Genotyping of canine MHC gene DLA-88 by next-generation sequencing reveals high frequencies of new allele discovery and gene duplication.

Dogs have served as one of the most reliable preclinical models for a variety of diseases and treatments, including stem/progenitor cell transplantation. At the genetic epicenter of dog transplantation models, polymorphic major histocompatibility complex (MHC) genes are most impactful on transplantation success. Among the canine class I and class II genes, DLA-88 has been best studied in transplantation matching and outcomes, with 129 DLA-88 alleles identified.

The canine gut microbiome is associated with higher risk of gastric dilatation-volvulus and high risk genetic variants of the immune system.

Background: Large and giant dog breeds have a high risk for gastric dilatation-volvulus (GDV) which is an acute, life-threatening condition. Previous work by our group identified a strong risk of GDV linked to specific alleles in innate and adaptive immune genes. We hypothesize that variation in the genes of the immune system act through modulation of the gut microbiome, or through autoimmune mechanisms, or both, to predispose dogs to this condition.

Associations between gastric dilatation-volvulus in Great Danes and specific alleles of the canine immune-system genes DLA88, DRB1, and TLR5.

OBJECTIVE To determine whether specific alleles of candidate genes of the major histocompatibility complex (MHC) and innate immune system were associated with gastric dilatation-volvulus (GDV) in Great Danes. ANIMALS 42 healthy Great Danes (control group) and 39 Great Danes with ≥ 1 GDV episode. PROCEDURES Variable regions of the 2 most polymorphic MHC genes (DLA88 and DRB1) were amplified and sequenced from the dogs in each group.

Isolation, genetic manipulation, and transplantation of canine spermatogonial stem cells: progress toward transgenesis through the male germ-line.

The dog is recognized as a highly predictive model for preclinical research. Its size, life span, physiology, and genetics more closely match human parameters than do those of the mouse model. Investigations of the genetic basis of disease and of new regenerative treatments have frequently taken advantage of canine models.

Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells.

The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors.

A preclinical model of double- versus single-unit unrelated cord blood transplantation.

Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5 x 10(7)/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements.

Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis.

Retroviral integration provides a unique and heritable genomic tag for a target cell and its progeny, enabling studies of clonal composition and repopulation kinetics after gene transfer into hematopoietic stem cells. The clonal tracking method, linear amplification-mediated polymerase chain reaction (LAM-PCR) is widely employed to follow the hematopoietic output of retrovirally marked stem cells. Here we examine the capabilities and limitations of conventional LAM-PCR to track individual clones in a complex multiclonal mix.

Stable trichimerism after marrow grafting from 2 DLA-identical canine donors and nonmyeloablative conditioning.

Although hematopoietic cell transplantation (HCT) is generally accomplished using a single donor, multiple donors have been used to enhance the speed of engraftment, particularly in the case of umbilical cord blood grafts. Here we posed the question in the canine HCT model whether stable dual-donor chimerism could be established using 2 DLA-identical donors. We identified 8 DLA-identical littermate triplets in which the marrow recipients received 2 Gy total body irradiation followed by marrow infusions from 2 donors and postgrafting immunosuppression.

The post-translational phenotype of collagen synthesized by SAOS-2 osteosarcoma cells.

The human osteosarcoma-derived cell line, SAOS-2, exhibits many of the phenotypic characteristics of osteoblasts including the deposition of types I and V collagens in an extracellular matrix. Lesser amounts of collagen XI chains were also detected. The cell layer collagen contains hydroxylysyl pyridinoline cross-links but without the accompanying lysyl pyridinoline typical of human bone collagen.

Growth factor receptors as regulators of hematopoiesis.

Nearly 15 years have elapsed since the US Food and Drug Administration last approved a major new hematopoietic cytokine. Promiscuous binding to multiple receptors, or to receptors expressed by multiple tissues, reduces growth factor specificity and promotes side effects. Here we show that hematopoiesis can be differentially regulated using receptors rather than ligands.

Anatomical compartments modify the response of human hematopoietic cells to a mitogenic signal.

Methods for specifically regulating transplanted cells have many applications in gene and cell therapy. We examined the response of human cord blood CD34+ cells to a specific mitotic signal in vivo. Using a conditional signaling molecule (F36VMpl) that is specifically activated by an artificial ligand called a chemical inducer of dimerization (CID), human hematopoietic cells transplanted into immune deficient mice were induced to proliferate.

Overexpression of glutathione-S-transferase, MGSTII, confers resistance to busulfan and melphalan.

A major obstacle to hematopoietic gene therapy is the lack of appropriate in vivo selection protocols that can raise the presently low numbers of gene-altered stem cells to therapeutically useful levels. Overexpression of glutathione-S-transferases (GST), in combination with busulfan treatment, may provide an exploitable selection mechanism for hematopoietic gene therapy strategies. GST provides a major route of detoxification of a variety of xenobiotics, including alkylating agents used for myeloablative chemotherapy.

Radiation dose determines the degree of myeloid engraftment after nonmyeloablative stem cell transplantation.

A multivariate analysis of 121 dogs conditioned with 200, 100, or 50 cGy of total body irradiation (TBI) followed by hematopoietic stem cell transplantation from matched littermates showed that TBI dose was the only factor examined that was statistically significantly associated with the percentage of donor myeloid engraftment in stable long-term chimeras ( P = .008). To understand the direct effects of low-dose irradiation on hematopoietic stem/progenitor cells, nonirradiated and irradiated human CD34 + cells were evaluated for competitive repopulating ability in nonobese diabetic/severe combined immunodeficiency beta2m -/- mice.

Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration.

HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.

The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors.

A promising and increasingly exploited property of hematopoietic stem cells is their ability to efflux the fluorescent dye Hoechst 33342. The Hoechst-negative cells are isolated by fluorescence-activated cell sorting as a so-called side "population" (SP) of bone marrow. This SP from bone marrow, as well as other tissues, is reported to contain immature stem cells with considerable plasticity.