Oxidative stress accelerates intestinal tumorigenesis by enhancing 8-oxoguanine-mediated mutagenesis in MUTYH-deficient mice.

Oxidative stress-induced DNA damage and its repair systems are related to cancer etiology; however, the molecular basis triggering tumorigenesis is not well understood. Here, we aimed to explore the causal relationship between oxidative stress, somatic mutations in pre-tumor-initiated normal tissues, and tumor incidence in the small intestines of MUTYH-proficient and MUTYH-deficient mice. MUTYH is a base excision repair enzyme associated with human colorectal cancer. Mice were administered different concentrations of potassium bromate (KBrO3; an oxidizing agent)-containing water for 4 wk for mutagenesis studies or 16 wk for tumorigenesis studies. All Mutyh -/- mice treated with >0.1% KBrO3 developed multiple tumors, and the average tumor number increased dose dependently. Somatic mutation analysis of Mutyh -/-/rpsL transgenic mice revealed that G:C  > T:A transversion was the only mutation type correlated positively with KBrO3 dose and tumor incidence. These mutations preferentially occurred at 5'G in GG and GAA sequences in rpsL This characteristic mutation pattern was also observed in the genomic region of Mutyh -/- tumors using whole-exome sequencing. It closely corresponded to signature 18 and SBS36, typically caused by 8-oxo-guanine (8-oxoG). 8-oxoG-induced mutations were sequence context dependent, yielding a biased amino acid change leading to missense and stop-gain mutations. These mutations frequently occurred in critical amino acid codons of known cancer drivers, Apc or Ctnnb1, known for activating Wnt signal pathway. Our results indicate that oxidative stress contributes to increased tumor incidence by elevating the likelihood of gaining driver mutations by increasing 8-oxoG-mediated mutagenesis, particularly under MUTYH-deficient conditions.

ROS control in human iPS cells reveals early events in spontaneous carcinogenesis.

Reactive oxygen species (ROS) generated during cellular respiration oxidize various cellular constituents, which cause carcinogenesis. Because most studies on the role of ROS in carcinogenesis have mainly been performed using tumor-derived cell lines, which harbor various types of mutation, it has been difficult to determine the molecular details that lead to cancer formation. To overcome this difficulty, we established human-induced pluripotent stem cell lines in which the intracellular ROS levels are controlled at various differentiation stages by manipulating the ROS-yielding mitochondria. By introducing a specific amino acid substitution (I69E) into the succinate dehydrogenase complex, subunit C protein, a component of mitochondrial respiratory chain complex II, the ROS level increased considerably. When ROS-overproducing cells at the early stage of endoderm differentiation were subcutaneously inoculated into the backs of nude mice, we observed tumor formation. These tumor-initiating cells were subjected to a comprehensive analysis by RNA sequencing. It was revealed that tumor-initiating cells showed 27 upregulated transcripts compared with control cells. The newly identified genes include those coding for PAX8 and FOSB (transcription factors) as well as FGF22, whose expressions are known to increase in developing embryos. These results suggest that these genes may play a pivotal role in cancer formation at the very early stages of cell differentiation.

Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing.

Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI+ phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome.

Celecoxib and 2,5-dimethylcelecoxib inhibit intestinal cancer growth by suppressing the Wnt/ß-catenin signaling pathway.

We previously reported that celecoxib, a selective COX-2 inhibitor, strongly inhibited human colon cancer cell proliferation by suppressing the Wnt/ß-catenin signaling pathway. 2,5-Dimethylcelecoxib (DM-celecoxib), a celecoxib analog that does not inhibit COX-2, has also been reported to have an antitumor effect. In the present study, we elucidated whether DM-celecoxib inhibits intestinal cancer growth, and its underlying mechanism of action. First, we compared the effect of DM-celecoxib with that of celecoxib on the human colon cancer cell lines HCT-116 and DLD-1. 2,5-Dimethylcelecoxib suppressed cell proliferation and inhibited T-cell factor 7-like 2 expression with almost the same strength as celecoxib. 2,5-Dimethylcelecoxib also inhibited the T-cell factor-dependent transcription activity and suppressed the expression of Wnt/ß-catenin target gene products cyclin D1 and survivin. Subsequently, we compared the in vivo effects of celecoxib and DM-celecoxib using the Mutyh-/- mouse model, in which oxidative stress induces multiple intestinal carcinomas. Serum concentrations of orally administered celecoxib and DM-celecoxib elevated to the levels enough to suppress cancer cell proliferation. Repeated treatment with celecoxib and DM-celecoxib markedly reduced the number and size of the carcinomas without showing toxicity. These results suggest that the central mechanism for the anticancer effect of celecoxib derivatives is the suppression of the Wnt/ß-catenin signaling pathway but not the inhibition of COX-2, and that DM-celecoxib might be a better lead compound candidate than celecoxib for the development of novel anticancer drugs.

BubR1 insufficiency inhibits neointimal hyperplasia through impaired vascular smooth muscle cell proliferation in mice.

OBJECTIVE: BubR1, a cell cycle-related protein, is an essential component of the spindle checkpoint that regulates cell division. Mice with BubR1 expression reduced to 10% of the normal level display a phenotype characterized by progeria; however, the involvement of BubR1 in vascular diseases is still unknown. We generated mice in which BubR1 expression was reduced to 20% (BubR1(L/L) mice) of that in wild-type mice (BubR1(+/+)) to investigate the effects of BubR1 on arterial intimal hyperplasia. APPROACH AND RESULTS: Ten-week-old male BubR1(L/L) and age-matched wild-type littermates (BubR1(+/+)) were used in this study. The left common carotid artery was ligated, and histopathologic examinations were conducted 4 weeks later. Bone marrow transplantation was also performed. Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aorta to examine cell proliferation, migration, and cell cycle progression. Severe neointimal hyperplasia was observed after artery ligation in BubR1(+/+) mice, whereas BubR1(L/L) mice displayed nearly complete inhibition of neointimal hyperplasia. Bone marrow transplantation from all donors did not affect the reconstitution of 3 hematopoietic lineages, and neointimal hyperplasia was still suppressed after bone marrow transplantation from BubR1(+/+) mice to BubR1(L/L) mice. VSMC proliferation was impaired in BubR1(L/L) mice because of delayed entry into the S phase. VSMC migration was unaffected in these BubR1(L/L) mice. p38 mitogen-activated protein kinase-inhibited VSMCs showed low expression of BubR1, and BubR1-inhibited VSMCs showed low expression of p38. CONCLUSIONS: BubR1 may represent a new target molecule for treating pathological states of vascular remodeling, such as restenosis after angioplasty.

Differentiation-inducing factor-3 inhibits intestinal tumor growth in vitro and in vivo.

Differentiation-inducing factor-1 (DIF-1) produced by Dictyostelium discoideum strongly inhibits the proliferation of various types of cancer cells by suppression of the Wnt/ß-catenin signal transduction pathway. In the present study, we examined the effect of differentiation-inducing factor-3 (DIF-3), a monochlorinated metabolite of DIF-1 that is also produced by D. discoideum, on human colon cancer cell lines HCT-116 and DLD-1. DIF-3 strongly inhibited cell proliferation by arresting the cell cycle at the G0/G1 phase. DIF-3 reduced the expression levels of cyclin D1 and c-Myc by facilitating their degradation via activation of GSK-3ß in a time and dose-dependent manner. In addition, DIF-3 suppressed the expression of T-cell factor 7-like 2, a key transcription factor in the Wnt/ß-catenin signaling pathway, thereby reducing the mRNA levels of cyclin D1 and c-Myc. Subsequently, we examined the in vivo effects of DIF-3 in Mutyh(-/-) mice with oxidative stress-induced intestinal cancers. Repeated oral administration of DIF-3 markedly reduced the number and size of cancers at a level comparable to that of DIF-1. These data suggest that DIF-3 inhibits intestinal cancer cell proliferation in vitro and in vivo, probably by mechanisms similar to those identified in DIF-1 actions, and that DIF-3 may be a potential novel anti-cancer agent.

Endogenous ROS production in early differentiation state suppresses endoderm differentiation via transient FOXC1 expression.

Oxidative stress plays a pivotal role in the differentiation and proliferation of cells and programmed cell death. However, studies on the role of oxidative stress in differentiation have mainly employed the detection of reactive oxygen species (ROS) during differentiation or generated by ROS inducers. Therefore, it is difficult to clarify the significance of endogenous ROS production in the differentiation of human cells. We developed a system to control the intracellular level of ROS in the initial stage of differentiation in human iPS cells. By introducing a specific substitution (I69E) into the SDHC protein, a component of the mitochondrial respiratory chain complex, the endogenous ROS level increased. This caused impaired endoderm differentiation of iPS cells, and this impairment was reversed by overproduction of mitochondrial-targeted catalase, an anti-oxidant enzyme. Expression of tumor-related FOXC1 transcription factor increased transiently as early as 4 h after ROS-overproduction in the initial stage of differentiation. Knockdown of FOXC1 markedly improved impaired endoderm differentiation, suggesting that endogenous ROS production in the early differentiation state suppresses endoderm differentiation via transient FOXC1 expression.

Structural and dynamic features of the MutT protein in the recognition of nucleotides with the mutagenic 8-oxoguanine base.

Escherichia coli MutT hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, an event that can prevent the misincorporation of 8-oxoguanine opposite adenine in DNA. Of the several enzymes that recognize 8-oxoguanine, MutT exhibits high substrate specificity for 8-oxoguanine nucleotides; however, the structural basis for this specificity is unknown. The crystal structures of MutT in the apo and holo forms and in the binary and ternary forms complexed with the product 8-oxo-dGMP and 8-oxo-dGMP plus Mn(2+), respectively, were determined. MutT strictly recognizes the overall conformation of 8-oxo-dGMP through a number of hydrogen bonds. This recognition mode revealed that 8-oxoguanine nucleotides are discriminated from guanine nucleotides by not only the hydrogen bond between the N7-H and Odelta (N119) atoms but also by the syn glycosidic conformation that 8-oxoguanine nucleotides prefer. Nevertheless, these discrimination factors cannot by themselves explain the roughly 34,000-fold difference between the affinity of MutT for 8-oxo-dGMP and dGMP. When the binary complex of MutT with 8-oxo-dGMP is compared with the ligand-free form, ordering and considerable movement of the flexible loops surrounding 8-oxo-dGMP in the binary complex are observed. These results indicate that MutT specifically recognizes 8-oxoguanine nucleotides by the ligand-induced conformational change.

Characteristic mutations induced in the small intestine of Msh2-knockout gpt delta mice.

BACKGROUND: Base pair mismatches in genomic DNA can result in mutagenesis, and consequently in tumorigenesis. To investigate how mismatch repair deficiency increases mutagenicity under oxidative stress, we examined the type and frequency of mutations arising in the mucosa of the small intestine of mice carrying a reporter gene encoding guanine phosphoribosyltransferase (gpt) and in which the Msh2 gene, which encodes a component of the mismatch repair system, was either intact (Msh2+/+::gpt/0; Msh2-bearing) or homozygously knockout (KO) (Msh2-/-::gpt/0; Msh2-KO). RESULTS: Gpt mutant frequency in the small intestine of Msh2-KO mice was about 10 times that in Msh2-bearing mice. Mutant frequency in the Msh2-KO mice was not further enhanced by administration of potassium bromate, an oxidative stress inducer, in the drinking water at a dose of 1.5 g/L for 28 days. Mutation analysis showed that the characteristic mutation in the small intestine of the Msh2-KO mice was G-to-A transition, irrespective of whether potassium bromate was administered. Furthermore, administration of potassium bromate induced mutations at specific sites in gpt in the Msh2-KO mice: G-to-A transition was frequently induced at two known sites of spontaneous mutation (nucleotides 110 and 115, CpG sites) and at nucleotides 92 and 113 (3'-side of 5'-GpG-3'), and these sites were confirmed to be mutation hotspots in potassium bromate-administered Msh2-KO mice. Administration of potassium bromate also induced characteristic mutations, mainly single-base deletion and insertion of an adenine residue, in sequences of three to five adenine nucleotides (A-runs) in Msh2-KO mice, and elevated the overall proportion of single-base deletions plus insertions in Msh2-KO mice. CONCLUSIONS: Our previous study revealed that administration of potassium bromate enhanced tumorigenesis in the small intestine of Msh2-KO mice and induced G-to-A transition in the Ctnnb1 gene. Based on our present and previous observations, we propose that oxidative stress under conditions of mismatch repair deficiency accelerates the induction of single-adenine deletions at specific sites in oncogenes, which enhances tumorigenesis in a synergistic manner with G-to-A transition in other oncogenes (e.g., Ctnnb1).

Oxidative-stress-driven mutagenesis in the small intestine of the gpt delta mouse induced by oral administration of potassium bromate.

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.

Resveratrol and its Related Polyphenols Contribute to the Maintenance of Genome Stability.

Genomic destabilisation is associated with the induction of mutations, including those in cancer-driver genes, and subsequent clonal evolution of cells with abrogated defence systems. Such mutations are not induced when genome stability is maintained; however, the mechanisms involved in genome stability maintenance remain elusive. Here, resveratrol (and related polyphenols) is shown to enhance genome stability in mouse embryonic fibroblasts, ultimately protecting the cells against the induction of mutations in the ARF/p53 pathway. Replication stress-associated DNA double-strand breaks (DSBs) that accumulated with genomic destabilisation were effectively reduced by resveratrol treatment. In addition, resveratrol transiently stabilised the expression of histone H2AX, which is involved in DSB repair. Similar effects on the maintenance of genome stability were observed for related polyphenols. Accordingly, we propose that polyphenol consumption can contribute to the suppression of cancers that develop with genomic instability, as well as lifespan extension.

MUTYH-null mice are susceptible to spontaneous and oxidative stress induced intestinal tumorigenesis.

MUTYH is a mammalian DNA glycosylase that initiates base excision repair by excising adenine opposite 8-oxoguanine and 2-hydroxyadenine opposite guanine, thereby preventing G:C to T:A transversion caused by oxidative stress. Recently, biallelic germ-line mutations of MUTYH have been found in patients predisposed to a recessive form of hereditary multiple colorectal adenoma and carcinoma with an increased incidence of G:C to T:A somatic mutations in the APC gene. In the present study, a systematic histologic examination revealed that more spontaneous tumors had developed in MUTYH-null mice (72 of 121; 59.5%) than in the wild type (38 of 109; 34.9%). The increased incidence of intestinal tumors in MUTYH-null mice (11 tumors in 10 of 121 mice) was statistically significant compared with the wild type (no intestinal tumors in 109 mice). Two adenomas and seven adenocarcinomas were observed in the small intestines, and two adenomas but no carcinomas were found in the colons. In MUTYH-null mice treated with KBrO(3), the occurrence of small intestinal tumors dramatically increased. The mean number of polyps induced in the small intestines of these mice was 61.88 (males, 72.75; females, 51.00), whereas it was 0.85 (males, 0.50; females, 1.00) in wild-type mice. The tumors developed predominantly in the duodenum and in the upper region of the (jejunum) small intestines. We conclude that MUTYH suppresses spontaneous tumorigenesis in mammals, thus providing experimental evidence for the association between biallelic germ-line MUTYH mutations and a recessive form of human hereditary colorectal adenoma and carcinoma.

Budding Uninhibited by Benzimidazole-1 Insufficiency Prevents Acute Renal Failure in Severe Sepsis by Maintaining Anticoagulant Functions of Vascular Endothelial Cells.

Severe sepsis is critical to health and can result in acute renal failure (ARF). Tissue factor (TF) and thrombomodulin (TM) play key roles in vascular endothelial functions by helping maintain microcirculation in the kidney. Budding uninhibited by benzimidazole-1 (Bub1) plays a role in Akt and JNK signaling, which control TF and TM, respectively. We hypothesized that Bub1 could control vascular endothelial function in sepsis. The aim of this study was to determine the role of Bub1 in septic ARF. We used Mouse cecum ligation and puncture (CLP) using low Bub1 expressing (Bub1) and wild-type (Bub1) mice in vivo and lipopolysaccharide (LPS) stimulation of human aortic endothelial cell (HAEC) in vitro. Bub1 mice had a higher survival rate after CLP than Bub1. Bub1 mice had more severe ARF after CLP than Bub1 with blood biochemical and pathological analyses. TF expression in Bub1 mice and control HAEC (control) significantly increased in the septic model compared with Bub1 and Bub1 silenced HAEC (siBub1). TM expression in the control significantly decreased after LPS stimulation compared with siBub1. Akt and JNK phosphorylation of siBub1 were attenuated after LPS stimulation. Associations of Bub1 with Akt or JNK after LPS stimulation of HAEC were detected using immunoprecipitation, suggesting that Bub1 is involved in the phosphorylation of Akt and JNK after LPS stimulation. Bub1 insufficiency attenuates TF expression and reduces TM suppression by blocking Akt and JNK phosphorylation, respectively, thus leading to the prevention of ARF and death caused by sepsis.

Replication stress triggers microsatellite destabilization and hypermutation leading to clonal expansion in vitro.

Mismatch repair (MMR)-deficient cancers are characterized by microsatellite instability (MSI) and hypermutation. However, it remains unclear how MSI and hypermutation arise and contribute to cancer development. Here, we show that MSI and hypermutation are triggered by replication stress in an MMR-deficient background, enabling clonal expansion of cells harboring ARF/p53-module mutations and cells that are resistant to the anti-cancer drug camptothecin. While replication stress-associated DNA double-strand breaks (DSBs) caused chromosomal instability (CIN) in an MMR-proficient background, they induced MSI with concomitant suppression of CIN via a PARP-mediated repair pathway in an MMR-deficient background. This was associated with the induction of mutations, including cancer-driver mutations in the ARF/p53 module, via chromosomal deletions and base substitutions. Immortalization of MMR-deficient mouse embryonic fibroblasts (MEFs) in association with ARF/p53-module mutations was ~60-fold more efficient than that of wild-type MEFs. Thus, replication stress-triggered MSI and hypermutation efficiently lead to clonal expansion of cells with abrogated defense systems.

Mismatch repair dependence of replication stress-associated DSB recognition and repair.

Most cancers develop with one of two types of genomic instability, namely, chromosomal instability (CIN) or microsatellite instability (MSI). Both are induced by replication stress-associated DNA double-strand breaks (DSBs). The type of genomic instability that arises is dependent on the choice of DNA repair pathway. Specifically, MSI is induced via a PolQ-dependent repair pathway called microhomology-mediated end joining (MMEJ) in a mismatch repair (MMR)-deficient background. However, it is unclear how the MMR status determines the choice of DSB repair pathway. Here, we show that replication stress-associated DSBs initially targeted by the homologous recombination (HR) system were subsequently hijacked by PolQ-dependent MMEJ in MMR-deficient cells, but persisted as HR intermediates in MMR-proficient cells. PolQ interacting with MMR factors was effectively loaded onto damaged chromatin in an MMR-deficient background, in which merged MRE11/γH2AX foci also effectively formed. Thus, the choice of DNA repair pathway according to the MMR status determines whether CIN or MSI is induced.

Improved gene correction efficiency with a tailed duplex DNA fragment.

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.

DNA repair dysfunction in gastrointestinal tract cancers.

The DNA repair system surveys the genome, which is always suffering from exposure to both exogenous as well as endogenous mutagens, to maintain the genetic information. The fact that the basis of this DNA repair system is highly conserved, from prokaryote to mammalian cells, suggests the importance of precise genome maintenance mechanisms for organisms. In the past 15 years, considerable progress has been made in understanding how repair processes interact and how disruptions of these mechanisms lead to the accumulation of mutations and carcinogenesis. In 1993, two groups reported that DNA mismatch repair could be associated with hereditary non-polyposis colorectal cancer, indicating a connection between faulty DNA repair function and cancer. More recently, an inherited disorder of DNA glycosylase, which removes mutagenic oxidized base from DNA, has been reported in individuals with a predisposition to multiple colorectal adenomas and carcinomas. This is the first report that directly indicates the role of the repair of oxidative DNA in human inherited cancer. Studies from gene knockout mice have elucidated the principal role of these repair systems in the process of carcinogenesis. Moreover, clinical samples derived from cancer patients have shown the direct involvement. This review focuses on the function of DNA mismatch repair and oxidative DNA/nucleotide repair among various DNA repair systems in cells, both of which are essentially involved in the carcinogenesis of gastrointestinal tract cancer.

Effects of target sequence and sense versus anti-sense strands on gene correction with single-stranded DNA fragments.

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.

Orpheus recombination : a comprehensive bacteriophage system for murine targeting vector construction by transplacement.

In recent years, methods to address the simplification of targeting vector (TV) construction have been developed and validated. Based on in vivo recombination in Escherichia coli, these protocols have reduced dependence on restriction endonucleases, allowing the fabrication of complex TV constructs with relative ease. Using a methodology based on phage-plasmid recombination, we have developed a comprehensive TV construction protocol dubbed Orpheus recombination (ORE). The ORE system addresses all necessary requirements for TV construction; from the isolation of genespecific regions of homology to the deposition of selection/disruption cassettes. ORE makes use of a small recombination plasmid, which bears positive and negative selection markers and a cloned homologous "probe" region. This probe plasmid may be introduced into and excised from phage-borne murine genomic clones by two rounds of single crossover recombination. In this way, desired clones can be specifically isolated from a heterogeneous library of phage. Furthermore, if the probe region contains a designed mutation, it may be deposited seamlessly into the genomic clone. The complete removal of operational sequences allows unlimited repetition of the procedure to customize and finalize TVs within a few weeks. Successful gene-specific clone isolation, point mutations, large deletions, cassette insertions, and finally coincident clone isolation and mutagenesis have all been demonstrated with this method.