Increased efficiency of oligonucleotide-mediated gene repair through slowing replication fork progression

Abstract

Targeted gene modification mediated by single-stranded oligonucleotides (SSOs) holds great potential for widespread use in a number of biological and biomedical fields, including functional genomics and gene therapy. By using this approach, specific genetic changes have been created in a number of prokaryotic and eukaryotic systems. In mammalian cells, the precise mechanism of SSO-mediated chromosome alteration remains to be established, and there have been problems in obtaining reproducible targeting efficiencies. It has previously been suggested that the chromatin structure, which changes throughout the cell cycle, may be a key factor underlying these variations in efficiency. This hypothesis prompted us to systematically investigate SSO-mediated gene repair at various phases of the cell cycle in a mammalian cell line. We found that the efficiency of SSO-mediated gene repair was elevated by approximately 10-fold in thymidine-treated S-phase cells. The increase in repair frequency correlated positively with the duration of SSO/thymidine coincubation with host cells after transfection. We supply evidence suggesting that these increased repair frequencies arise from a thymidine-induced slowdown of replication fork progression. Our studies provide fresh insight into the mechanism of SSO-mediated gene repair in mammalian cells and demonstrate how its efficiency may be reliably and substantially increased.

Fig. 1.

Illustration for SSO-mediated gene repair in reporter system. (A) In the WT EGFP reporter gene, the initiation codon of ATG contributes to the proper translation. The conversion from “A” in the WT to “T” in the mutant type of EGFP gene inactivates the initiation codon (ATG). Translation of the EGFP gene can occur only if the initiation codon is restored. An upstream ACCGGT in WT to GAATTC in mutant type converts an AgeI restriction site to an EcoRI. This alteration is included to identify the repaired EGFP gene from the WT EGFP. (B) SSO sequences. E6 is designed to be complementary to the untranscribed strand except a”T–T“mismatch, and RE6 is designed to be complementary to the transcribed strand except an”A–A“mismatch. EC6 is a control SSO that has the totally complementary sequence to the untranscribed strand of the mEGFP gene. The underlined residues indicate the target site. All SSOs have six phosphorothioate modifications at both terminals. (C) Sequence analysis of the EGFP gene isolated from the repaired clones, revealing a targeted gene correction (”A“in the initiation codon) and a new ATG-EcoRI haplotype, which indicates a successful repair event. WT contamination would have an ATG–AgeI combination.

Fig. 2.

Evaluation of SSO-mediated repair frequency during different phases of the cell cycle. The cells were sustained in specific phases by corresponding inhibitors for 8 h after SSO transfection, then released by changing medium and detected in 2 days. The x-axis indicates the different phases in which cells were synchronized. Unsynchronized cells were treated at the same time as control (n = 3).

Fig. 3.

Repair efficiency of E6 in the conditions with or without thymidine. (A) We incubated 2 mM of thymidine in medium 2 h before and 48 h after SSO transfection. The x-axis indicates the SSO/thymidine incubation time. The y-axis indicates the frequency of repaired cells. Compared with the control graph, where no thymidine was added before or after SSO transfection, the thymidine-administered group represents obviously higher frequency (P < 0.01, n = 5). (B) Repaired cells detected under UV light. (Left) Without thymidine, there is only one green cell in several eye fields. (Right) With thymidine there are about six green cells in almost every eye field. (C) FACS results comparing frequencies of SSO-mediated repair 48 h after transfection by E6 without (1) or with (2) thymidine incubation.

Fig. 4.

Effects of different types of S-phase inhibitors on the repair efficiency. Three different inhibitors were kept in the medium 2 h before and 48 h after transfection with E6. The x-axis indicates the SSO/inhibitor incubation time after transfection; the y-axis indicates the frequency of repaired cells (n = 3).

Fig. 5.

Efficiencies of E6-SSO in different concentration of thymidine. Thymidine was kept in the medium 2 h before and 48 h after transfection with E6. The x-axis indicates the thymidine concentration used; the y-axis indicates the frequency of repaired cells (n = 3).

Fig. 6.

Effect of preinhibition time on the SSO-mediated repair efficiency. Preinhibition time refers to the duration of thymidine incubation time for cells before transfection. The x-axis indicates the duration of pretransfection thymidine coincubation. Cells continued to incubate with thymidine/E6 for 12 h after transfection in one group and for 48 h for the other group. Results showed that frequencies of both groups decreased gradually as the duration of preinhibition increased from 6 to 24 h (n = 3).