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Targeted DNA editing has great potential to cure some genetic diseases; however, the use of artificial nucleases such as CRISPR-Cas9 and TALEN in gene therapy can potentially cause severe side effects due to off-target DNA cleavages. Single-stranded (ss) DNAs and 5'-tailed duplexes (TDs) can achieve target base substitutions when introduced without artificial nucleases into cultured cells and mouse liver. In this study, ss DNA and TD were separately co-introduced into human U2OS cells, together with a target plasmid DNA bearing an inactivated lacZα gene, and the gene correction efficiencies were compared. Unlike the genes examined in previous studies, ss DNA and TD showed similar efficiencies. Therefore, ss DNAs might be as useful as TD for gene correction, depending on the target sequence.

Base sequence conversion in target DNA is achieved when a 5'-tailed duplex (TD) is introduced into cells. In this study, the effects of target DNA cleavage on sequence conversion with a TD were examined. Plasmid DNAs with and without cleavage near the target position were each introduced into HeLa cells, together with the TD. The cleavage promoted the sequence alteration efficiency by ca. 7-fold. These results suggested that the sequence conversion efficiency with the TD fragment is increased when an artificial nuclease introduces cleavage near the target site.

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies. A comprehensive analysis of the transcription of 4'-thioDNA templates to natural RNA is herein reported. The modifications of DNA with 2'-deoxy-4'-thionucleosides, so-called 4'-thioDNA, were found to act as templates for transcription with RNA polymerases both in vitro and in mammalian cells.

The transience of transgene expression is a major obstacle in the development of nonviral vectors. The CpG-free and pLIVE plasmids reportedly achieve long-term transgene expression in mouse liver. In this work, the anti-silencing elements within these plasmids were studied. The effects of plasmid that was being silenced on transgene expression from the CpG-free plasmid and those of transgene expression at early time points on silencing were also examined. The results suggested that the backbone sequence of the CpG-free plasmid and the 3' untranslated region of the albumin gene of the pLIVE plasmid contribute to durable expression. In addition, no influence of the silencing of another plasmid on the duration of CpG-free plasmid expression or of transgene expression at early time points on silencing was detected., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Plasmid DNA was chromatinized with core histones (H2A, H2B, H3, and H4) in vitro and was delivered into mouse liver by hydrodynamics-based administration. Transgene expression from the chromatinized plasmid DNA was more efficient than that from plasmid DNA delivered in the naked form. The use of acetylation-enriched histones isolated from cells treated with a histone deacetylase inhibitor (trichostatin A) seemed to be more effective. These results indicated that chromatinized plasmid DNA is useful for efficient transgene expression in vivo., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Tailed duplex (TD) DNAs, prepared by annealing an oligonucleotide to a several-hundred-base single-stranded (ss) DNA fragment, correct a base-substitution mutation with high efficiency. In the present study, the abilities of TD fragments to correct single-base insertion and deletion mutations were examined, using hygromycin-resistance and enhanced green fluorescent protein fusion (Hyg-EGFP) genes inactivated by +G and -C frameshift mutations. The 5'-TD and 3'-TD DNA fragments were co-transfected with plasmid DNA containing the inactivated Hyg-EGFP gene into CHO-K1 cells, and the gene correction efficiencies were determined by introducing the plasmid DNA recovered from the transfected cells into Escherichia coli cells. In contrast to their efficiencies for the substitution mutation, the gene correction abilities of the TD fragments were relatively low. The correction efficiencies by the TD fragments were apparently higher than that by a ss DNA fragment, one of the DNA fragments employed for gene correction. These results suggest that the TD fragments have the potential to correct frameshift mutations, although further improvement is required.

The intranuclear disposition of plasmid DNA is extremely important for transgene expression. Exogenous histones have been used as carriers of plasmid DNA in histone-mediated gene delivery. In this study, the effects of exogenous histone H3 complexed with plasmid DNA on transgene expression efficiency were examined. The plasmid-histone complexes in various ratios were transfected into HeLa cells by osmotic pressure. Histone H3 suppressed transgene expression in the nucleus in a dose-dependent manner. Our results suggest that the histone-mediated gene delivery is unlikely to be useful, from the viewpoint of the intranuclear disposition., (Copyright 2010 Elsevier B.V. All rights reserved.)

DNA conformation may be an important factor affecting gene transcription. In this study, we examined how DNA sequences with unusual conformations affect transgene expression. A(30) and (CG)(15) sequences that can adopt the B' and Z conformations, respectively, were introduced into a beta-actin promoter. Luciferase plasmids containing the manipulated promoter were transfected into NIH3T3 cells by electroporation and were delivered into mouse livers with a hydrodynamics-based injection. Expression from plasmid with the (CG)(15) sequence was multiple times higher than expression from control plasmid DNA. The A(30) sequence also tended to enhance expression. These results suggest that non-B DNA sequences could improve transgene expression in cells.

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.

In a previous study, we hypothesized that some type 2 diabetes mellitus susceptible genes may be up/down-regulated in white blood cells (WBC) of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, reflecting their up/down-regulation in major insulin-target tissues such as the liver before the onset of diabetes. We identified 57 potential candidate genes for predicting diabetes. In this study, we examined this hypothesis further by extending the experimental conditions from before the onset (6 weeks) to after the onset (24 weeks) of diabetes that type 2 diabetes mellitus susceptible genes are co-regulated in WBC, reflecting their expression in the liver. Using rat oligo DNA microarrays, we found that 48 genes are up/down-regulated in OLETF rats compared to control Long-Evans Tokushima Otsuka (LETO) rats in WBC and liver under fasting or insulin administration conditions. Twenty nine and 33 genes were up/down-regulated in both WBC and livers, respectively, under fasting and insulin administration conditions, respectively. Eight out of 29 genes in fasting condition and 12 out of 33 genes in insulin administration conditions have been reported to be type 2 diabetes mellitus susceptible genes and the remainder have not been reported to be related to type 2 diabetes mellitus. These results support our hypothesis that the expression levels of type 2 diabetes mellitus related genes in WBC are reflective of those in the liver after the onset of diabetes.

Background: Heterogeneity of transgene expression, the presence or absence (below the limit of detection) of transgene expression on a cell-by-cell basis, is a severe disadvantage in the use of cationic lipid-mediated gene vectors for gene therapy and experiments in molecular biology. Understandings of intracellular trafficking and the function (transgene expression) of vectors related to cellular physiology are essential in terms of clarifying the mechanism underlying the heterogeneity., Methods: To distinguish the contribution of nuclear transfer efficiency and subsequent intranuclear transcription efficiency to the overall heterogeneity in transgene expression, a novel imaging system was established for the dual visualization of the nuclear transfer of pDNA and marker gene expression (lacZ) in single cells., Results: The expression of LacZ occurred in only approximately 30% of HeLa cells of the nuclear pDNA-positive cells, indicating that intranuclear transcription efficiency contributed to the heterogeneity. Dual imaging against synchronized cells further revealed that the efficiency of nuclear delivery was comparable irrespective of cell cycle status, which is contrary to the generally accepted hypothesis that nuclear import of pDNA is enhanced during cell division when the nuclear membrane structure is perturbed. The most significant finding in the present study is that nuclear transcription efficiency in terms of the ratio of LacZ-positive cells to nuclear pDNA-positive cells drastically increased in the late S and G2/M phase., Conclusions: This is the first demonstration to show that cell cycle dependent intranuclear transcription appears to be responsible for the overall heterogeneity of transgene expression., (Copyright 2007 John Wiley & Sons, Ltd.)

The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was transfected into cultured cells including HeLa by electroporation, and the amounts of intranuclear plasmid DNA and luciferase were quantitated at various time points. Decrease in expression efficiency from one copy of the exogenous DNA over time occurred as the case of mouse liver, and its degrees varied between cell lines. These results suggest that exogenous DNA is 'silenced' in the cultured cells as well as in mouse hepatocytes.

Cationic lipid-mediated transfer of DNA is promising in gene therapy. However, one disadvantage with this approach is the induction of an inflammatory response, which may decrease transgene expression. Recently, we found that plasmid DNA containing N6-methyladenine (N6-MeA), a bacterium-specific modified base, induced cytokine twice as efficiently as plasmid DNA without N6-MeA, when complexed with cationic lipids. Thus, plasmid DNA without N6-MeA might express a transgene more efficiently than that containing N6-MeA in vivo. To evaluate the effects of adenine methylation on transgene expression in vivo, we injected luciferase-encoding plasmid DNA, complexed with cationic lipids or a cationic polymer, intravenously into mice. When the plasmid DNA-cationic lipid complexes were injected, the luciferase expression from the methylated and unmethylated plasmids was similar, although cytokine was more efficiently elicited by the methylated DNA than the unmethylated DNA. Hydrodynamics-based injections of plasmid DNA-cationic polymer complexes did not induce cytokine, and the luciferase expression from the unmethylated plasmid was slightly lower than that from the methylated plasmid DNA. These results suggest that the presence of N6-MeA did not reduce transgene expression in vivo.

The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8x10(4)- and 4x10(3)-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively. The rate of elimination of luciferase activity upon DNA transfection was smaller than that for the luciferase protein itself (k(el) for DNA transfection

Oligodeoxyribonucleotides containing 8-hydroxyguanine and 2-hydroxyadenine, purine lesions produced in cells by reactive oxygen species, were synthesized and inserted into vector DNAs to introduce each lesion at a predetermined site. The manipulated DNAs were transfected into living cells, and the mutants induced by each DNA lesion were collected and analyzed. In addition, the mutations induced by damaged DNA precursors with the two oxidized purine bases were studied by the use of chemically synthesized nucleoside triphosphates. In this review article, the author summarizes the mutagenic potentials of the two oxidized purine bases, by focusing on experiments examined by the author and his collaborators.

Efficient nuclear entry of exogenous DNA is one of the key factors toward gene therapy success with nonviral vectors. To re-address the effects of a nuclear localization signal (NLS) peptide attached directly to DNA, we prepared three dumbbell-shaped, green fluorescent protein (GFP)-encoding DNAs containing one or two NLS peptides. The peptide was conjugated to the loop-forming oligodeoxyribonucleotides by cross-linking reactions between the peptide and a modified uracil base with a dioxaoctylamino linker, and the oligonucleotides were then ligated to the DNA molecules. The NLS-conjugated DNA dumbbells were microinjected into the cytosols and nuclei of simian COS-7 cells. In addition, unconjugated DNA dumbbells, with or without a modified uracil base, were also examined for comparison. The GFP gene was expressed with efficiencies in the order of the unmodified DNA >or= the NLS-conjugated DNA > the unconjugated DNA with the base modification, with both cytosolic and intranuclear microinjections. Thus, we concluded that (i) one or two NLS peptide(s) did not dramatically improve the nuclear entry of DNA and that (ii) chemical modification of DNA reduced the transcription efficiency or stability in the nucleus.

DNA and DNA precursors (deoxyribonucleotides) suffer damage by reactive oxygen/nitrogen species. They are important mutagens for organisms, due to their endogenous formation. Damaged DNA and nucleotides cause alterations of the genetic information by the mispairing properties of the damaged bases, such as 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) and 2-hydroxyadenine. Here, the author reviews the mutagenic potentials of damaged bases in DNA and of damaged DNA precursors formed by reactive oxygen/nitrogen species, focusing on the results obtained with synthetic oligonucleotides and 2'-deoxyribonucleoside 5'-triphosphates.

To visualize the intracellular trafficking of exogenous DNAs delivered by cationic liposomes, rhodamine-labeled DNAs were transfected into NIH3T3 cells and observed by confocal laser microscopy. After 0.5- to 1-h incubations, the DNAs reached the nucleus with a much higher frequency than that expected from the cell division rate. This result suggests that DNAs can enter the nucleus in the presence of the nuclear membrane. Interestingly, some DNAs appeared to extend through the nuclear membrane in the aggregated form which were much larger than the nuclear pore complex. The DNAs which have passed through the nuclear membrane were stained with SYTO 24, a DNA labeling reagent. The stained part may be "naked" DNA that is free of lipids or proteins. This observation indicates that a complex containing DNA fuses with the nuclear membrane and then naked DNA is released into the nucleus.

Different DNA polymerases misinsert 2-hydroxy-dATP differently, according to a study of the alpha subunit of E. coli DNA polymerase III and the exonuclease-deficient Klenow fragment of DNA polymerase I. The polymerases misinserted the compound opposite T and G rather than T and C and were more likely to insert it opposite T than G.

An oxidatively damaged base, 2-hydroxyadenine (2-OH-Ade), weakly blocked DNA replication during lagging strand synthesis in simian COS-7 cells. The base did not affect the efficiency of leading strand synthesis. The frequency of mutation for 2-OH-Ade in the COS-7 cells were 0.6% - 0.1%, depending on the sequence and strand location. Despite the differences in mutation spectra of 2-OH-Ade due to the variations in sequences and strands, the base caused substitution and deletion mutations in mammalian cells, indicating mutagenicity in eukaryotic as well as in prokaryotic cells.

G:C sites distant from 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) are frequently mutated when the lesion-bearing plasmid DNA is replicated in human cells with reduced Werner syndrome (WRN) protein. To detect the untargeted mutations preferentially, the oxidised guanine base was placed downstream of the reporter supF gene and the plasmid DNA was introduced into WRN-knockdown cells. The total mutant frequency seemed higher in the WRN-knockdown cells as compared to the control cells. Mutation analyses revealed that substitution mutations occurred at the G:C pairs of 5′-GpA-3′/5′-TpC-3′ sites, the preferred sequence for the apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3)-family cytosine deaminases, in the supF gene in both control and knockdown cells. These mutations were observed more frequently at G sites than C sites on the DNA strand where the GO base was originally located. This tendency was promoted by the knockdown of the WRN protein. The present results imply the possible involvement of APOBEC3-family cytosine deaminases in the action-at-a-distance (untargeted) mutations at G:C (or G) sites induced by GO and in cancer initiation by oxidative stress. [ABSTRACT FROM AUTHOR]

Reactive oxygen species generate 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine), which induces G:C→T:A transversion mutations. The knockdowns of the protein responsible for Werner syndrome (WRN), a cancer-associated DNA helicase, and DNA polymerase (pol) λ, a WRN-interacting DNA pol, cause untargeted base-substitution mutations (action-at-a-distance mutations). To examine the consequences of the dual reductions of WRN and pol λ for the mutations caused by GO, siRNAs against both proteins were introduced into human U2OS cells. A replicable plasmid DNA with the oxidised nucleobase in a unique position of the supF gene was then introduced into the double knockdown cells. The amplified DNA recovered from the cells was used to transform a bacterial indicator strain. The mutant frequency and the subsequent sequence analysis revealed that the double knockdown additively promoted the G:C→T:A substitution at the GO position and increased the action-at-a-distance mutations to a level similar to that of the single WRN knockdown. Thus, WRN and DNA pol λ seem to suppress the targeted G:C→T:A mutation at least in part independently and reduce the untargeted mutations via an identical pathway. [ABSTRACT FROM AUTHOR]

The correction of an inactivated hygromycin resistance and enhanced green fluorescent protein (Hyg–EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and anti-sense ssDNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ssDNA fragments. The gene correction efficiency varied (0.8–9.3%), depending on target positions and sense/anti-sense strands. Sense ssDNA fragments corrected the target gene with equal or higher efficiencies as compared to their anti-sense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the anti-sense fragments. These results suggest that the sense ssDNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA. [ABSTRACT FROM PUBLISHER]

Abstract: The intranuclear disposition of exogenous DNA is highly important for the therapeutic effects of the administrated DNA. Naked luciferase-plasmid DNA was delivered into mouse liver by a hydrodynamics-based injection, and the amounts of intranuclear plasmid DNA, luciferase, and its mRNA were quantitated at various time points. Methylation of the promoter of the luciferase gene was also analyzed. Expression efficiency from one copy of the exogenous DNA dramatically decreased over time, and the DNA was methylated and degraded into fragments. Unexpectedly, methylation of the intact plasmid DNA was low and did not increase over time. Rather, the fragmented DNA was methylated more frequently than the intact plasmid. These results suggest that the CpG methylation and the degradation of exogenous DNA, and its ‘silencing’, occurred in parallel in the nucleus. [Copyright &y& Elsevier]

During gene therapy the concentration of plasmid DNA or oligonucleotides in the plasma can be quite different from their concentrations in the nucleus or cytosol where they exert their actions. For a better understanding of the apparent discrepancies between pharmacokinetics (PK) and pharmacodynamics (PD), a new concept for intracellular PK with an emphasis on the final efficacy of gene transcription is needed. Here, the conventional PK and intracellular PK and PD of non-viral gene delivery systems are discussed, together with a new concept, referred to as controlled intracellular disposition, which integrates these factors to gain a better understanding of gene expression in the nucleus. The importance of optimizing the system from a transcriptional point of view in the nucleus is also discussed. These new concepts must be integrated to develop an optimized non-viral gene delivery system. [Copyright &y& Elsevier]

To examine the mutagenicity of 5-formylcytosine (5-fC), an oxidation product of 5-meth-ylcytosine (5-mC), 5-fC was incorporated into predetermined sites of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5'-AFGCGT-3' and 5'-ACGFGT-3' (F represents 5-fC), the recognition site for the restriction enzyme MluI (5'-ACGCGT-3)-5-fC was incorporated into the template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and analyzed after a second transfection into Escherichia coli. 5-fC weakly blocked DNA replication in mammalian cells. The 5-fC residues were mutagenic, with mutation frequencies in double-stranded vectors of 0.03–0.28%. The mutation spectrum of 5-fC was broad, and included targeted (5-fC↑G, 5-fC↑A, and 5-fC↑T) and untargeted mutations. These results suggest that the oxidation of 5-mC results in mutations at and around the modified sites. [ABSTRACT FROM AUTHOR]

Background: Abasic sites are formed spontaneously and by nucleobase chemical modifications and base excision repair. A chemically stable abasic site analog was site-specifically introduced into replicable plasmid DNAs, which were transfected into human U2OS cells. The amplified DNAs were recovered from the cells and used for the transformation of a bacterial indicator strain. Results: Large deletion mutations were induced by the analog, in addition to point mutations at the modified site. No apparent sequence homology at the deletion junctions was found. Conclusion: These results suggested that the large deletions induced by the abasic site analog are formed by homology-independent events. [ABSTRACT FROM AUTHOR]

Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase η that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase t incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase η may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis. [ABSTRACT FROM AUTHOR]

Abstract: A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486–493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5′-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5′-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment (∼600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA. [Copyright &y& Elsevier]

In this paper, we describe the synthesis of the 3′-3′-linked oligonucleotides connected with pentaerythritol composed of asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oligopurine domains on alternate strands was investigated using the electrophoretic mobility shift assay (EMSA) and DNase I footprinting experiment. It was found that the 3′-3′-linked oligonucleotides composed of asymmetrical sequences formed the stable antiparallel triplexes with the DNA targets as compared with the unlinked oligonucleotides. Thus, oligonucleotides linked with pentaerythritol would be useful as antigene oligonucleotides for DNA targets consisting of the alternating oligopyrimidine–oligopurine sequences. [Copyright &y& Elsevier]

Various diseases, including cancer, are caused by genetic mutations. A 5′-tailed duplex (TD) DNA, consisting of a long single-stranded (ss) editor DNA and a short (∼35-base) ss assistant oligodeoxyribonucleotide, can introduce a base-substitution in living cells and thus correct mutated genes. Previously, several hundred-base DNAs were employed as the editor DNAs. In this study, 5′-TDs were prepared from various editor DNAs with different lengths and examined for their gene correction abilities, using plasmid DNA bearing a mutated copepod green fluorescent protein (copGFP) gene, in human cells. High-throughput analysis was performed by the reactivated fluorescence of the wild-type protein encoded by the corrected gene as the indicator. The analysis revealed that 5′-TDs with ∼100-base ss editor DNAs enabled gene editing at least as efficiently as those with longer editor DNAs. Moreover, the antisense strand was more effective as the editor than the sense strand, in contrast to the 5′-TDs with longer editor strands. These results indicated that the 5′-TD fragments with shorter editor strands than those used in previous studies are useful nucleic acids for gene correction. [ABSTRACT FROM AUTHOR]

Abstract: The effects of a genetic insulator (chicken cHS4) on transgene expression in a positive feedback system using the GAL4-VP16 activator and luciferase reporter plasmids were examined. The introduction of cHS4 enhanced luciferase expression in mouse liver, indicating that this system would be useful for efficient transgene expression. [Copyright &y& Elsevier]

An abstract of the article "Silencing of Plasmid DNA after Hydrodynamics-Based Transfection in Mice," by Hiroshi Ochiai, Hideyoshi Harashima and Hiroyuki Kamiya is presented.

An abstract of the article "Various Aspects of the Single-Stranded DNA Fragment-Mediated Gene Correction," by Hiroyuki Tsuchiya, Hideyoshi Harashima, and Hiroyuki Kamiya is presented.

A 606-base single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, corrects a hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene more efficiently than a PCR fragment, which is the conventional type of DNA fragment used in gene correction. Here, a tailed duplex, obtained by annealing an oligonucleotide to the ss DNA fragment, was used in the correction. The tailed duplex may be a good substrate for the RAD51 protein, an important enzyme in homologous recombination, which could be the gene correction pathway. The annealing of the oligonucleotides enhanced the correction efficiency of the Hyg-EGFP gene, especially when annealed in the 3′-region of the ss DNA fragment. Both the length and backbone structure of the oligonucleotides affected the gene correction efficiency. This type of gene correction device was also effective for another target gene, the rpsL gene. The results obtained in this study indicate that tailed duplex DNA fragments are effective nucleic acids for gene correction. [ABSTRACT FROM AUTHOR]

Abstract: Oxidatively damaged DNA precursors (deoxyribonucleotides) are formed by reactive oxygen species. After the damaged DNA precursors are incorporated into DNA, they might be removed by DNA repair enzymes. In this study, to examine whether a nucleotide excision repair enzyme, Escherichia coli UvrABC, could suppress the mutations induced by oxidized deoxyribonucleotides in vivo, oxidized DNA precursors, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate and 2-hydroxy-2′-deoxyadenosine 5′-triphosphate, were introduced into uvrA, uvrB, and uvrC E. coli strains, and mutations in the chromosomal rpoB gene were analyzed. Unexpectedly, these oxidized DNA precursors induced mutations only slightly in the uvrA and uvrB strains. In contrast, effect of the uvrC-deficiency was not observed. Next, mutT, mutT/uvrA, and mutT/uvrB E. coli strains were treated with H2O2, and the rpoB mutant frequencies were calculated. The frequency of the H2O2-induced mutations was increased in all of the strains tested; however, the increase was three- to four-fold lower in the mutT/uvrA and mutT/uvrB strains than in the mutT strain. Thus, UvrA and UvrB are involved in the enhancement, but not in the suppression, of the mutations induced by these oxidized deoxyribonucleotides. These results suggest a novel role for UvrA and UvrB in the processing of oxidative damage. [Copyright &y& Elsevier]

Abstract: Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N6-methyladenine (N6-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N6-MeA induced cytokines when injected into mice. Co-injection of N6-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N6-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N6-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine. [Copyright &y& Elsevier]