caa a22 4500 469033843 CHVBK 20180323132751.0 cr unu---uuuuu 170328e19920601xx s 000 0 eng 10.1007/BF00215284 doi (NATIONALLICENCE)springer-10.1007/BF00215284 A new approach to the cloning of genes encoding T-cell epitopes [Elektronische Daten] [D. Scott, P. Dyson, E. Simpson] The molecular structure of antigens recognized exclusively by T cells, such as minor histocompatibility antigens and some antigens that provoke autoimmune responses, has proved difficult to determine. Recently, several antigens induced on tumor cells by mutagen treatment have been cloned by transfection of genomic DNA libraries into P1.HTR cells, screening for antigen expression using T-cell clones, and subsequent recovery of the integrated DNA by cosmid rescue. We have modified this techniques and have stably transfected P1. HTR cell lines with polyoma T antigen, which allows episomal replication of the shuttle vector, pCDM8. Using pCDM8-CAT constructs, we have determined the frequency of transfection and plasmid copies taken up per cell under optimal transfection conditions. Using a pCDM8 construct which expresses the tumor-specific antigen, P91A (pCDM8-tum-), that is recognized by a T-cell clone, we have found that cells transfected with this antigen can be recognized by the T-cell clone when they are present at only 1%-3% of a mixed population. Progeny of a single cell transfected with pCDM8-tum-: pCDM8-CAT at proportions of 1:10, 1:25, and 1:50 are recognized by the T-cell clone. Furthermore, Hirt extracted plasmid DNA from transfectants expressing the tum- antigen can be amplified in bacteria, transfected back into P1.HTR recipients, and recognized by the T-cell clone. This approach should enable reasonably rapid screening of cDNA libraries for even relatively low abundance messages encoding, for example, minor histocompatibility and allonatigens, and allow their subsequent cloning. Springer-Verlag, 1992 Scott D. Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow, HA1 3UJ, Middlesex, UK aut Dyson P. Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow, HA1 3UJ, Middlesex, UK aut Simpson E. Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow, HA1 3UJ, Middlesex, UK aut Immunogenetics Springer-Verlag 36/2(1992-06-01), 86-94 0093-7711 36:2<86 1992 36 251 https://doi.org/10.1007/BF00215284 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00215284 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Scott D. Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow, HA1 3UJ, Middlesex, UK aut NATIONALLICENCE 700 1- Dyson P. Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow, HA1 3UJ, Middlesex, UK aut NATIONALLICENCE 700 1- Simpson E. Transplantation Biology Section, Clinical Research Centre, Watford Road, Harrow, HA1 3UJ, Middlesex, UK aut NATIONALLICENCE 773 0- Immunogenetics Springer-Verlag 36/2(1992-06-01), 86-94 0093-7711 36:2<86 1992 36 251 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer