RESUMEN
Mice carrying human immunoglobulin transloci were immunised with HIV-1 gp140 antigen to gain insight into the range and nature of human monoclonal antibodies (mAbs) that can be elicited from such humanised mice. Using five-feature mice that harbour YAC-based germline-configuration human IgM, Igκ and Igλ transloci in a mouse background disrupted for endogenous mouse IgH and Igκ expression, gp140-specific human IgM mAbs were readily elicited following serial immunisation. These mAbs were converted to human IgG1 format and were found to bind diverse epitopes within gp140, exhibiting high functional affinity for the antigen-typically in the nanomolar or sub-nanomolar range. The number of specific, stable hybridomas per mouse was, however, low (typically around five) with the hybridomas within individual mice often being clonally related. Nevertheless, different mice used B cell clones expressing varied V(D)J combinations, with affinity maturation through somatic hypermutation making a critical contribution. Thus, a wide range of distinct high-affinity mAbs can be obtained by immunising multiple animals. The results confirm the utility of the translocus-mouse approach and give insight into strategies for possible future improvement.
Asunto(s)
Anticuerpos Monoclonales/genética , Genes de Inmunoglobulinas , Inmunoglobulina M/genética , Translocación Genética/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/metabolismo , Síndrome de Inmunodeficiencia Adquirida/patología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Cromosomas Artificiales de Levadura/genética , Humanos , Hibridomas/citología , Hibridomas/inmunología , Hibridomas/metabolismo , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Transgénicos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Elements with insulator/border activity have been characterized most extensively in Drosophila melanogaster. In vertebrates, the first example of such an element was provided by a hypersensitive site of the chicken beta-globin locus, cHS4. It has been proposed that the homologous site in humans, HS5, functions as a border of the human beta-globin locus. Here, we have characterized HS5 of the human beta-globin locus control region. We have examined its tissue-specificity and assessed its insulating properties in transgenic mice using a lacZ reporter assay. Most importantly, we have tested its enhancer blocking activity in the context of the full beta-globin locus. Our results show that HS5 is erythroid-specific rather than ubiquitous in human tissues. Furthermore, HS5 does not fulfil the criteria of a general in vivo insulator in the transgene protection assay. Finally, a HS5 conditional deletion from the complete locus demonstrates that HS5 has no discernable activity in adult erythroid cells. Surprisingly, HS5 functions as an enhancer blocker in embryonic erythroid cells. We conclude that HS5 is a developmental stage-specific border in erythroid cells.
