Michael J. Owen

The Application of Transgenic Mice for Therapeutic Antibody Discovery

In 2006, panitumumab, the first fully human antibody generated from transgenic mice, was approved for clinical use by the US Food and Drug Administration (FDA). Since then, a further seven such antibodies have been approved. In this chapter, we discuss how transgenic mice technologies can provide a powerful platform for creating human therapeutic antibodies.

DNase hypersensitivity and methylation of the humanCD3G andD genes during T-cell development

The mouse and humanCD3G andD genes are organized in opposite transcriptional orientation, their 5′ ends being separated by about 1.6 kilobases (kb) of DNA. The molecular basis of the tissue-specific regulation of expression of the humanCD3G andD genes was examined using DNase I hypersensitivity and CpG methylation analysis. Two T cell-specific DNase I hypersensitivity sites were defined within the intergenic region. A third hypersensitive site (DHS3) was detected 0.4 kb 3′ to theCD3D gene. This latter site was present in all T cells, but was absent in all other committed cell types examined. DHS3 was also detected in the lympho-myeloid progenitor cell KG1, but was absent when this line was induced to differentiate to the macrophage lineage. The intergenic region was undermethylated in T cells expressingCD3, but was in general more extensively methylated in other cell types. Importantly, however, in KG1 sublines which express theCD3 genes the intergenic region remains extensively methylated. These results define areas 3′ to theD gene and within the intergenic region which contain regulatory elements that influence bothCD3D andG expression. They further show that transcription form theCD3D andG genes may occur initially from a methylated promoter. Significantly, the 3′ regulatory region was shown to adopt an open chromatin structure prior to lineage commitment and beforeCD3 transcription.

Transfection of the ChloramphenicolAcetyltransferase Gene into Eukaryotic Cells Using Diethyl-Aminoethyl (DEAE)-Dextran

The study of gene regulation in eukaryotic cells involves a practical requirement for two distinct techniques: first, a transfection system, or more simply, a way of getting DNA into a cell; and second, a reporter system, which, as the name suggests, is a means of finding out what the transfected DNA does from its new location inside the cell. These two requirements are amply met by transfecting cells, in the presence of the polycation diethyl aminoethyl-dextran (DEAE-dextran), with a plasmid vector that has regulatory sequences adjacent to the chloramphenicol acetyl transferase (CAT) gene. This type of gene transfer is often referred to as a transient expression system. Production of the CAT enzyme peaks at around 40-48 h and thereafter the level falls as the plasmid DNA is diluted out in a growing population of cells. The general strategy of subcloning putative regulatory regions followed by transfection and quantification of CAT activity is outlined in a flow diagram (Fig. 1) Fig. 1. Flow diagram showing the key steps in transient transfection analysis. (1) Putative regulatory regions are excised from genomic DNA by restriction enzyme digestion. Fragments are (2) subcloned into the CAT vector and (3) transfected into eukaryotic cells using DEAE dextran. (4) About 40 h after transfection, the cells are lysed and the lysates incubated with chloramphenicol and radiolabeled acetyl coenzyme A. (5) Radiolabeled chloramphenicol is extracted from the mixture and (6) the level of radioactivity related to the function of the subcloned fragment.

T-cell receptor Vβ5 usage defines reactivity to a human T-cell receptor monoclonal antibody

The monoclonal antibody 3D6 reacts with the T-cell receptor (Tcr) on the T-leukemic line HPBALL and with 2–13% of human peripheral blood T lymphocytes. In this study Vα and Vβ expression in a panel of T-cell populations and clones expressing the 3D6 epitope was determined by Southern and northern hybridization analysis. The results demonstrate that these 3D6-positive T cells, regardless of CD4/CD8 phenotype or function, express a gene of the Vβ5 family, also expressed by HPBALL. No correlation of the HPBALL Vα gene. The results demonstrate that 3D6 recognizes an epitope solely on the Tcrβ chain and that the use of this β chain, together with an appropriate Vα, can impart a diverse pattern of reactivity to a T cell.