Table 1.
Summary of screening of ES clones electroporated with the Tec/Rlk-modified BAC
| Antibiotic selection | Number of clones | Positives by PCR |
Positives by FISH (%) |
Positives by real-time PCR (%) |
|||
|---|---|---|---|---|---|---|---|
| neo (Tec) | bsd (Rlk) | 2 spots | 3 spots | Tec (±) | Rlk (±) | ||
| G418 | 25/1 plate | 25 | 2 | – | – | 5 (20) | 0 |
| Blasticidin | 23/1 plate | 1 | 23 | – | – | 0 | 3 (13) |
| G418+blasticidin | 11/10 plates | 11 | 10 | 4 (36) | 7 | 3 (27) | 1 (9) |
ES cells were electroporated with BAC RPCI-23 65-18 modified by recombineering and selected in G418 (one plate), blasticidin (one plate) or G418 and blasticidin (10 plates). Clones were screened by FISH, PCR for the presence of the neo and bsd markers and q-PCR (examining the copy number of the targeted Tec and Rlk exons, as well as the neo and bsd resistance markers). DNA from WT ES cells (containing two copies of the deleted Tec and Rlk region) was used as a calibrator and from ES cells that were either targeted in the same position in Tec and Rlk locus by conventional constructs used as both calibrators and positive controls. Only three clones had disrupted Tec and one clone (indicated in red) had disrupted both genes, although four clones appeared to be positive when screened by FISH.