Inbred Sublines Research Articles

Development of transplantable B-cell lymphomas in the MHC-defined miniature swine model

BackgroundEstablishment of transplantable tumors in clinically relevant large animals allows translational studies of novel cancer therapeutics.MethodsHere we describe the establishment, characterization, and serial transplantation of a naturally occurring B-cell lymphoma derived from a unique, highly inbred sub-line of Massachusetts General Hospital (MGH) major histocompatibility complex (MHC)-defined miniature swine.ResultsThe lymphoblastic cell line (LCL) originated from peripheral blood of a 2.5 year old female swine leukocyte antigen (SLA)dd-inbred miniature swine breeder demonstrating clinical signs of malignancy. Flow cytometric phenotypic analysis of subclones derived from the original cell line revealed surface markers commonly expressed in a B-cell lineage neoplasm. A subclone of the original LCL was transplanted into mildly-conditioned histocompatible miniature swine and immunocompromised NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Tissue and blood samples harvested 2 weeks following subcutaneous and intravenous injection in a highly inbred SLAdd pig were cultured for tumor growth and phenotypic analysis before serial transfer into NSG mice. Evidence of tumor growth in vivo was found in all tumor cell recipients. In vitro growth characteristics and surface phenotype were comparable between the original and serially transplanted tumor cell lines.ConclusionsThese results indicate the feasibility of developing a large-animal transplantable tumor model using cells derived from spontaneously occurring hematologic malignancies within the highly inbred miniature swine herd.

Conserved, breed-dependent, and subline-dependent innate immune responses of Fayoumi and Leghorn chicken embryos to Newcastle disease virus infection

Newcastle disease virus (NDV) is a threat to the global poultry industry, but particularly for smallholder farmers in low- and middle-income countries. Previous reports suggest that some breeds of chickens are less susceptible to NDV infection, however, the mechanisms contributing to this are unknown. We here examined the comparative transcriptional responses of innate immune genes to NDV infection in inbred sublines of the Fayoumi and Leghorn breeds known to differ in their relative susceptibility to infection as well as at the microchromosome bearing the major histocompatability complex (MHC) locus. The analysis identified a set of five core genes, Mx1, IRF1, IRF7, STAT1, and SOCS1, that are up-regulated regardless of subline. Several genes were differentially expressed in a breed- or subline-dependent manner. The breed-dependent response involved TLR3, NOS2, LITAF, and IFIH1 in the Fayoumi versus IL8, CAMP, and CCL4 in the Leghorn. Further analysis identified subline-dependent differences in the pro-inflammatory response within the Fayoumi breed that are likely influenced by the MHC. These results have identified conserved, breed-dependent, and subline-dependent innate immune responses to NDV infection in chickens, and provide a strong framework for the future characterization of the specific roles of genes and pathways that influence the susceptibility of chickens to NDV infection.

Differential organization of male copulatory patterns in high- and low-yawning-frequency sublines versus outbred Sprague–Dawley rats

The temporal organization of masculine sexual behavior in rats is highly stereotyped; involving a sequence of mounts, intromissions and ejaculations. Sexual behavior has been described in exogamic and genetically manipulated rodent species. In this work, we compare the male sexual behavior of outbred Sprague–Dawley (SD) to those of rats inbred for high (HY)- and low (LY)- spontaneous yawning frequency. In the first experiment, the percentage of inexperienced rats' ejaculatory behavior is significantly lower in the HY and LY respect to Sprague–Dawley rats. The latency to ejaculate for inexperienced HY was shorter than the LY and SD rats. In the second experiment, we examined the differences between inbred sublines and Sprague–Dawley rats once the subjects had become sexually experienced after four copulatory sessions. HY rats still have slower proportion of ejaculators respect to LY and SD rats. Additionally, postejaculatory latencies were longer for HY rats, with longer intercopulatory intervals and higher number of copulatory bouts that delayed ejaculation. Both sublines show lower copulatory efficiency respect to SD rats. In conclusion, both sublines show alterations in the temporal organization of sexual motor pattern that are due at least partially to strong inbreeding process to select them

Candidate Transcriptomic Sources of Inbreeding Depression in Drosophila melanogaster

The genomic causes of inbreeding depression are poorly known. Several studies have found widespread transcriptomic alterations in inbred organisms, but it remains unclear which of these alterations are causes of the depression and which are mere responses to the ensuing physiological stress induced by increased homozygosity due to inbreeding. Attempting to differentiate causes from responses, we made a c-DNA microarray analysis of inbreeding depression in Drosophila melanogaster. The rationale of the experiment was that, while depression is a general phenomenon involving reductions in fitness in different inbred lines, its first genetic causes would be different for each inbred line, as they are expected to be caused by the fixation of rare deleterious genes. We took four sets of inbred sublines, each set descending from a different founding pair obtained from a large outbred stock, and compared the expression of the three most depressed sublines and the three least depressed sublines from each set. Many changes in expression were common to all sets, but fourteen genes, grouped in four expression clusters, showed strong set-specific changes, and were therefore possible candidates to be sources of the inbreeding depression observed.

Gene-Expression Changes Caused by Inbreeding Protect Against Inbreeding Depression in Drosophila

We present a transcriptomic analysis aimed at investigating whether the changes in gene expression that occur under inbreeding generally reduce or enhance inbreeding depression. Discerning between these two alternatives can be addressed only when both changes in expression due to inbreeding and to inbreeding depression are estimated simultaneously. We used Affymetrix 2.0 arrays to study the changes in gene expression associated with both inbreeding and inbreeding depression for fitness in four sets of inbred sublines of Drosophila melanogaster. We found that for most genes showing changes in expression associated with inbreeding, the least depressed sublines were those showing the largest departures in expression from that of the outbred control. This suggests a pattern consistent with a protective role of expression changes against inbreeding effects, and would reveal a new dimension of the transcriptomics of inbreeding. The variation in depression observed could then be due not only to the genetic damages primarily originating that depression, but also possibly to differences in the ability to carry out the appropriate adjustments in gene expression to cope with the inbreeding. We also found that these expression changes with a putative protective role against inbreeding effects show a clear specificity on RNA synthesis and splicing and energy derivation functions.

Myelogenous leukemia in adult inbred MHC-defined miniature swine: A model for human myeloid leukemias

This manuscript reports on five cases of spontaneous myelogenous leukemia, similar to human disease, occurring within highly inbred, histocompatible sublines of Massachusetts General Hospital (MGH) MHC-defined miniature swine. In cases where a neoplasm was suspected based on clinical observations, samples were obtained for complete blood count, peripheral blood smear, and flow cytometric analysis. Animals confirmed to have neoplasms were euthanized and underwent necropsy. Histological samples were obtained from abnormal tissues and suspect lesions. The phenotype of the malignancies was assessed by flow cytometric analysis of processed peripheral blood mononuclear cells and affected tissues. Five cases of spontaneous myeloid leukemia were identified in adult animals older than 30 months of age. All animals presented with symptoms of weight loss, lethargy, and marked leukocytosis. At autopsy, all animals had systemic disease involvement and presented with severe hepatosplenomegaly. Three of the five myelogenous leukemias have successfully been expanded in vitro. The clustered incidence of disease in this closed herd suggests that genetic factors may be contributing to disease development. Myelogenous leukemia cell lines established from inbred sublines of MGH MHC-defined miniature swine have the potential to be utilized as a model to evaluate therapies of human leukemia.

Establishment of transplantable porcine tumor cell lines derived from MHC- inbred miniature swine

AbstractThe lack of transplantable tumors has limited assessment of graft-versus-tumor effects following hematopoietic cell transplantation in clinically relevant large-animal models. We describe the derivation and characterization of porcine tumor cell lines with initial efforts of tumor transplantation using immunocompromised mice and highly inbred sublines of Massachusetts General Hospital major histocompatibility complex (MHC)–inbred miniature swine. Autopsies were performed routinely on swine that died unexpectedly or had suspicion of malignancy based on clinical symptoms or peripheral blood analysis. Tissue samples were obtained for pathology, phenotyped by flow cytometry, and placed in culture. Based on growth, lines were selected for passage into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and miniature swine. Porcine tumor recipients were preconditioned with total body irradiation from 0 to 500 cGy or with a 30-day course of oral cyclosporine. We identified 19 cases of hematologic tumors. Nine distinct tumor cell lines were established from 8 of these cases, including 3 derived from highly inbred sublines. In vivo tumor growth and serial transfer were observed in immunocompromised mice for one tumor cell line and in miniature swine for 1 of 2 tumor cell lines expanded for this purpose. These results suggest the possibility of developing a transplantable tumor model in this large-animal system.

Estimating numbers of EMS-induced mutations affecting life history traits in Caenorhabditis elegans in crosses between inbred sublines

Inbred lines of the nematode Caenorhabditis elegans containing independent EMS-induced mutations were crossed to the ancestral wild-type strain (N2). Replicated inbred sublines were generated from the F1 offspring under conditions of minimal selection and, along with the N2 and mutant progenitor lines, were assayed for several fitness correlates including relative fitness (w). A modification of the Castle-Wright estimator and a maximum-likelihood (ML) method were used to estimate the numbers and effects of detectable mutations affecting these characters. The ML method allows for variation in mutational effects by fitting either one or two classes of mutational effect, and uses a Box-Cox power transformation of residual values to account for a skewed distribution of residuals. Both the Castle-Wright and the ML analyses suggest that most of the variation among sublines was due to a few (approximately 1.5-2.5 on average) large-effect mutations. Under ML, a model with two classes of mutational effects, including a class with small effects, fitted better than a single mutation class model, although not significantly better. Nonetheless, given that we expect there to be many mutations induced per line, our results support the hypothesis that mutations vary widely in their effects.

Development of genetic differentiation and postzygotic isolation in experimental metapopulations of spider mites.

We studied the development of genetic differentiation and postzygotic isolation in experimental metapopulations of the two-spotted spider mite, Tetranychus urticae Koch. A genetically diverse starter population was made by allowing six inbred sublines to interbreed. Then three migration patterns were tested: no migration, or one or three immigrants per subpopulation per generation. Variations in four traits were investigated: allozymes, acaricide resistance, diapause, and hatchability. In the allozymes, acaricide resistance, and diapause, genetic variation among subpopulations became high in metapopulations with no migration, but not in the others, which showed that one immigrant is enough to prevent genetic differentiation. Hatchability, which was decreased by interbreeding among the six sublines, gradually recovered in succeeding generations. In metapopulations with no migration, hatchability was reduced again after in-migration at the 15th generation. Different karyotypes or coadapted gene complexes can survive in different subpopulations by genetic drift, and both Wolbachia-infected and -noninfected subpopulations may be selected, which would lead to postzygotic isolation between isolated subpopulations. Our results indicate that sampling effects such as genetic drift or stochastic loss of Wolbachia produce postzygotic isolation in laboratory populations of spider mite.

Specific Expression of Gene di (diabetes insipidus) in Homologous Inbred Rat Lines

The diabetes insipidus mutation is displayed in homozygotes in the form of diabetes insipidus with water consumption from 30 to 100% of body weight per day. We developed two inbred sublines of the di/di Brattleboro rats as well as the recombinant inbred subline by integrating genes of August rats into the di/di mutant genome. Changes in the genetic background proved to have no effect on the quantitative parameters of the diabetes insipidus. The intensity of the secondary immune response and the content of tropomyosin in the medulla of the rat kidney can serve as additional marker traits of the di/di genotype.

Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, delta-sarcoglycan, in hamster: an animal model of disrupted dystrophin-associated glycoprotein complex.

Cardiomyopathy (CM) is a primary degenerative disease of myocardium and is traditionally categorized into hypertrophic and dilated CMs (HCM and DCM) according to its gross appearance. Cardiomyopathic hamster (CM hamster), a representative model of human hereditary CM, has HCM and DCM inbred sublines, both of which descend from the same ancestor. Herein we show that both HCM and DCM hamsters share a common defect in a gene for delta-sarcoglycan (delta-SG), the functional role of which is yet to be characterized. A breakpoint causing genomic deletion was found to be located at 6.1 kb 5' upstream of the second exon of delta-SG gene, and its 5' upstream region of more than 27.4 kb, including the authentic first exon of delta-SG gene, was deleted. This deletion included the major transcription initiation site, resulting in a deficiency of delta-SG transcripts with the consequent loss of delta-SG protein in all the CM hamsters, despite the fact that the protein coding region of delta-SG starting from the second exon was conserved in all the CM hamsters. We elucidated the molecular interaction of dystrophin-associated glycoproteins including delta-SG, by using an in vitro pull-down study and ligand overlay assay, which indicates the functional role of delta-SG in stabilizing sarcolemma. The present study not only identifies CM hamster as a valuable animal model for studying the function of delta-SG in vivo but also provides a genetic target for diagnosis and treatment of human CM.

Accumulation of mutations affecting body weight in inbred mouse lines

The variation from spontaneous mutations for 6-week body weight in the mouse was estimated by selection from a cross of two inbred sublines, C57BL/6 and C57BL/10, separated about 50 years previously from the same inbred line. Selection was practised high and low for 12 generations from the F2, followed by one generation of relaxation. The lines diverged by approximately 1.7 g or 0.7 sd. The additive genetic variance was estimated in the F2 by restricted maximum likelihood and from the selection response, and from this variance the mutational heritability hM2 was estimated using the number of generations since divergence. Estimates of hM2 range from 0.08 to 0.10% depending on the method of analysis. These estimates are similar to those found for other species, but lower than other estimates for the mouse. It is concluded that substantial natural and, perhaps, artificial selection operated during the maintenance of the sublines.

Effects of multiple retrovirus insertions on quantitative traits of mice.

To assess the potential to generate quantitative genetic variation by insertional mutagenesis in a vertebrate, lines of mice in which many provirus vector inserts segregated at a low initial frequency on an inbred background (insert lines) were subjected to divergent artificial selection on body weight at 6 weeks and responses and heritability estimates compared to control lines lacking inserts. Heritability estimates were more than 1.5 times greater in the insert lines than in the controls, but because the phenotypic variance was substantially higher in the insert lines the genetic variance was about 3 times greater. Realized heritability estimates tended to be lower than heritabilities estimated by an animal model which utilizes information in covariances between all relatives in the data set. A surprisingly large response to selection occurred in the inbred control line. Insert lines were about 20% less fertile than controls. Division of the selection lines into inbred sublines in the later generations of the experiment revealed substantially greater variation among sublines of the insert lines than among the controls. Heritabilities were similar to typical estimates for the trait in outbred populations. In conclusion, there was clear evidence of extra variation deriving from inserts, which has yet to be attributed to individual genes.

Accounting for bias in estimates of the rate of polygenic mutation.

Experimental data on the rate of response to artificial selection in initially inbred lines or the rate of divergence among inbred sublines can be used to estimate the rate of increase in variance of quantitative traits from new mutations. So far estimates have been based on the infinitesimal model of many genes with small additive effects which imply a rate of increase in heritability for Drosophila melanogaster bristle number traits of about 0.1% per generation. Such estimates are biased because mutants tend to have large effects, to have non-additive gene action, and to be deleterious. Here, recent information on the distribution of effects of new mutations on Drosophila melanogaster bristle number and viability is used to infer the direction and magnitude of this bias. The infinitesimal model tends to underestimate the mutational variance, typically by a factor of about 3, but this factor depends on the experimental design. Averages of revised estimates, accounting for this bias, of the per generation increment in heritability from mutation are 0.36% and 0.21% for abdominal and sternopleural bristle number, respectively, in experiments involving M strains, and 1.4% and 0.7% for abdominals and sternopleurals, respectively, in P strains.

Variable Prevalence of Lymphocytic Thyroiditis among Diabetes-Prone Sublines of BB/Wor Rats*

The BB/Wor rat develops spontaneous autoimmune insulin-dependent diabetes mellitus (DM) and lymphocytic thyroiditis (LT). Six different inbred sublines of this rat model have been selected for studying the pathogenesis of DM and, thereby, the prevalence of DM has been carefully monitored and found to be relatively constant. In contrast, we have observed a striking difference in the prevalence and intensity of LT in these six sublines, varying from 100% in NB subline rats to 4.9% in BE subline rats at 105-110 days of age. Excess iodide administration frequently increases the prevalence of LT but did not do so in the two sublines (BB and BE) with the lowest frequency of spontaneous LT. In view of this variable prevalence of LT in the different BB/Wor sublines, it is imperative that investigators studying the pathogenesis and modulation of LT in this rat model select those sublines which express a desired frequency of spontaneous LT.

Increase in liver protein phosphatase-1 in spontaneously diabetic Chinese hamsters

Two broad-specifically protein phosphatases, termed protein phosphatase-1 (PrP-1) and protein phosphatase-2A (PrP-2A), accounting for all the hepatic activity regulating glycogen phosphorylase, were measured in spontaneously diabetic Chinese hamsters exhibiting persistent glycosuria. When compared with genetically related inbred sublines free of glycosuria, diabetic animals demonstrated approximately 25% increase in PrP-1 activity measured either in crude tissue extracts or in cytosols fractionated by ion-exchange chromatography. No significant alteration in total PrP-2A activity was observed in the diabetic animals. These findings indicate that a specific change in hepatic PrP-1 is associated with genetically acquired diabetes in Chinese hamsters. In contrast to reported data using animals with experimentally induced diabetes mellitus, hepatic PrP-1 was increased in the spontaneously diabetic Chinese hamsters. The data suggests that distinct alterations in PrP-1 and associated metabolic consequences are exhibited by different types of diabetes.

A deletion in a rat major histocompatibility complex class I gene is linked to the absence of beta 2-microglobulin-containing serum molecules.

Class I major histocompatibility antigens are composed of a heavy chain that is noncovalently associated with beta 2-microglobulin (beta 2m). Most class I molecules are membrane bound, but mouse and rat cDNA clones and genes without a functional code for the transmembrane amino acids have been identified. The membrane-associated class I molecules are important in the control of cell-mediated cytotoxicity, while the function of the soluble molecules remains unclear. Previous studies have shown that beta 2m circulates in rat serum in three different molecular weight classes. The first is free beta 2m (Mr, 12,000), the second is about Mr 70,000, and the third is roughly Mr 200,000. In an inbred subline of immunodeficient, diabetes-prone BioBreeding rats (BioBreeding/Hagedorn), previous work detected two restriction fragment polymorphisms in class I major histocompatibility complex genes, one of them a gene deletion on a 7-kilobase BamHI fragment and the other on a 2-kilobase BamHI fragment. In these rats we have found that the third serum beta 2m-binding size class is absent. Analysis of F1 and F2 individuals following cross-breeding between BioBreeding/Hagedorn rats and genetically related (nondiabetic) control BioBreeding w-subline rats demonstrated that the large-size serum peak of beta 2m was associated with the presence of the class I restriction fragments.

Using the triple test cross to investigate the genetics of behavior in wild populations. II. Escape-avoidance conditioning inRattus norvegicus

The detailed results of a triple test cross using wild rats crossed to partially inbred sublines of the Roman High and Low Avoidance selection strains clearly indicated considerable amounts of additive genetic variation in the wild population for avoidance performance while there is a general absence of nonadditive variation, with the exception of some dominance variation together with duplicate type epistasis. Where present, directional dominance for this phenotype is toward high performance only for later trials, and the effects of any epistasis reinforce this trend. The overall conformity of these results to the predictions from previous laboratory rat studies suggests no reason to reject the representativeness assumption necessary for arguments from genetic architecture in laboratory populations to adaptive significance in contemporary wild populations.

Insulin and glucagon release in the diabetic Chinese hamster: differences among inbred sublines.

Release of insulin and glucagon from perfused pancreases in vitro of 40 normal male and female Chinese hamsters (from one inbred subline) and 110 male and female diabetic hamsters (from three inbred sublines) was measured in response to glucose plus arginine, theophylline alone, or potassium alone, in order to determine if differences in hormone secretion exist among different diabetic sublines. Glucose plus arginine and potassium produced subnormal insulin responses in all three diabetic sublines, whereas theophylline induced 'normal' or above normal insulin responses. Excessive glucagon release was consistently seen in only one diabetic subline. The female normal animals showed greater insulin release than the male normal hamsters in response to glucose plus arginine. This sex difference was not seen in the diabetic animals.

The Chinese hamster as a model for the study of diabetes mellitus.

Selection for and against diabetes and subsequent inbreeding of Chinese hamsters started in 1963. Currently there are six inbred sublines that have greater than 85% incidence of glycosuria and two control inbred nondiabetic sublines that are essentially free of glycosuria. At birth, hamsters from inbred sublines are considered prediabetic. There is phenotypic variation between diabetic sublines. Onset time, incidence of ketonuria, blood glucose, plasma insulin, glucagon and glycohydrolase levels vary from subline to subline, but pancreatic insulin and glucagon levels are consistently low and high, respectively, in all diabetic sublines compared with nondiabetics. Experimental breeding data suggest a minimum of two homozygous recessive genes for diabetes. It is not known if the inbred lines are similar diabetic genotypes, but the probability is high that modifier background genes vary from subline to subline. Chinese hamsters have diabetes ranging from mild to severe. Animals weighing 25 g can excrete up to 75 ml of urine containing 3 g of glucose per day. Fasting blood glucose as high as 500 mg/dl and 10 mumol/ml of beta-hydroxybutyrate have been reported. Gluconeogenesis is elevated, and some glycolytic enzymes are decreased in severe diabetes. Low levels of renal acid glycohydrolase enzymes may contribute to glomerular capillary loop basement membrane thickening in diabetic hamsters. Caloric restriction per se or reduction of dietary fat prevented onset of hyperglycemia and hyperinsulinemia in prediabetics. Morphologic changes have been observed in pancreatic islets, kidney, nerve, blood vessels, eyes, brain, and genito-urinary systems of diabetic Chinese hamsters. Pathogenesis of diabetes in this animal appears to be related to an increased demand for insulin. Initially there is a positive response to this demand by beta cells, but exhaustion occurs. This is followed by a decrease in beta-cell mass and relative or absolute insulin deficiency.