V(D)J recombination assays in human primary dermal fibroblasts
. | Positive control . | -RAG2 . | -ART . | +ART . | +ARM27 (H35D) . | +ARM32 (M1T) . | +ARM29 (aa 8-692) . |
|---|---|---|---|---|---|---|---|
| ARTEMIS-positive fibroblasts | |||||||
| Experiment 1 | 54.09 | 0.02 | 1.71 | 1.99 | |||
| Experiment 2 | 65.52 | 0.08 | 2.61 | 2.51 | |||
| Experiment 3 | 67.43 | 0.06 | 2.48 | 2.84 | |||
| Omenn syndrome fibroblasts | |||||||
| Experiment 1 | 61.97 | 0.05 | 0.66 | 5.07 | |||
| Experiment 2 | ND | ND | ND | ND | |||
| Experiment 3 | 70.88/72.47* | 0.02/0.04* | 0.68/0.50* | 5.43/5.10* | |||
| ARTEMIS-negative fibroblasts | |||||||
| Experiment 1 | 54.15 | 0.05 | 0.01 | 5.66 | 0.14 | 2.09 | 0.04 |
| Experiment 2 | 64.35 | 0.06 | 0.06 | 4.77 | 0.07 | 2.30 | 0.10 |
| Experiment 3 | 65.22 | 0.05 | 0.02 | 6.50 | 0.05 | 2.65 | 0.09 |
. | Positive control . | -RAG2 . | -ART . | +ART . | +ARM27 (H35D) . | +ARM32 (M1T) . | +ARM29 (aa 8-692) . |
|---|---|---|---|---|---|---|---|
| ARTEMIS-positive fibroblasts | |||||||
| Experiment 1 | 54.09 | 0.02 | 1.71 | 1.99 | |||
| Experiment 2 | 65.52 | 0.08 | 2.61 | 2.51 | |||
| Experiment 3 | 67.43 | 0.06 | 2.48 | 2.84 | |||
| Omenn syndrome fibroblasts | |||||||
| Experiment 1 | 61.97 | 0.05 | 0.66 | 5.07 | |||
| Experiment 2 | ND | ND | ND | ND | |||
| Experiment 3 | 70.88/72.47* | 0.02/0.04* | 0.68/0.50* | 5.43/5.10* | |||
| ARTEMIS-negative fibroblasts | |||||||
| Experiment 1 | 54.15 | 0.05 | 0.01 | 5.66 | 0.14 | 2.09 | 0.04 |
| Experiment 2 | 64.35 | 0.06 | 0.06 | 4.77 | 0.07 | 2.30 | 0.10 |
| Experiment 3 | 65.22 | 0.05 | 0.02 | 6.50 | 0.05 | 2.65 | 0.09 |
ARTEMIS-positive fibroblasts, ARTEMIS-negative fibroblasts, and Omenn syndrome fibroblasts were tested in 3 independent experiments. Human primary dermal fibroblasts were cotransfected with V(D)J recombination substrate vector and expression plasmids coding for RAG1, RAG2, wild-type ARTEMIS (+ART), or mutant ARTEMIS proteins (+ARM27, 32, 29), respectively. As controls the same cotransfections without the expression plasmids coding for RAG2 (-RAG2) or wild-type ARTEMIS (-ART) were performed. As a positive control, a pre-recombined substrate vector was transfected. In each assay 5 × 104 fibroblasts were analysed by FACS. The percentages of recombination-positive cells out of the subpopulation of transfected fibroblasts are depicted.
ND indicates not determined.
Two independent values were ascertained