Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells

Abstract

We have investigated the use of single-stranded oligodeoxynucleotides (ssODN) to produce specific single-base alterations in episomal and chromosomal DNA in mouse embryonic stem (ES) cells. Two different reporter genes, EGFP and LacZ, each with a single point mutation that inactivates reporter activity, were used. ssODN homologous to the target sequence, except for a single mismatch at the mutant base, were used to correct the mutant reporter genes. When tested in CHO-K1 cells, the ssODN showed correction rates of 0.5-1.0%, consistent with prior reports. ssODN in the antisense orientation provided higher rates of gene conversion than those in the sense orientation for both reporter genes. Nuclear extracts from mouse ES cells exhibited nearly the same correction activity as extracts from CHO-K1 cells. ssODN corrected the mutant bases of both episomal and chromosomal mutant reporter genes in mouse ES cells. Although the efficiency of gene correction observed in ES cells is low, approximately 10 À4, these results demonstrate that ssODN can produce single-base alterations in the genomic DNA of mouse ES cells. As conversion efficiency is improved by the continued development of oligonucleotide structure and DNA delivery methods, ssODN could be used to produce ES cells with specific mutations in any gene in a single step. The targeted ES cells could in turn be used to create accurate mouse models of inherited diseases.

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

“…8,9 Unfortunately, targeting frequencies in mouse ESCs range from 10 À7 to 10 À4 , which is relatively low compared with those found in other cell types. [10][11][12][13][14] A better understanding of the reaction parameters governing ssODN-mediated gene targeting in ESCs 15,16 is imperative to improve the applicability of the technique. Although different DNA repair pathways may impinge on the targeting process, we will particularly address the role of the DNA mismatch repair (MMR) system that imposes a strong barrier to effective application of ssODN-mediated gene targeting.…”

Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells

“…All of the developed systems have provided clear evidences of gene correction by ODN in the episome and chromosome of many mammalian cells, including CHO-K1, 31 primary human keratinocytes, 38 melanocytes 37 and mouse ES cells. 39,40 As a first step toward gene targeting, several mouse MSC lines with defective selectable marker genes mimicking Gene modification by ODNs in MSCs K Flagler et al endogenous targets have been established to provide more than one system for testing. Analysis of clonal subpopulations of MSCs with transduced Puro r -Neo s cassette showed that all generated clones were capable of undergoing differentiation which is highly indicative of the pluripotent, progenitor characteristics of generated stable lines.…”

“…Our clonal experiments in ES cells also showed that the number of LacZ-corrected cells at 48 h after transfection (1 out of 5 Â 10 4 ) was similar to the number of viable LacZ-positive colonies obtained by limiting dilution meaning that most of the mutant LacZ-corrected ES cells expanded to make viable colonies. 39 Very recently, we showed that targeting ESNeo s cells with a correcting ODN resulted in about 50-100 Neo r clones from the initial 1 Â 10 6 ES cells. 40 Detection of EGFP fluorescence, a priori, requires that correction has taken place and been completed so that transcription and translation of the corrected gene product were detectable.…”

“…In the past 8 years, we have made significant progress in our understanding of the mechanism responsible for gene alteration by ODNs [32][33][34][35][36] and provided clear evidence that this approach can be used to introduce permanent site-specific changes into episomal and chromosomal DNA of somatic cells 31,37,38 and embryonic stem (ES) cells. 39,40 The goal of the present study was to assess the use of ODN-directed gene repair in MSCs. To achieve this goal, first, we developed the procedure for isolation of adult murine MSCs and demonstrated that established cultures represent morphologically homogenous population of cells with phenotypic and functional features of mesenchymal progenitors.…”

A LacZ-based transgenic mouse for detection of somatic gene repair events in vivo

“…This low level of gene repair probably reflects the low transfection efficiency associated with ES cells (approximately 1-5%) and previous work has shown ES cells to have very low levels of gene repair activity. 5,21 In addition, ES cells have an intrinsically lower rate of mutation frequency compared to somatic cells, which might also influence gene repair frequencies. 22 The lack of detectable gene repair events in these mice is probably due to the inefficiency of gene delivery in vivo.…”