Analysis of Hirt and genomic DNA isolated from SB transposon-nucleofected human primary T cells and clones
. | Total colonies . | No. of colonies analyzed . | pT2/DsRed . | pUbSB10 . | Unknown . |
|---|---|---|---|---|---|
| Hirt DNA | |||||
| Donor 1: pT2/DsRed, day 1 | 3.7 × 107* | 20 | 19 | 0 | 1 |
| Donor 1: pT2/DsRed + pUbSB10, day 1 | 6.7 × 107* | 20 | 8 | 12 | 0 |
| Donor 1: pT2/DsRed, day 7 | 2 × 105* | 20 | 18 | 0 | 2 |
| Donor 1: pT2/DsRed + pUbSB10, day 7 | 1 × 106* | 20 | 9 | 11 | 0 |
| Donor 1: pT2/DsRed + pUbSB10, sorted, more than 50 days | 0† | — | — | — | — |
| Donor 2: pT2/DsRed + pUbSB10, sorted, more than 50 days | 0† | — | — | — | — |
| T-cell clones | |||||
| NO71 | 0† | — | — | — | — |
| L6 | 2† | 2 | 0 | 1 | 1 |
| L22 | 2† | 0 | 0 | 0 | 2 |
| O9 | 4† | 0 | 0 | 0 | 4 |
| O21 | 19† | 0 | 0 | 0 | 19 |
| O56 | 1† | 0 | 0 | 0 | 1 |
| O69 | 4† | 0 | 0 | 1 | 3 |
| pUC19 control | 7.5 × 1010 | — | — | — | — |
| Genomic DNA | |||||
| Donor 1: pT2/DsRed, day 1 | 3.8 × 108‡ | 20 | 20 | 0 | 0 |
| Donor 1: pT2/DsRed + pUbSB10, day 1 | 3.4 × 108‡ | 20 | 11 | 9 | 0 |
| Donor 1: pT2/DsRed, day 7 | 2.5 × 105‡ | 20 | 20 | 0 | 0 |
| Donor 1: pT2/DsRed + pUbSB10, day 7 | 1.2 × 106‡ | 20 | 5 | 15 | 0 |
| Donor 1: pT2/DsRed + pUbSB10, sorted, more than 50 days | 0§ | — | — | — | — |
| Donor 2: pT2/DsRed + pUbSB10, sorted, more than 50 days | 0§ | — | — | — | — |
| T-cell clones | |||||
| NO71 | 1§ | 1 | 1 | 0 | 0 |
| L6 | 0§ | — | — | — | — |
| L22 | 0§ | — | — | — | — |
| O9 | 0§ | — | — | — | — |
| O21 | 0§ | — | — | — | — |
| O56 | 0§ | — | — | — | — |
| O69 | 0§ | — | — | — | — |
| pUC19 control | 1 × 1011 | — | — | — | — |
. | Total colonies . | No. of colonies analyzed . | pT2/DsRed . | pUbSB10 . | Unknown . |
|---|---|---|---|---|---|
| Hirt DNA | |||||
| Donor 1: pT2/DsRed, day 1 | 3.7 × 107* | 20 | 19 | 0 | 1 |
| Donor 1: pT2/DsRed + pUbSB10, day 1 | 6.7 × 107* | 20 | 8 | 12 | 0 |
| Donor 1: pT2/DsRed, day 7 | 2 × 105* | 20 | 18 | 0 | 2 |
| Donor 1: pT2/DsRed + pUbSB10, day 7 | 1 × 106* | 20 | 9 | 11 | 0 |
| Donor 1: pT2/DsRed + pUbSB10, sorted, more than 50 days | 0† | — | — | — | — |
| Donor 2: pT2/DsRed + pUbSB10, sorted, more than 50 days | 0† | — | — | — | — |
| T-cell clones | |||||
| NO71 | 0† | — | — | — | — |
| L6 | 2† | 2 | 0 | 1 | 1 |
| L22 | 2† | 0 | 0 | 0 | 2 |
| O9 | 4† | 0 | 0 | 0 | 4 |
| O21 | 19† | 0 | 0 | 0 | 19 |
| O56 | 1† | 0 | 0 | 0 | 1 |
| O69 | 4† | 0 | 0 | 1 | 3 |
| pUC19 control | 7.5 × 1010 | — | — | — | — |
| Genomic DNA | |||||
| Donor 1: pT2/DsRed, day 1 | 3.8 × 108‡ | 20 | 20 | 0 | 0 |
| Donor 1: pT2/DsRed + pUbSB10, day 1 | 3.4 × 108‡ | 20 | 11 | 9 | 0 |
| Donor 1: pT2/DsRed, day 7 | 2.5 × 105‡ | 20 | 20 | 0 | 0 |
| Donor 1: pT2/DsRed + pUbSB10, day 7 | 1.2 × 106‡ | 20 | 5 | 15 | 0 |
| Donor 1: pT2/DsRed + pUbSB10, sorted, more than 50 days | 0§ | — | — | — | — |
| Donor 2: pT2/DsRed + pUbSB10, sorted, more than 50 days | 0§ | — | — | — | — |
| T-cell clones | |||||
| NO71 | 1§ | 1 | 1 | 0 | 0 |
| L6 | 0§ | — | — | — | — |
| L22 | 0§ | — | — | — | — |
| O9 | 0§ | — | — | — | — |
| O21 | 0§ | — | — | — | — |
| O56 | 0§ | — | — | — | — |
| O69 | 0§ | — | — | — | — |
| pUC19 control | 1 × 1011 | — | — | — | — |
Hirt and genomic DNA were isolated as described in “Materials and methods” from transiently nucleofected PBLs on days 1 and 7, and from stably transposed bulk T cells as well as T-cell clones. Freshly isolated PBLs (120 × 106 cells) were nucleofected with pT2/DsRed (5 × 106 cells per 5 μg) or pT2/DsRed plus pUbSB10 (5 μg each) and incubated with human T-cell medium for 24 hours at 37°C. After FACS analysis to confirm DsRed expression, half of the transfected PBLs were washed once with PBS and used to isolate Hirt and genomic DNA, and the other half of the cells were cocultured with anti-CD3/28 beads for an additional 3 days at 37°C. Stable DsRed+ (> 95%) bulk T cells also were established using FACS sorting from 2 donors' PBLs 4 weeks after nucleofection with pT2/DsRed plus pUBSB10. The Wilcoxon rank sum test was used to analyze the difference between control (* or †) and experimental groups (‡ or §) (all P < .01).
— indicates not done.