The splinker ligation technique increases the yield of homologous recombinant ES cell clones
| . | No. of colonies/plate . | Enrichment factor . | No. of homologous recombinant clones/ no. of clones analyzed (%) . | |
|---|---|---|---|---|
| G418 . | G418 plus ganciclovir . | |||
| Nonsplinked | 820 | 360 | 2.3 | 2/25 (8) |
| Splinked | 560 | 80 | 7 | 22/42 (28) |
| . | No. of colonies/plate . | Enrichment factor . | No. of homologous recombinant clones/ no. of clones analyzed (%) . | |
|---|---|---|---|---|
| G418 . | G418 plus ganciclovir . | |||
| Nonsplinked | 820 | 360 | 2.3 | 2/25 (8) |
| Splinked | 560 | 80 | 7 | 22/42 (28) |
The targeting construct was linearized with NotI and either transfected without further treatment (nonsplinked) or after ligation to NotI-compatible splinkers (splinked). The cells were then transfected, and colonies were selected in the presence of G418 with or without negative selection by ganciclovir. The enrichment factor was calculated by dividing the number of colonies obtained by G418 selection over the number of colonies obtained by selection in G418 plus ganciclovir. Finally, surviving clones were isolated and analyzed by Southern blot analysis for homologous recombination.