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At least 109 records · Page 6Linked to original sources
PubMed2003

Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks.

The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.

Blotting, Southern↗
PubMed2002

Dystrophin gene repair in mdx muscle precursor cells in vitro and in vivo mediated by RNA-DNA chimeric oligonucleotides.

Point mutations in the dystrophin gene cause dystrophin deficiency and muscular dystrophy in the mdx mouse and a subset of patients with Duchenne muscular dystrophy. As an approach to gene therapy for muscular dystrophies due to point mutations, we have studied the ability of RNA-DNA chimeric oligonucleotides (chimeraplasts) to induce repair of the dystrophin gene in mdx mice. We have previously demonstrated that targeting chimeraplasts can repair the exon 23 point mutation in differentiated myofibers in vivo after intramuscular injection. For long-term benefit to patients with muscular dystrophy, any gene therapy technology must target not only differentiated myofibers but also undifferentiated muscle precursor cells that are involved in ongoing muscle repair. The focus of the current studies was to test whether chimeraplasts could repair the dystrophin mutation in mdx muscle precursor cells. Initial studies were done by transfecting a targeting chimeraplast into mdx myoblasts in vitro. Gene repair was demonstrated at the DNA, RNA, and protein levels in these cells, whereas treatment of the cells with a control chimeraplast resulted in no gene correction. After differentiation of mdx cells that had been treated with a targeting chimeraplast, immunoblot analysis demonstrated full-length dystrophin expression. By quantitative analysis of independent cultures, the amount of dystrophin expressed ranged from 2 to 15% of that expressed in wild-type cells, providing a measure of the efficacy of gene conversion in vitro. To extend the assessment to muscle precursor cells in vivo, we injected targeting and control chimeraplasts into muscles of mdx mice. When muscle precursor cells were subsequently derived from muscles injected with a targeting chimeraplast, we found that gene repair had occurred in these cells as well. These results, taken together, further demonstrate that chimeraplast-mediated gene repair may be effective as an approach to gene therapy for muscular dystrophies due to point mutations.

Animals↗
PubMed1999

Gene targeted DNA double-strand break induction by (125)I-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells.

A parallel binding motif 16mer triplex-forming oligonucleotide (TFO) complementary to a polypurine-polypyrimidine target region near the 3'-end of the SupF gene of plasmid pSP189 was labeled with [5-(125)I]dCMP at position 15. Following triplex formation and decay accumulation, radiation-induced site-specific double-strand breaks (DSBs) were produced in the pSP189 SupF gene. Bulk damaged DNA and the isolated site-specific DSB-containing DNA were separately transfected into human WI38VA13 cells and allowed to repair prior to recovery and analysis of mutants. Bulk damaged DNA had a relatively low mutation frequency of 2.7 x 10(-3). In contrast, the isolated linear DNA containing site-specific DSBs had an unusually high mutation frequency of 7.9 x 10(-1). This was nearly 300-fold greater than that observed for the bulk damaged DNA mixture, and >1.5 x 10(4)-fold greater than background. The mutation spectra displayed a high proportion of deletion mutants targeted to the(125)I binding position within the SupF gene for both bulk damaged DNA and isolated linear DNA. Both spectra were characterized by complex mutations with mixtures of changes. However, mutations recovered from the linear site-specific DSB-containing DNA presented a much higher proportion of complex deletion mutations.

Base Sequence↗
PubMed2002

Mutations at coding repeat sequences in mismatch repair-deficient human cancers: toward a new concept of target genes for instability.

Because the discovery of a link between mismatch repair deficiency and sporadic or inherited human cancers characterized by microsatellite instability (MSI-H tumors), genes containing coding repeat sequences have been found to be mutated at these repeats in MSI-H tumors from different primary sites as reported in the present review. Accumulation of such alterations appears to be the main molecular mechanism by which MSI-H cells accumulate functional changes with putative oncogenic effects. These mutations occur in many genes at variable frequencies. They can affect genes with a putative role in human carcinogenesis involved in different or similar pathways and are thus thought to be inactivating or activating events selected for in these cancers in a recessive or dominant manner. However, because of the high level of instability characterizing these cancers, they are also likely to occur in genes without any expected role in MSI-H carcinogenesis. In light of these recent data, the concept of target genes for instability and their possible role in MSI-H cancers is reconsidered here.

Base Pair Mismatch↗
PubMed1998

Mismatch repair genes and mononucleotide tracts as mutation targets in colorectal tumors with different degrees of microsatellite instability.

Microsatellite instability occurs in 15% of colorectal carcinomas and may be due to replication errors (RER). The pattern of instability--'severe' vs 'mild'--and the tumorigenic pathway, as reflected by the involvement of functionally important genes, may vary according to the underlying gene(s). We defined 'mild' RER as mono- or tetranucleotide repeat instability in the absence of widespread instability at dinucleotide repeats and studied 15 colorectal tumors with this phenotype for mutations in the DNA mismatch repair genes MSH2, MLH1, MSH3, and MSH6. No mutations were found, suggesting that these genes were not implicated. We then compared colorectal cancers with 'mild' RER (n = 15), and those with 'severe' RER without (n = 11) or with (n = 22) detectable mutations in MSH2 or MLH1 to assess the involvement of mononucleotide repeats contained in the coding regions of MSH3, MSH6, BAX, and TGFbeta RII. The combined mutation rates of the above mentioned loci varied significantly between the three groups of tumors, being 0%, 25% and 52%, respectively. Furthermore, the individual genes showed specific patterns of involvement; for example, among tumors with 'severe' RER, TGFbeta RII displayed uniformly high mutation rates while MSH3, MSH6, and BAX were more frequently altered in tumors that also showed MSH2 or MLH1 mutations. Our findings suggest that different subcategories exist among unstable tumors, defined by the RER pattern on the one hand and tumorigenic pathway on the other, and structural changes of MSH2 and MLH1 are likely to explain only a proportion of these cases.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Analysis of one-sided marker segregation patterns resulting from mammalian gene targeting.

The double-strand break repair (DSBR) model is currently accepted as the paradigm for acts of double-strand break (DSB) repair that lead to crossing over between homologous sequences. The DSBR model predicts that asymmetric heteroduplex DNA (hDNA) will form on both sides of the DSB (two-sided events; 5:3/5:3 segregation). In contrast, in yeast and mammalian cells, a considerable fraction of recombinants are one sided: they display full conversion (6:2 segregation) or half-conversion (5:3 segregation) on one side of the DSB together with normal 4:4 segregation on the other side of the DSB. Two mechanisms have been proposed to account for these observations: (i) hDNA formation is restricted to one side of the DSB or the other, and (ii) recombination is initially two sided, but hDNA repair directed by Holliday junction cuts restores normal 4:4 segregation on that side of the DSB in which the mismatch is closest to the cut junction initiating repair. In this study, we exploited a well-characterized gene-targeting assay to test the predictions that these mechanisms make with respect to the frequency of recombinants displaying 4:4 marker segregation on one side of the DSB. Unexpectedly, the results do not support the predictions of either mechanism. We propose a derivation of mechanism (ii) in which the nicks arising from Holliday junction cleavage are not equivalent with respect to directing repair of adjacent hDNA, possibly as a result of asynchronous cleavage of the DSBR intermediate.

Animals↗
PubMed2006

Differentiation and injury-repair signals modulate the interaction of E2F and pRB proteins with novel target genes in keratinocytes.

E2F transcription factors are central to epidermal morphogenesis and regeneration after injury. The precise nature of E2F target genes involved in epidermal formation and repair has yet to be determined. Identification of these genes is essential to understand how E2F proteins regulate fundamental aspects of epidermal homeostasis and transformation. We have conducted a genome-wide screen using CpG island microarray analysis to identify novel promoters bound by E2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, and determined that multiple E2F and retinoblastoma (pRb) family proteins associate with them in exponentially proliferating cells. We also assessed the effect on E2F and pRb binding to those genes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or to activation of repair mechanisms induced by transforming growth factor-beta (TGF-beta). These studies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/or pRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter in cells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter in the presence of TGF-beta. Functionally, these different interactions resulted in transcriptional repression by BMP-6 and TGF-beta of the N-myc gene, via mechanisms that involved E2F binding to the promoter and association with pRb-family proteins. Thus, multiple combinations of E2F and pRb family proteins may associate with and transcriptionally regulate a given target promoter in response to differentiation and injury-repair stimuli in epidermal keratinocytes.

Animals↗
PubMed2001

Importance of DNA repair in carcinogenesis: evidence from transgenic and gene targeting studies.

We have generated transgenic mice by introducing copies of the E. coli O6-methylguanine-DNA methyltransferase gene, ada. Liver extracts from homozygotes demonstrate about three times the control enzyme activity and increase up to about eight-fold can be induced by treatment with zinc, since the metal-responsive metallothionein promoter is attached to the ada gene. Furthermore, studies of liver carcinogenesis in our transgenic mice demonstrated significantly reduced rates of development of hepatocellular tumors after treatment with dimethylnitrosamine or diethylnitrosamine. It is well known that xeroderma pigmentosum (XP) patients are deficient in DNA repair. The availability of XPA (XP group A complementing) knockout mice has enabled us to investigate the functional role of the XPA nucleotide excision repair gene in carcinogenesis in vivo, first using the mouse skin as a model system. XPA-/- mice demonstrated skin ulcers 5-7 days after 7,12-dimethylbenz[a]anthracene (DMBA) treatment and papilloma development within 4 weeks prior to promotion, skin tumor incidence being also much higher than in heterozygous and wild-type mice. Experiments targeting the lung, liver and tongue have also been conducted to answer the question of whether the internal organs of these mice are also susceptible to chemical carcinogens. For lung carcinogenesis, mice were instilled intratracheally with a small dose of benzo[a]pyrene. The pulmonary tumor incidence in XPA-/- mice was significantly higher than in XPA+/- and XPA+/+ mice. XPA-/- mice were also found to be have enhanced sensitivity to aflatoxin B1 regarding liver tumor induction. In addition, administration of 4-nitroquinoline-1-oxide in drinking water for 50 weeks resulted in tongue tumors only in XPA-/- mice. These studies, thus, provided convincing evidence that XPA mice are also sensitive to carcinogenesis in organs other than the skin.

Animals↗
PubMed2003

A comparison of gene repair strategies in cell culture using a lacZ reporter system.

Synthetic oligonucleotides and DNA fragments of less than 1 kilobase (kb) have been shown to cause site-specific genetic alterations in mammalian cells in culture and in vivo. We have used a lacZ reporter gene system to compare the efficiency of episomal and chromosomal gene repair in human embryonic kidney epithelial cells (HEK293), Chinese Hamster Ovary fibroblasts (CHOK1), human bronchial epithelial cells (16HBE), and mouse embryonic stem (ES) cells. The lacZ gene contains a G to A nucleotide change, (Glu to Lys mutation) that abrogates beta-galactosidase activity. We compared the efficiency of different gene repair methods to correct this mutation and restore beta-galactosidase activity. We evaluated PCR-generated double-stranded DNA fragments of 0.52-1.9 kb, single-stranded DNA oligonucleotides of 20, 35, or 80 bases containing internal phosphorothioate links, and a 68 base RNA:DNA oligonucleotide. All of the oligonucleotides and DNA fragments showed some gene repair ability with an episomal plasmid. Short DNA fragments of 0.52 kb or greater gave the highest frequencies of episomal gene repair while single-stranded DNA oligonucleotides gave the highest frequency of chromosomal repair. In the context of a chromosomal target, antisense DNA oligonucleotides gave 5-fold higher frequencies of gene repair than their sense counterparts. The RNA:DNA chimeric oligonucleotide gave little or no gene repair on either a chromosomal or episomal target.

Blotting, Southern↗
PubMed2004

Activation of the transcription factor HIF-1 and its target genes, VEGF, HO-1, iNOS, during fracture repair.

One of the immediate sequelae of bone fracture is regional hypoxia resulting from vasculature disruption. Hypoxia stabilizes and activates the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha), which ultimately leads to HIF-1-regulated gene expression. Because nothing is known about HIF-1 involvement in bone regeneration, we performed a series of experiments to elucidate the expression pattern of HIF-1alpha and selected HIF-1 target genes using a rat femoral fracture model. Callus samples were obtained on postfracture days (PFD) 3, 5, 7, 10, 14, and 21. Quantitative RT-PCR (qRT-PCR) was employed to quantify the temporal mRNA expression patterns of HIF-1alpha, vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1). Elevated HIF-1alpha and VEGF expression was seen at all time points, with peak increases of approximately 6- and 2-fold relative to the intact bone present on PFD 10 for HIF-1alpha and VEGF, respectively. Robust activation of iNOS was detected solely on PFD 10 (6.8-fold) with all other time points showing slight downregulation. HO-1 expression peaked on PFD 3 (4.5-fold) with no significant changes on any other PFD. Western blot analysis verified the temporal expression patterns with HIF-1alpha protein expression showing a steady rise to a PFD 10 peak of approximately 18-fold. Similarly, the expression patterns for VEGF and HO-1 showed increases of approximately 4-fold at their PFD 10 and PFD 3 peaks, respectively. Immunohistochemical analysis of PFD 10 callus sections revealed coexpression of HIF-1alpha and VEGF in proliferating chondrocytes and active osteoblasts. Immunostaining for HO-1 on PFD 3 callus sections demonstrated strong expression in hematoma macrophages and vascular endothelial cells. Taken together, these experiments demonstrate for the first time that HIF-1alpha is upregulated at both transcriptional and translational levels in the fracture callus and indicate that PFD 10 may be a key angiogenic time point in the developing rat fracture callus.

Animals↗
PubMed2002

Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type.

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction.

A variety of technological advances in recent years have made permanent genetic manipulation of an organism a technical possibility. As the details of natural biological processes for genome modification are elucidated, the enzymes catalyzing these events (transposases, recombinases, integrases and DNA repair enzymes) are being harnessed or modified for the purpose of intentional gene modification. Targeted integration and gene repair can be mediated by the DNA-targeting specificity inherent to a particular enzyme, or rely on user-designed specificities. Integration sites can be defined by using DNA base-pairing or protein-DNA interaction as a means of targeting. This review will describe recent progress in the development of 'user-targetable' systems, particularly highlighting the application of custom DNA-binding proteins or nucleic acid homology to confer specificity.

DNA Repair↗
PubMed1994

Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.

We established a mouse Ltk- cell line that contains within its genome a herpes simplex virus thymidine kinase gene (tk) that had been disrupted by the insertion of the recognition sequence for yeast endonuclease I-SceI. The artificially introduced 18 bp I-SceI recognition sequence was likely a unique sequence in the genome of the mouse cell line. To assess whether an induced double-strand break (DSB) in the genomic tk gene would be repaired preferentially by gene targeting or non-homologous recombination, we electroporated the mouse cell line with endonuclease I-SceI alone, one of two different gene targeting constructs alone, or with I-SceI in conjunction with each of the two targeting constructs. Each targeting construct was, in principle, capable of correcting the defective genomic tk sequence via homologous recombination. tk+ colonies were recovered following electroporation of cells with I-SceI in the presence or absence of a targeting construct. Through the detection of small deletions at the I-SceI recognition sequence in the mouse genome, we present evidence that a specific DSB can be introduced into the genome of a living mammalian cell by yeast endonuclease I-SceI. We further report that a DSB in the genome of a mouse Ltk- cell is repaired preferentially by non-homologous end-joining rather than by targeted homologous recombination with an exogenous donor sequence. The potential utility of this system is discussed.

Animals↗
PubMed2003

MBD4 mutations are rare in gastric carcinomas with microsatellite instability.

MBD4 encodes a protein that interacts with the mismatch repair protein hMLH1. Therefore, it has been postulated that mutations in MBD4 may result in mismatch repair deficiency. Furthermore, it was shown that MBD4 is a target gene in mismatch repair-deficient tumors. We searched for mutations of MBD4 in sporadic gastric carcinomas (SGC) and compared them with the MSI status of the tumors by screening a series of 42 SGC [22 high microsatellte instability (MSI-H) and 20 microsatellite stable (MSS)] for mutations in both the poly(A)10 and (A)6 tracts of the gene as well as in its entire coding sequence. Mutations of the poly(A)10 tract of MBD4 were also screened in 126 MSI-H colon carcinomas. One of the 22 MSI-H gastric carcinomas and 12 of 126 MSI-H colon carcinomas showed MBD4 mutations in the poly(A)10 tract of exon 3. In addition, seven sequence variants were found in SGC, all of them having a polymorphic nature. We conclude that MBD4 mutations in the entire coding sequence, including the two poly(A) tracts, are rare in MSI gastric carcinomas. MBD4 is likely to be a bystander target gene in MSI-H tumors.

Adenosine↗
PubMed1997

Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.

Repair of DNA damage resulting in double-strand breaks (DSBs) is controlled by gene products executing homologous recombination or end-joining pathways. The MRE11 gene has previously been implicated in DSB repair in the yeast Saccharomyces cerevisiae . Here we have developed a methodology to study the roles of the murine Mre11 homolog in pluripotent embryonic stem cells. Using a gene targeting approach, a triple LoxP site cassette was inserted into a region of MRE11 genomic DNA flanking conserved phosphodiesterase motifs. The addition of Cre recombinase activity promotes deletions of three types that can be scored. We find that deletion at phosphodiesterase motif III encoded in the N-terminus of Mre11 is acheived in the presence of a wild-type MRE11 allele. However, when the wild-type MRE11 allele is inactivated by gene targeted insertion of a neo marker, only Cre recombination events that allow expression of wild-type Mre11 protein are observed. Therefore, Mre11 is required for normal cell proliferation. This methodology introduces a means to study important regions of essential genes in cell culture models.

Amino Acid Sequence↗
PubMed2000

Measuring gene-specific nucleotide excision repair in human cells using quantitative amplification of long targets from nanogram quantities of DNA.

We have been developing a rapid and convenient assay for the measurement of DNA damage and repair in specific genes using quantitative polymerase chain reaction (QPCR) methodology. Since the sensitivity of this assay is limited to the size of the DNA amplification fragment, conditions have been found for the quantitative generation of PCR fragments from human genomic DNA in the range of 6-24 kb in length. These fragments include: (1) a 16.2 kb product from the mitochondrial genome; (2) 6.2, 10.4 kb, and 15.4 kb products from the hprt gene, and (3) 13.5, 17.7, 24.2 kb products from the human beta-globin gene cluster. Exposure of SV40 transformed human fibroblasts to increasing fluences of ultraviolet light (UV) resulted in the linear production of photoproducts with 10 J/m(2) of UVC producing 0.085 and 0.079 lesions/kb in the hprt gene and the beta-globin gene cluster, respectively. Kinetic analysis of repair following 10 J/m(2) of UVC exposure indicated that the time necessary for the removal of 50% of the photoproducts, in the hprt gene and beta-globin gene cluster was 7.8 and 24.2 h, respectively. Studies using lymphoblastoid cell lines show very little repair in XPA cells in both the hprt gene and beta-globin locus. Preferential repair in the hprt gene was detected in XPC cells. Cisplatin lesions were also detected using this method and showed slower rates of repair than UV-induced photoproducts. These data indicate that the use of long targets in the gene-specific QPCR assay allows the measurement of biologically relevant lesion frequencies in 5-30 ng of genomic DNA. This assay will be useful for the measurement of human exposure to genotoxic agents and the determination of human repair capacity.

Cell Line↗
PubMed1997

Homology requirements for targeting heterologous sequences during P-induced gap repair in Drosophila melanogaster.

The effect of homology on gene targeting was studied in the context of P-element-induced double-strand breaks at the white locus of Drosophila melanogaster. Double-strand breaks were made by excision of P-w(hd), a P-element insertion in the white gene. A nested set of repair templates was generated that contained the 8 kilobase (kb) yellow gene embedded within varying amounts of white gene sequence. Repair with unlimited homology was also analyzed. Files were scored phenotypically for conversion of the yellow gene to the white locus. Targeting of the yellow gene was abolished when all of the 3' homology was removed. Increases in template homology up to 51 base pairs (bp) did not significantly promote targeting. Maximum conversion was observed with a construct containing 493 bp of homology, without a significant increase in frequency when homology extended to the tips of the chromosome. These results demonstrate that the homology requirements for targeting a large heterologous insertion are quite different than those for a point mutation. Furthermore, heterologous insertions strongly affect the homology requirements for the conversion of distal point mutations. Several aberrant conversion tracts, which arose from templates that contained reduced homology, also were examined and characterized.

ATP-Binding Cassette Transporters↗