| Popis: |
The pathway of targeted gene repair may consist of two major steps, the pairing of a short modified single-stranded oligonucleotide with the target site followed by a repair event that reverses a point mutation. In this study, we show that the level of correction is influenced by the position of the mismatched base pair at the target site relative to the oligonucleotide. Vectors were designed to target an eGFP gene containing a single point mutation and integrated stability into DLD-1 cells. 72-mers are introduced into these cells by electroporation and expression of the corrected eGFP mutation is detected by FACS analyses. Oligonucleotides with a number of structural variants were designed to target the mutant eGFP gene. Appreciable levels of gene repair occur when the oligonucleotide contains the base that creates the mismatch with the target site in the center, whereas the efficiency of correction decreases less than two fold when the mismatch is located near the 3′ end of the oligonucleotide. However, correction directed by an oligonucleotide which creates a mismatch near its 5′ end is reduced up to 17 fold, depending on the vector design. The same directionality is observed even when these positioned mismatches are separated by 18 bases of sequence heterology. These results lead us to propose a directional specific repair pathway consisting of proteins involved in both homologous recombination and mismatch repair. Thus, the pairing stability of the repair complex likely plays a role in the successful application of this technique and these types of factors must be taken into consideration when designing protocols for targeted gene repair. |
