V(D)J recombinase activity in primary and secondary murine lymphoid organs: assessment by a PCR assay with extrachromosomal plasmids.
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 DQ52 and JH2 regions of the murine IgH gene, or of the D beta 2-1 and J beta 2.6 regions of the murine TCR beta 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 Camr assay that we prepared with the DQ52 and JH2 regions of the murine IgH 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 Camr 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.
Abe
M
M
Department of Cell Biology, Radiation Effects Research Foundation, Nagasaki, Japan.
Hayashida
K
K
Takayama
K
K
Shiku
H
H
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000032:analysis*; D004274:DNA, Recombinant / Q000235:genetics; D004268:DNA-Binding Proteins; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D018398:Homeodomain Proteins; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics*; D019426:Integrases; D008221:Lymphoid Tissue / Q000201:enzymology*; D051379:Mice; D009687:Nuclear Proteins; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000235:genetics; D045522:Recombinases; D011995:Recombination, Genetic; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012680:Sensitivity and Specificity; D014162:Transfection / Q000235:genetics
D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007136:Immunoglobulins; D009687:Nuclear Proteins; D011506:Proteins; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; D045522:Recombinases; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C115006:integron integrase IntI1