All candidate mutations were resequenced on the opposite strand for verification

All candidate mutations were resequenced on the opposite strand for verification. RESULTS Methylation Analysis of a Mouse Transgene After 5azaCdR Administration. inactivate the enzyme before its conversation with DNA, are needed for chemoprevention or long term therapy. to demonstrate the inverse relationship between DNA methylation and gene expression and to reactivate epigenetically silenced genes (2). The potential reversibility of epigenetic silencing of tumor suppressor genes in tumor cells has renewed desire for the use of 5azaCdR for chemotherapy (3). In fact, we recently showed that 5azaCdR significantly reduces intestinal tumor multiplicity in Min mice by reducing genomic methylation levels (4). Mechanistically, 5azaCdR is usually a cytosine analogue that functions as a suicide substrate for DNA methyltransferase when incorporated into DNA at the target site for DNA methylation, CpG dinucleotides (5). Santi transgene (12) isolated from colonic DNA from mice with and without 5azaCdR administration, we show that mutations induced by 5azaCdR are predominantly C:G??G:C transversions. Moreover, three-quarters of the mutations in 5azaCdR-treated mice occur at CpG dinucleotides. We propose a model for how the DNA methyltransferase enzyme may be involved in facilitating 5azaCdR-induced mutagenesis. The mutagenicity of this drug calls into question its utility as PTP1B-IN-1 a chemopreventive agent for colon cancer and highlights the need to identify novel inhibitors of DNA methyltransferase PTP1B-IN-1 for this purpose. MATERIALS AND METHODS Mice. The substrain was managed in the C57BL/6 strain background [Stratagene (12)]. 5azaCdR Administration. 5azaCdR (Sigma, catalog no. A-3656) was solubilized in sterile PBS at 2.5 mg/ml and stored in aliquots at ?80C until use. Mice were injected s.c. using a 30-gauge needle on a 0.1-ml Hamilton 700 series syringe with a Luer tip and a PB600 dispenser attachment that delivers 50 discrete models of 2 l (4). Weekly injections were started at 7 days after birth and continued for a total of 14 weeks. Increments of 2 l (5 g)/5 g mouse body weight (rounded to the nearest 5 g) were delivered. Genomic DNA Isolation. Tissues were isolated from 100-day-old mice, frozen immediately on dry ice, and stored at ?80C until use. High molecular excess weight DNA was prepared by proteinase K digestion of colons pulverized in liquid nitrogen. Two extractions with phenolCchloroform and one chloroform extraction were done, and the DNA was precipitated with ethanol by spooling onto a flame-sealed pasteur pipet. Isolated DNA was resuspended in 10 mM Tris, pH 7.5/0.1 mM EDTA. Methylation Analysis. Genomic DNA (5 g) was digested with gene (spanning from nucleotide ?50 in the Packaging and Mutant Isolation. phages were packaged from your genomic DNAs using Transpack extracts (Stratagene) as recommended by the manufacturer. Packaged DNAs were used to infect the SCS-8 strain of (Stratagene) and were plated on NZY agar plates using top agarose made up of 1.5 mg/ml 5-bromo-4-chloro-3-indolyl -d-galactoside (Stratagene). Blue plaques of all intensities were isolated and replated at low density in the presence of 5-bromo-4-chloro-3-indolyl -d-galactoside for plaque purification. Sequence Analysis. Isolated plaques were either utilized for excision of the pLIZ plasmid made up of the Rabbit Polyclonal to MAP3K7 (phospho-Thr187) gene as explained (14) or used as themes for PCR amplification of the gene. Plasmid DNAs or PCR fragments were cycle-sequenced using dye terminators on an automated Applied Biosystems sequencer (Whitehead Institute Sequencing Facility). Sequences were compared with the wild-type gene sequence using the megalign program (DNAstar, Madison, WI) to identify mutations. All candidate mutations were resequenced on the opposite strand for verification. RESULTS Methylation Analysis of a Mouse Transgene After 5azaCdR Administration. To examine the spectrum of mutations induced by 5azaCdR gene in a bacteriophage shuttle vector provides an attractive assay for mutation screening (12). Inactivating mutations in the transgene are scored as blue plaques in a background of colorless plaques after packaging of genomic DNA and plating on indication bacteria. The gene contains 94 CpG dinucleotides and a total of 299 cytosine residues in 1083 bp of coding sequence that could serve as targets for 5azaCdR mutagenesis. The loss-of-function allele (13) was launched into the transgenic strain to genetically alter the levels of DNA methyltransferase. The recessive embryonic lethal phenotype of the mutation precluded analysis of homozygous mutants. heterozygous and wild-type transgenic offspring either were injected weekly with 5 g of 5azaCdR/5 g body weight or were left untreated for 14 weeks to reproduce conditions from our previous study of polyp figures in Min mice. As we have shown previously, this regimen results in four unique classes of genomic methylation as measured by Southern blot hybridization.?, The position of the completely methylated sequences. inhibitors of DNA methyltransferase, which can inactivate the enzyme before its conversation with DNA, are needed for chemoprevention or long term therapy. to demonstrate the inverse relationship between DNA methylation and gene expression and to reactivate epigenetically silenced genes (2). The potential reversibility of epigenetic silencing of tumor suppressor genes in tumor cells has renewed desire for the use of 5azaCdR for chemotherapy (3). In fact, we recently showed that 5azaCdR significantly reduces intestinal tumor multiplicity in Min mice by reducing genomic methylation levels (4). Mechanistically, 5azaCdR is usually a cytosine analogue that functions as a suicide substrate for DNA methyltransferase when incorporated into DNA at the target site for DNA methylation, CpG dinucleotides (5). Santi transgene (12) isolated from colonic DNA from mice with and without 5azaCdR administration, we show that mutations induced by 5azaCdR are predominantly C:G??G:C transversions. Moreover, three-quarters of the mutations in 5azaCdR-treated mice occur at CpG dinucleotides. We propose a model for how the DNA methyltransferase enzyme may be involved in facilitating 5azaCdR-induced mutagenesis. The mutagenicity of this drug calls into question its utility as a chemopreventive agent for colon cancer and highlights the need to identify novel inhibitors of DNA methyltransferase for this purpose. MATERIALS AND METHODS Mice. The substrain was managed in the C57BL/6 strain background [Stratagene (12)]. 5azaCdR Administration. 5azaCdR (Sigma, catalog no. A-3656) was solubilized in sterile PBS at 2.5 mg/ml and stored in aliquots at ?80C until use. Mice were injected s.c. using a 30-gauge needle on a 0.1-ml Hamilton 700 series syringe with a Luer tip and a PB600 dispenser attachment that delivers 50 discrete models of 2 l (4). Weekly injections were started at 7 days after birth and continued for a total of 14 weeks. Increments of 2 l (5 g)/5 g mouse body weight (rounded to the nearest 5 g) were delivered. Genomic DNA Isolation. Tissues were isolated from 100-day-old mice, frozen immediately on dry ice, and stored at ?80C until use. High molecular excess weight DNA was prepared by proteinase K digestion of colons pulverized in liquid nitrogen. Two extractions with phenolCchloroform and one chloroform extraction were done, and the DNA was precipitated with ethanol by spooling onto a flame-sealed pasteur pipet. Isolated DNA was resuspended in 10 mM Tris, pH 7.5/0.1 mM EDTA. Methylation Analysis. Genomic DNA (5 g) was digested with PTP1B-IN-1 gene (spanning from nucleotide ?50 in the Packaging and Mutant Isolation. phages were packaged from your genomic DNAs using Transpack extracts (Stratagene) as recommended by the manufacturer. Packed DNAs had been utilized to infect the SCS-8 stress of (Stratagene) and had been plated on NZY agar plates using best agarose including 1.5 mg/ml 5-bromo-4-chloro-3-indolyl -d-galactoside (Stratagene). Blue plaques of most intensities had been isolated and replated at low denseness in the current presence of 5-bromo-4-chloro-3-indolyl -d-galactoside for plaque purification. Series Evaluation. Isolated plaques had been either useful for excision from the pLIZ plasmid including the gene as referred to (14) or utilized as web templates for PCR amplification from the gene. Plasmid DNAs or PCR fragments had been cycle-sequenced using dye terminators with an computerized Applied Biosystems sequencer (Whitehead Institute Sequencing Service). Sequences had been weighed against the wild-type gene series using the megalign system (DNAstar, Madison, WI) to recognize mutations. All applicant mutations had been resequenced on the contrary strand for confirmation. RESULTS Methylation Evaluation of the Mouse Transgene After 5azaCdR Administration. To examine the spectral range of mutations induced by 5azaCdR gene inside a bacteriophage shuttle vector has an appealing assay for mutation testing (12). Inactivating mutations in the transgene are obtained as blue plaques inside a history of colorless plaques.