Recovery of cell cycle delay following targeted gene repair by oligonucleotides

Abstract

We have previously shown that activation of the homologous recombinational repair pathway leads to a block of cell division in corrected cells, possibly through the activity of checkpoint proteins Chk1 and Chk2. In this study, we examine the long term impact of this stalling on the growth of cells that have enabled gene repair events. Using a mutated eGFP gene as an episomal reporter, we show that corrected (eGFP positive) cells contain only a few active replication templates two weeks after electroporation, yet do not display an apoptotic or senescent phenotype. By six weeks after electroporation, cells resume active replication with a cell cycle profile that is comparable to that of the non-corrected (eGFP negative) population. These results indicate that the initial stalling is transient and eGFP positive cells eventually resume a normal phenotypic growth pattern, allowing for passaging and expansion in vitro.

DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides

“…4B and suggest an ODN-independent single-strand break activity since the control (No ODN) exhibits the same pattern described above. However, it is pertinent to consider that cells treated with ODN experience a significant delay in replication; a process that can generate transient single-stranded breaks [13, 16, 18, 34, 35]. Therefore, the demonstrated lag in replication in ODN-treated cells, as seen with both the BrdU incorporation assay (Figure 4C) and cell counts after recovery (data not shown), implicate that ODN treatment is responsible for some degree of single-stranded break occurrence independent of normal cellular replication.…”

Progress and Prospects: targeted gene alteration (TGA)

“…Some of these problems were best characterized by Olsen et al 12 First, as reported by many labs, 17,21,22 little correction activity occurs in the G1 phase of the cell cycle. Second, it is now clear that proteins involved in homologous recombination participate in TGA because transcription itself enhances homologous recombination.…”

“…This issue was examined in greater detail however, and it was found that corrected cells regain replicative activity fully. 22 During this lag period, the non-corrected cells advance through several rounds of replication and cell divisions initially 'outgrowing' the corrected population. As a result the correction frequency appears to diminish until the replication potential of corrected cells is restored.…”

Combinatorial gene editing in mammalian cells using ssODNs and TALENs

“…The frequency with which these events occur is highly dependent on the introduction of a large amount of ssODN into the target cell, a type of mass action effect45. The frequency of the process, known as gene editing, can be enhanced by the pre-treatment with reagents or drugs that induce double strand (ds) DNA breakage678.…”