TABLE 1.
Gene targeting in ES cells with diverged repair substrates
| Transfected plasmid(s) | Frequency of neo+ coloniesa
|
||||
|---|---|---|---|---|---|
| Expt 1 (% WT)b | Expt 2 (% WT) | Expt 3 (% WT) | Expt 4 (% WT) | Expt 5 (% WT) | |
| pneo-WT + pPGK3xnlsI-SceI | 1.40 × 10−4 (100) | 1.44 × 10−4 (100) | 1.15 × 10−4 (100) | 4.69 × 10−4 (100) | 4.63 × 10−4 (100) |
| pneo-5mu + pPGK3xnlsI-SceI | 0.39 × 10−4 (28) | 0.71 × 10−4 (49) | 0.61 × 10−4 (53) | 1.77 × 10−4 (38) | 1.90 × 10−4 (41) |
| pneo-7mu + pPGK3xnlsI-SceI | 0.28 × 10−4 (20) | 0.58 × 10−4 (40) | 0.33 × 10−4 (29) | 1.14 × 10−4 (24) | 1.22 × 10−4 (26) |
| pneo-8mu + pPGK3xnlsI-SceI | 0.18 × 10−4 (13) | 0.27 × 10−4 (19) | 0.27 × 10−4 (23) | 0.72 × 10−4 (15) | 0.73 × 10−4 (16) |
| pneo-WT + pPGKlacZ | <1 × 10−7 | NDc | ND | <1 × 10−7 | ND |
| pPGK3xnlsI-SceI | <1 × 10−7 | ND | ND | <1 × 10−7 | ND |
| pMC1neod | 5.4 × 10−4 | ND | 4.8 × 10−4 | 7.8 × 10−4 | 1.4 × 10−3 |
a
The frequency of neo+ colonies was determined by dividing the number of neo+ colonies obtained by the number of electroporated cells (2 × 107), correcting for 50% viability of cells generally observed following electroporation under these conditions.
b
% WT, percent frequency of neo+ colonies derived from transfection of the diverged pneo repair substrate with pPGK3xnlsI-SceI divided by the frequency of neo+ colonies derived from transfection of pneo-WT with pPGK3xnlsI-SceI.
c
ND, not determined.
d
Total number of colonies from pMC1neo transfection were estimated by plating dilutions of electroporated cells (1:6 to 1:60).