammatory bowel disease (IBD). We have created a series of rat strains carrying targeted genetic alterations within three IBD susceptibility genes: Nod2, Atg16l1, and Il23r, using CRISPR/Cas9 genome editing technology. Knock-out alleles and alleles with known human susceptibility polymorphisms were generated on three different genetic backgrounds: Fischer, Lewis and Sprague Dawley. The availability of these rat models will contribute to our understanding of the basic biological roles of these three genes as well as provide new potential IBD animal models.

The Atg16l1 gene: characterization of wild type, knock-in, and knock-out phenotypes in rats.

ATG16L1 is a ubiquitous autophagy gene responsible, in part, for formation of the double-membrane bound autophagosome that delivers unwanted cellular debris and intracellular pathogens to the lysosome for degradation. A single, nonsynonymous adenine to guanine polymorphism resulting in a threonine to alanine amino acid substitution (T300A) directly preceded by a caspase cleavage site (DxxD) causes an increased susceptibility to Crohn's disease (CD) in humans. The mechanism behind this increased susceptibility is still being elucidated, however, the amino acid change caused by this point mutation results in increased ATG16L1 protein sensitivity to caspase 3-mediated cleavage. To generate novel rat strains carrying genetic alterations in the rat Atg16l1 gene, we first characterized the wild-type rat gene. We identified four alternative splice variants with tissue-specific expression. Using CRISPR-Cas9 genome editing technology, we developed a knock-in rat model for the human ATG16L1 T300A CD risk polymorphism, as well as a knock-out rat model to evaluate the role of Atg16l1 in autophagy as well as its potential effect on CD susceptibility. These are the first reported rat strains with alterations of the Atg16l1 gene. Consistent with studies of the effects of human ATG16L1 polymorphisms, models exhibit morphological abnormalities in both Paneth and goblet cells, but do not develop spontaneous intestinal permeability or inflammatory bowel disease. Analysis of the gut microbiota does not show inherent differences in bacterial composition between wild-type and genetically modified animals. These Atg16l1 strains are valuable new animal models for the study of both autophagy and CD susceptibility.

Using Vaginal Impedance Measurement to Identify Proestrus in Rats Given Luteinizing Hormone Releasing Hormone (LHRH) Agonist.

Vaginal cytology is the most common method of monitoring the estrous cycle in rats; however, this test requires specific technical training and can be subject to interpretation. Vaginal impedance offers a quicker and less technically challenging alternative and has been used successfully to identify estrus in normally cycling breeder rats. We hypothesize that vaginal impedance can also be used to stage the estrous cycle in rats that have been given luteinizing hormone releasing hormone (LHRH) for timed mating. Vaginal impedance measurements and vaginal cytology were performed in LHRH-primed female rats (n = 36) at the expected peak of proestrus and paired with proven stud males. Breeding success was determined by gross necropsy to detect embryo implantation sites in the female rats. We found that the predictive rates of vaginal cytology and impedance measurement for proestrus were similar; however, both methods resulted in high proportions of false positive and false negative determinations (28% and 31%, respectively). We further hypothesized that females respond to LHRH at variable rates, resulting in variable times of peak proestrus. To test this, vaginal impedance measurements were performed multiple times throughout the expected day of proestrus in LHRH-primed female rats (n = 36). Females were either paired with a male 24 h after reaching the proestrus threshold (n = 18) or paired according to our standard protocol at 1300 h on the day after the expected proestrus (n = 18). Sequential measurements reduced false positive and negative rates (14% and 8%, respectively). Pregnancy rates did not differ based on the time of pairing during expected estrus. Overall, we determined vaginal impedance can be more successful than vaginal cytology at identifying proestrus in the rat, but only if multiple measurements are taken.

A novel conditional ZsGreen-expressing transgenic reporter rat strain for validating Cre recombinase expression.

The Cre/loxP recombination system has revolutionized the ability to genetically manipulate animal genomes in order to conditionally control gene expression. With recent advances in genome editing, barriers to manipulating the rat genome have been overcome and it is now possible to generate new rat strains (Cre drivers) in which Cre recombinase expression is carefully controlled temporally and/or spatially. However, the ability to evaluate and characterize these Cre driver strains is limited by the availability of reliable reporter rat strains. Here, we describe the generation and characterization of a new transgenic rat strain in which conditional expression of the ZsGreen fluorescent protein gene requires the presence of exogenous Cre recombinase. Breeding Cre-expressing rat strains to this stable ZsGreen reporter strain provides an ideal method for validating new rat Cre driver lines and will greatly accelerate the characterization pipeline.