| Abstrakt: |
We developed a highly specific and sensitive polymerase chain reaction (PCR) assay to measure V(D)J recombinase activity using extrachromosomal plasmids and PCR. Extrachromosomal plasmids were prepared by eukaryotic replication origin, and a combination of the D52 and J2 regions of the murine lgH gene, or of the D2–1 and J2.6 regions of the murine TCRβ gene, both with recombination signal sequences. Plasmids, transfected into cells to be examined and recovered after 48 h, were processed to detect recombined molecules by PCR with primers for the expected sequences produced by the precise signal joint. The PCR assay, when compared with a Cam assay that we prepared with the D52 and J2 regions of the murine lH gene, seems to have the following advantages. It detects only the recombined products produced by V(D)J recombinase activity and is therefore highly specific. It detects V(D)J recombinase activity in cells, including those with low replication frequency, which our Cam assay failed to do. This also enables detection of the recombinase activity not only in murine cell lines, but also in cells of murine lymphoid organs. The assay detects V(D)J recombinase activity in cell lines of human origin by replacing the eukaryotic replication origin of plasmids. High V(D)J recombinase activity was detected in bone marrow cells followed by thymic cells, and apparently lower activity was detected in cells of the lymph node and spleen of normal mice. [ABSTRACT FROM PUBLISHER] |
