Production of monoclonal antibody against recombinant bovine sex-determining region Y (SRY) and their preferential binding to Y chromosome-bearing sperm.

Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.

Isolation and characterization of pediatric canine bone marrow CD34+ cells.

Historically, the dog has been a valuable model for bone marrow transplantation studies, with many of the advances achieved in the dog being directly transferable to human clinical bone marrow transplantation protocols. In addition, dogs are also a source of many well-characterized homologues of human genetic diseases, making them an ideal large animal model in which to evaluate gene therapy protocols. It is generally accepted that progenitor cells for many human hematopoietic cell lineages reside in the CD34+ fraction of cells from bone marrow, cord blood, or peripheral blood. In addition, CD34+ cells are the current targets for human gene therapy of diseases involving the hematopoietic system. In this study, we have isolated and characterized highly enriched populations of canine CD34+ cells isolated from dogs 1 week to 3 months of age. Bone marrow isolated from 2- to 3-week-old dogs contained up to 18% CD34+ cells and this high percentage dropped sharply with age. In in vitro 6-day liquid suspension cultures, CD34+ cells harvested from 3-week-old dogs expanded almost two times more than those from 3-month-old dogs and the cells from younger dogs were also more responsive to human Flt-3 ligand (Flt3L). In culture, the percent and number of CD34+ cells from both ages of dogs dropped sharply between 2 and 4 days, although the number of CD34+ cells at day 6 of culture was higher for cells harvested from the younger dogs. CD34+ cells harvested from both ages of dogs had similar enrichment and depletion values in CFU-GM methylcellulose assays. Canine CD34+/Rho123lo cells expressed c-kit mRNA while the CD34+/Rhohi cells did not. When transplanted to a sub-lethally irradiated recipient, CD34+ cells from 1- to 3-week-old dogs gave rise to both myeloid and lymphoid lineages in the periphery. This study demonstrates that canine CD34+ bone marrow cells have similar in vitro and in vivo characteristics as human CD34+ cells. In addition, ontogeny-related functional differences reported for human CD34+ cells appear to exist in the dog as well, suggesting pediatric CD34+ cells may be better targets for gene transfer than adult bone marrow. The demonstration of similarities between canine and human CD34+ cells enhances the dog as a large, preclinical model to evaluate strategies for improving bone marrow transplantation protocols, for gene therapy protocols that target CD34+ cells, and to study the engraftment potential of various cell populations that may contain hematopoietic progenitor cell activity.

Identification of HLA class II-restricted H-Y-specific T-helper epitope evoking CD4+ T-helper cells in H-Y-mismatched transplantation.

BACKGROUND: Stem-cell grafts between HLA-identical siblings are less likely to succeed when there is a sex mismatch. This lack of success can be interpreted as enhanced activity directed against minor histocompatibility antigens encoded by the Y chromosome (H-Y). So far, in man, only cytotoxic T lymphocytes (CTLs) specific for several minor histocompatibility antigens have been reported. We aimed to identify and clarify the role of MHC class II-restricted H-Y-specific T-helper cells in these transplant settings. METHODS: H-Y-specific MHC class II-restricted CD4+ T cells were isolated from blood of a female patient who rejected an HLA-identical male stem-cell transplant. By molecular cloning of H-Y genes and functional T-helper experiments, we elucidated antigen specificity and the functional properties of these H-Y-specific T-helper cells. FINDINGS: CD4+ T-helper cells recognise the Y gene-encoded peptide VIKVNDTVQI presented by HLA-DRbeta3*0301. These T-helper cells mature dendritic cells and enhance expansion of minor histocompatibility antigen-specific MHC class I-restricted CD8+ CTLs. INTERPRETATION: Characterisation of an MHC class II-restricted H-Y epitope that evoked CD4+ T-helper responses adds a novel cellular component to the alloimmune response against Y chromosome-encoded minor histocompatibility antigens. This component completes the H-Y-directed alloimmune response and aids understanding of the poorer outcome of sex-mismatched transplants.