Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats

Animal models for cystic fibrosis (CF) have contributed significantly to our understanding of disease pathogenesis. Here we describe development and characterization of the first cystic fibrosis rat, in which the cystic fibrosis transmembrane conductance regulator gene (CFTR) was knocked out using a pair of zinc finger endonucleases (ZFN). The disrupted Cftr gene carries a 16 base pair deletion in exon 3, resulting in loss of CFTR protein expression. Breeding of heterozygous (CFTR+/−) rats resulted in Mendelian distribution of wild-type, heterozygous, and homozygous (CFTR−/−) pups. Nasal potential difference and transepithelial short circuit current measurements established a robust CF bioelectric phenotype, similar in many respects to that seen in CF patients. Young CFTR−/− rats exhibited histological abnormalities in the ileum and increased intracellular mucus in the proximal nasal septa. By six weeks of age, CFTR−/− males lacked the vas deferens bilaterally. Airway surface liquid and periciliary liquid depth were reduced, and submucosal gland size was abnormal in CFTR−/− animals. Use of ZFN based gene disruption successfully generated a CF animal model that recapitulates many aspects of human disease, and may be useful for modeling other CF genotypes, including CFTR processing defects, premature truncation alleles, and channel gating abnormalities

Structural and functional annotation of the porcine immunome

Background: The domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.[br/] Results: The Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.[br/] Conclusions: This extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response

Analyses of pig genomes provide insight into porcine demography and evolution

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model

Reduced bone length, growth plate thickness, bone content, and IGF-I as a model for poor growth in the CFTR-deficient rat

<div><p>Background</p><p>Reduced growth and osteopenia are common in individuals with cystic fibrosis (CF). Additionally, improved weight and height are associated with better lung function and overall health in the disease. Mechanisms for this reduction in growth are not understood. We utilized a new CFTR knockout rat to evaluate growth in young CF animals, via femur length, microarchitecture of bone and growth plate, as well as serum IGF-I concentrations.</p><p>Methods</p><p>Femur length was measured in wild-type (WT) and SD-<i>CFTR</i>^{<i>tm1sage</i>} (<i>Cftr-/-</i>) rats, as a surrogate marker for growth. Quantitative bone parameters in <i>Cftr-/-</i> and WT rats were measured by micro computed tomography (micro-CT). Bone histomorphometry and cartilaginous growth plates were analyzed. Serum IGF-I concentrations were also compared.</p><p>Results</p><p>Femur length was reduced in both <i>Cftr-/-</i> male and female rats compared to WT. Multiple parameters of bone microarchitecture (of both trabecular and cortical bone) were adversely affected in <i>Cftr-/-</i> rats. There was a reduction in overall growth plate thichkness in both male and female <i>Cftr-/-</i> rats, as well as hypertrophic zone thickness and mean hypertrophic cell volume in male rats, indicating abnormal growth characteristics at the plate. Serum IGF-I concentrations were severely reduced in <i>Cftr-/-</i> rats compared to WT littermates.</p><p>Conclusions</p><p>Despite absence of overt lung or pancreatic disease, reduced growth and bone content were readily detected in young <i>Cftr-/-</i> rats. Reduced size of the growth plate and decreased IGF-I concentrations suggest the mechanistic basis for this phenotype. These findings appear to be intrinsic to the CFTR deficient state and independent of significant clinical confounders, providing substantive evidence for the importance of CFTR on maintinaing normal bone growth.</p></div

IGF-I is reduced in <i>Cftr-/-</i> rats.

<p>Serum IGF-I concentrations were reduced in <i>Cftr-/-</i> (•) male (261.5 ± 65.7 vs. 850.8 ± 61.1 ng/mL, p<0.0001, (A) and female (512.6 ± 70.1 vs. 1139 ± 149.7 ng/mL, p = 0.001, (B) rats compared to WT (▯). Age and weight adjusted IGF-I concentrations are found in the supplemental figure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.s001" target="_blank">S1 Fig</a>).</p

Cartilaginous growth plate analysis.

<p>Cartilaginous growth plates were evaluated based on overall growth plate thickness, average proliferating zone thickness, average number of proliferating cells/column, average hypertropic zone thickness, mean volume of hypertrophic cells, and mean hypertrophic cell volume standard deviation. Pictured growth plates are from the rats included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g003" target="_blank">Fig 3</a> (as well as microCT images in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g002" target="_blank">Fig 2</a>). They include 38 day old WT (A) and 42 day old <i>Cftr-/-</i> (C) male rats. Female bone images are from 42 day old <i>Cftr-/-</i> (B) and WT (D) rats. Each larger image is at 2X, with inserts demonstrating areas of measurement at 20X. Green outlined cells represent the proliferative zone and blue outlined cells are the hypertrophic zone. Both male and female <i>Cftr-/-</i> rats demonstrated a reduction in overall growth plate thickness (43% reduction in males and 35% in females). However, in males there was also a reduction in the hypertrophic zone thickness, mean volume of hypertrophic cells and hypertrophic cell volume standard deviation in the <i>Cftr-/-</i> rats, but not in the proliferative zone thickness or average number of proliferating cells/column. These findings are suggestive of a difference in the maturation from the proliferative zone into the hypertrophic zone, or differences in cellular activity of the hypertrophic zone, between the <i>Cftr-/-</i> and the WT rats.</p

Histomorphometric analysis.

<p>Representative histology images demonstrate histomorphologic findings of reduced bone content in <i>Cftr-/-</i> rats (more predominantly in females and detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.t002" target="_blank">Table 2</a>). Each figure demonstrates a blue line of 1000 microns in length for size consistency and photographed at 1.25X magnification. Pictured images obtained from 38 day old WT (A) and 42 day old <i>Cftr-/-</i> (C) male rats. Female bone images are from 42 day old <i>Cftr-/-</i> (B) and WT (D) rats. Histology images are from the male and female rat femurs pictured in microCT images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g002" target="_blank">Fig 2</a>).</p

<i>Cftr</i>-/- rats have reduced size and femur length.

<p><b>(</b>A) <i>Cftr-/-</i> rats are notably smaller in size compared to their wild type (WT) littermates. Animal total body weights are demonstrated versus age of the animals in <i>Cftr-/-</i> rats (•) compared to WT rats (▯). These findings are consistent with previously published data by Tuggle et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.ref013" target="_blank">13</a>]. (B) Excised femurs obtained at sacrifice were measured by digital calipers. Femur length is reduced in <i>Cftr-/-</i> rats (•) compared to WT rats (▯) regardless of age or gender (p<0.0001).</p