Improvement of SSO-mediated gene repair efficiency by nonspecific oligonucleotides.

Targeted gene repair mediated by single-stranded DNA oligonucleotides (SSOs) is a promising method to correct the mutant gene precisely in prokaryotic and eukaryotic systems. We used a HeLa cell line, which was stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome, to test the efficiency of SSO-mediated gene repair. We found that the mEGFP gene was successfully repaired by specific SSOs, but the efficiency was only approximately 0.1%. Then we synthesized a series of nonspecific oligonucleotides, which were single-stranded DNA with different lengths and no significant similarity with the SSOs. We found the efficiency of SSO-mediated gene repair was increased by 6-fold in nonspecific oligonucleotides-treated cells. And this improvement in repair frequency correlated with the doses of the nonspecific oligonucleotides, instead of the lengths. Our evidence suggested that this increased repair efficiency was achieved by the transient alterations of the cellular proteome. We also found the obvious strand bias that antisense SSOs were much more effective than sense SSOs in the repair experiments with nonspecific oligonucleotides. These results provide a fresh clue into the mechanism of SSO-mediated targeted gene repair in mammalian cells.

[Transfection and expression of hRI gene on human umbilical blood stem cells and gene therapy for mouse melanoma].

In order to explore the transfection and expression of hRI gene on human umbilical blood stem cells, and observe it's effect on the tumor growth. After enriching human umbilical cord blood CD34+ cells with a high-gradient magnetic cell sorting system (MACS), transfected them with supernatant of retrovirus containing human Ribonuclease inhibitor (hRI) cDNA. Hematopoietic progenitor clonogenic assay and PCR were used to evaluate transfection efficiency, and Western-blot and immune fluorescence were used to evaluate the expression quantity of hRI gene after transfection. Observe the effect of RI on the growth of melonoma in B16C57BL mice. The results showed that human umbilical blood CD34+ cells were highly purified by MACS, which made the purity of human umbilical blood CD34+ cells average 96.15%. hRI can be transfected on umbilical blood CD34+ cells, and the transfection efficiency was 35%. The positive expression of hRI gene on transfected CD34+ cells is identified by Western-blot and immune fluorescence assay. Mice injected with transfected CD34+ cells show a significant restraint of the tumor growth, a lower efficiency of tumor formation, a lower weight of the tumor and a longer incubation period of tumor formation with respect to the control groups. The results demonstrated the capacity of RI to inhibited the tumor growth by blocking the vasculature in tumor.

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

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.