Mouse DNA Research Articles

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.

Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson\u2019s disease individuals

BackgroundRecent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples.MethodsUsing 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR.ResultsAmplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination.We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences.ConclusionsTaxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.4jqTESRfTS1PdZkk_4BnPMVideo abstract

Aerobic exercise ameliorates survival, clinical score, lung inflammation, DNA and protein damage in septic mice

Background and objectiveSepsis is a potentially deadly organic dysfunction, and one of the main causes of mortality in intensive care units (ICU). Aerobic exercise (AE) is a preventive intervention in the establishment of inflammatory conditions, such as chronic lung diseases, but its effects on sepsis remain unclear. Therefore, this study aimed to evaluate the effects of AE on health condition, mortality, inflammation, and oxidative damage in an experimental model of pneumosepsis induced by Klebsiella pneumoniae (K.p). MethodsAnimals were randomly allocated to Control; Exercise (EXE); Pneumosepsis (PS) or Exercise + Pneumosepsis (EPS) groups. Exercised animals were submitted to treadmill exercise for 2 weeks, 30 min/day, prior to pneumosepsis induced by K.p tracheal instillation. ResultsPS produced a striking decrease in the health condition leading to massive death (85%). AE protected mice, as evidenced by better clinical scores and increased survival (70%). AE alleviated sickness behavior in EPS mice as evaluated in the open field test, and inflammation (nitrite + nitrate, TNF-α and IL-1β levels) in broncoalveolar fluid. Catalase activity, oxidative damage to proteins and DNA was increased by sepsis and prevented by exercise. ConclusionOverall, the beneficial effects of exercise in septic animals encompassed a markedly improved clinical score and decreased mortality, along with lower inflammation markers, less DNA and protein damage, as well as preserved antioxidant enzyme activity. Neural network risk analysis revealed exercise had a considerable effect on the overall health condition of septic mice.

Suppression of Peritoneal Fibrosis by Sonoporation of Hepatocyte Growth Factor Gene-Encoding Plasmid DNA in Mice.

Gene therapy is expected to be used for the treatment of peritoneal fibrosis, which is a serious problem associated with long-term peritoneal dialysis. Hepatocyte growth factor (HGF) is a well-known anti-fibrotic gene. We developed an ultrasound and nanobubble-mediated (sonoporation) gene transfection system, which selectively targets peritoneal tissues. Thus, we attempted to treat peritoneal fibrosis by sonoporation-based human HGF (hHGF) gene transfection in mice. To prepare a model of peritoneal fibrosis, mice were intraperitoneally injected with chlorhexidine digluconate. We evaluated the preventive and curative effects of sonoporation-based hHGF transfection by analyzing the following factors: hydroxyproline level, peritoneum thickness, and the peritoneal equilibration test. The transgene expression characteristics of sonoporation were also evaluated using multicolor deep imaging. In early-stage fibrosis in mice, transgene expression by sonoporation was observed in the submesothelial layer. Sonoporation-based hHGF transfection showed not only a preventive effect but also a curative effect for early-stage peritoneal fibrosis. Sonoporation-based hHGF transfection may be suitable for the treatment of peritoneal fibrosis regarding the transfection characteristics of transgene expression in the peritoneum under fibrosis.

Cytoplasmic Transfer Methods for Studying the Segregation of Mitochondrial DNA in Mice.

Heteroplasmic mice represent a valuable tool to study the segregation of different mtDNA haplotypes (mtDNAs with differing alleles) in vivo against a defined nuclear background. We describe two methods for the creation of such models, differing in the resulting initial heteroplasmy levels: (a) transfer of ooplasm and (b) fusion of two blastomeres. These methods result in typical heteroplasmy of 5% and 50% donor mtDNA , respectively. The choice of method depends on the aim of the study. By means of breeding even 100% donor mtDNA can be reached within a few generations.

Study of clustered DNA lesions in the liver in acute toxic injury

Objective: to test and optimize a method for detecting clustered DNA lesions with an assessment of the quantitative characteristics of hepatic changes using an experimental model of acute toxic hepatitis.Material and methods: Laboratory C57Bl/6 mice at the age of 10 weeks were used for the study. Acute toxic liver injury was induced by means of a single intraperitoneal injection of 30 % CCI4 solution in olive oil. The withdrawal of the animals from the experiment was made after 72 hours. For the detection of clustered DNA lesions, a liver fragment was resected.Results. The electrophoretic parameters which are the most optimal for obtaining primary data for the subsequent calculation of the number of clustered DNA lesions have been proposed. The number of clusters in the DNA samples from the animals of the control group is significantly lower than in the experimental group and amounts to 54.80 [37.65; 59.24] and 76.82 [60.95; 92.41] APE1 clusters per million bp., respectively.Conclusion. Successful testing and optimization of the OCDL method for the detection of clustered lesions in liver DNA have been performed. The study has shown an increase in the number of APE1 clusters and double-strand breaks in the DNA of the C57Bl laboratory mice with induced acute toxic hepatitis, which indicates significant derangement of DNA integrity and a high risk of developing progressive liver diseases in its toxic damage.

Inside Front Cover: Chloroplast gene markers detect diatom DNA in a drowned mice establishing drowning as a cause of death

Inside Front Cover: Chloroplast gene markers detect diatom DNA in a drowned mice establishing drowning as a cause of death

Low-Level Mouse DNA in Conditioned Medium Generates False Positive Cross-Species Contamination Results in Human Organoid Cultures.

Cell line authentication is critical for preventing the use of mixed or misidentified cell lines in research. Current efforts include short tandem repeat (STR) analysis and PCR-based assays to detect mixed species cultures. Using PCR analysis with mouse-specific primers, we identified contaminating mouse DNA in growth factor conditioned medium (CM) derived from the L-WRN cell line (L-WRN CM), as well as in human organoid cultures maintained in the L-WRN CM. DNA isolated from L-WRN CM matched the L-WRN cell signature by STR analysis. Organoid lines that were positive for murine DNA by PCR were further analyzed via bulk RNA-sequencing and transcripts were aligned to the human and mouse genomes. RNA analysis failed to detect mouse-specific gene expression above background levels, suggesting no viable murine cells were present in the organoid cultures. We interpret our data to show conclusive evidence that mouse cell-derived CM can be a source of contaminating murine DNA detected in human organoid cultures, even though live, transcriptionally-active murine cells are not present. Together, our findings suggest that multiple methods may be required to authenticate human organoid or cell lines and urges cautious interpretation of DNA-based PCR cell line authentication results.

CRISPR Turbo Accelerated KnockOut (CRISPy TAKO) for Rapid in vivo Screening of Gene Function.

The development of CRISPR/Cas9 technology has vastly sped up the process of mammalian genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous functional knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes an adaptation of the CRISPR/Cas9-mediated method to develop a cohort of homozygous gene-targeted KO animals in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of KO mice as proof-of-concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9 protein. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. Behavioral testing confirmed the hypothesis that successful one-generation KO of MyD88 would reproduce the decreased ethanol-induced sedative/hypnotic effects and increased ethanol consumption in males that were observed in previous studies. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.

In Memoriam: Mortimer L. Mendelsohn: (1 December, 1925-8 January, 2020).

In Memoriam: Mortimer L. Mendelsohn: (1 December, 1925-8 January, 2020).

Chloroplast gene markers detect diatom DNA in a drowned mice establishing drowning as a cause of death.

Diatoms are unicellular microalgae with cell wall made up of rigid silica found in all open water bodies. They thus resist degradation and hence are important tool to diagnose cause of death in drowned bodies. The nitric acid digestion method practiced conventionally in forensic science laboratories has limitations due to manual error. Plant chloroplast genes found in diatoms such as ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL-3P) and rbcL, universal plastidic amplicon (UPA), and photosynthesis I P700 apoprotein chlorophyll Al (psaA), which play an important role in photosystems I and II of photosynthesis, are tested to diagnose drowning in experimental mice. It was seen that psaA-2 showed amplification at 150bp in all biological samples. The sequences of psaA-2 gene marker showed 100% proximity to Thalassiosira weissflogii and rbcL-3P showed 99% resemblance to Pseudo-nitzschia multiseries. On the other hand, in postmortem drowned biological samples, the chloroplast-based gene marker failed to show any amplification.

Plasma DNA Profile Associated with DNASE1L3 Gene Mutations: Clinical Observations, Relationships to Nuclease Substrate Preference, and In Vivo Correction

SummaryPlasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a “CC” end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.

Large domains of heterochromatin direct the formation of short mitotic chromosome loops.

During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here, we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine nine trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes.

Genome editing with the donor plasmid equipped with synthetic crRNA-target sequence

CRISPR/Cas-mediated genome editing is a powerful tool for generating genetically mutated cells and organisms. Linearisation of donor cassettes with this system has been shown to facilitate both transgene donor insertion and targeted knock-in. Here, we developed a donor plasmid that we name pCriMGET (plasmid of synthetic CRISPR coded RNA target sequence-equipped donor plasmid-mediated gene targeting), in which an off-target free synthetic CRISPR coded RNA-target sequence (syn-crRNA-TS) is incorporated with a multi-cloning site, where a donor cassette can be inserted. With co-expression of Cas9 and the syn-crRNA-TS guide RNA (gRNA), pCriMGET provides a linearised donor cassette in vivo, thereby promoting the transgene donor insertion and targeted knock-in. When co-injected with Cas9 protein and gRNA into murine zygotes, pCriMGET yielded around 20% transgene insertion in embryos. This method also achieved more than 25% in-frame knock-in at the mouse Tbx3 gene locus without predicted insertion–deletion mutations using a transgene donor with 400-bp homology arms. pCriMGET is therefore useful as a versatile CRISPR/Cas9-cleavable donor plasmid for efficient integration and targeted knock-in of exogenous DNA in mice.

Abstract 5056: Quality control efforts in a large-scale, preclinical trial of rare cancer PDXs by the National Cancer Institute's patient-derived models repository (NCI PDMR)

Abstract The National Cancer Institute's Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) is performing a large-scale multi-year preclinical study with 39 PDX models of rare cancers (mesothelioma, MPNST, osteosarcoma, Merkel cell carcinoma, etc) treated with 56 novel therapeutic combinations in an exploratory, n-of-4 arm, study design. Combinations that show promising responses (e.g., regression or durable inhibition of tumor growth) will be repeated along with the single agent arms to determine if the response is driven by the combination or only one of the agents. In order to do this in a timely fashion, relatively speaking, the PDX tumors are serially passaged and each passage is treated with a set of 8 combinations plus relevant vehicle control(s) while in parallel enough PDXs are retained to be expanded for the next passage and drug set. Every serial passage undergoes several quality control assessments that serve as go/no-go criteria including pathology assessment, human:mouse DNA content assessment, and low pass whole genome sequencing to determine the average fraction of genome changed compared to the original donor material. If there is a QC failure, the PDX model is restarted from early passage cryo-material (passage 1-2). An additional quality control effort is to bookend the combination studies with the first set of agents to see if tumor response is similar across passages. To date, most of the models have demonstrated a high degree of stability, though a couple of models have moved toward murine content and have been restarted from early passage material so all drug combinations can be tested. DNA and RNA are retained from all passages so a full NGS evaluation can be performed at a later date. This effort has been ongoing for over a year and the first bookend studies are beginning to be tested to determine if response at first and last passage of the study are consistent with each other, given the constraints of the inherent heterogeneity of the models themselves. Single agent studies of drug combinations that demonstrated a response in 30%-50% of the models tested are also underway to determine which combinations have a more than additive effect compared to the single agents. Promising combinations will be moved forward to early phase clinical trials for these rare cancers. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Biswajit Das, Sergio Y. Alcoser, Suzanne Borgel, Devynn Breen, John Carter, Tiffanie Chase, Alice Chen, Lily Chen, Kristen Cooley, Emily Delaney, Raymond Divelbiss, Lyndsay Dutko, Thomas Forbes, Kyle Georgius, Michelle Gottholm-Ahalt, Tara Grinnage-Pulley, Sierra Hoffman, Chris Karlovich, Shahanawaz Jiwani, Justine Mills, Malorie Morris, Michael Mullendore, Dianne Newton, Rajesh Patidar, Gloryvee Rivera, Howard Stotler, Jesse Stottlemyer, Savanna Styers, Debbie Trail, Shannon Uzelac, Thomas Vilimas, Abigail Walke, Thomas Walsh, Nicole Walters, Peng Wang, P. Mickey Williams, Melinda Hollingshead, James H. Doroshow. Quality control efforts in a large-scale, preclinical trial of rare cancer PDXs by the National Cancer Institute's patient-derived models repository (NCI PDMR) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5056.

Regulatory-Compliant Validation of a Highly Sensitive qPCR for Biodistribution Assessment of Hemophilia A Patient Cells

The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 × 106 intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 × 103 to 1:1 × 106 human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies.

Improvement of cognitive and motor performance with mitotherapy in aged mice.

Changes in mitochondrial structure and function are mostly responsible for aging and age-related features. Whether healthy mitochondria could prevent aging is, however, unclear. Here we intravenously injected the mitochondria isolated from young mice into aged mice and investigated the mitotherapy on biochemistry metabolism and animal behaviors. The results showed that heterozygous mitochondrial DNA (mtDNA) of both aged and young mouse coexisted in tissues of aged mice after mitochondrial administration, and meanwhile, ATP content in tissues increased while reactive oxygen species (ROS) level reduced. Besides, the mitotherapy significantly improved cognitive and motor performance of aged mice. Our study, at the first report in aged animals, not only provides a useful approach to study mitochondrial function associated with aging, but also a new insight into anti-aging through mitotherapy.

Features of regenerative processes in case of extreme factors influencing the body

Changes in the specific liver mass, number of single and binucleate cells of different ploidy, mitotic index and alteration index, DNA content and inclusion of 3H-thymidine in DNA of Wistar rats and CBA mice in the following conditions: at 0°C for 3, 7, 14 days for 23 hours per day, and in the highlands conditions (3, 200 m above sea level) for 3 and 30 days. Activation of regenerative processes has been established — proliferation of hepatocytes (mainly diploid) in combination with polyploid cell share reduction, as well as cellular hypertrophy development. Cell dynamics shifts persisted throughout the observation duration and were opposite in direction when growing up and aging.

Update from the laboratory: mechanistic studies of pathways of cancer-associated venous thrombosis using mouse models.

Cancer patients have an increased risk of venous thromboembolism (VTE). The rate of VTE varies with cancer type, with pancreatic cancer having one of the highest rates, suggesting that there are cancer type-specific mechanisms of VTE. Risk assessment scores, such as the Khorana score, have been developed to identify ambulatory cancer patients at high risk of VTE. However, the Khorana score performed poorly in discriminating pancreatic cancer patients at risk of VTE. Currently, thromboprophylaxis is not recommended for cancer outpatients. Recent clinical trials showed that factor Xa (FXa) inhibitors reduced VTE in high-risk cancer patients but also increased major bleeding. Understanding the mechanisms of cancer-associated thrombosis should lead to the development of safer antithrombotic drugs. Mouse models can be used to study the role of different prothrombotic pathways in cancer-associated thrombosis. Human and mouse studies support the notion that 2 prothrombotic pathways contribute to VTE in pancreatic cancer patients: tumor-derived, tissue factor-positive (TF+) extracellular vesicles (EVs), and neutrophils and neutrophil extracellular traps (NETs). In pancreatic cancer patients, elevated levels of plasma EVTF activity and citrullinated histone H3 (H3Cit), a NET biomarker, are independently associated with VTE. We observed increased levels of circulating tumor-derived TF+ EVs, neutrophils, cell-free DNA, and H3Cit in nude mice bearing human pancreatic tumors. Importantly, inhibition of tumor-derived human TF, depletion of neutrophils, or administration of DNAse I to degrade cell-free DNA (including NETs) reduced venous thrombosis in tumor-bearing mice. These studies demonstrate that tumor-derived TF+ EVs, neutrophils, and cell-free DNA contribute to venous thrombosis in a mouse model of pancreatic cancer.

N6-methylated adenine on the target sites of mamA from Mycobacterium bovis BCG enhances macrophage activation by CpG DNA in mice

CpG motifs in DNA sequences are recognized by Toll-like receptor 9 and activate immune cells. Bacterial genomic DNA (gDNA) has modified cytosine bases (5-methylcytosine [5 mC]) and modified adenine bases (6-methyladenine [6 mA]). 5 mC inhibits immune activation by CpG DNA; however, it is unclear whether 6 mA inhibits immune activation by CpG DNA. Mycobacterium bovis BCG (BCG) has three adenine methyltransferases (MTases) that act on specific target sequences. In this study, we examined whether the 6 mA at the target sites of adenine MTases affected the immunostimulatory activity of CpG DNA. Our results showed that only 6 mA located at the target sequence of mamA, an adenine MTase from BCG, enhanced interleukin (IL)-12p40 production from murine bone marrow-derived macrophages (BMDMs) stimulated with CpG DNA. Enhancement of IL-12p40 production in BMDMs was also observed when BMDMs were stimulated with CpG DNA ligated to oligodeoxynucleotides (ODNs) harboring 6 mA. Accordingly, we then evaluated whether gDNA from adenine MTase-deficient BCG was less efficient with regard to stimulation of BMDMs. Indeed, gDNA from a mamA-deficient BCG had less ability to activate BMDMs than that from wild-type BCG. We concluded from these results that adenine methylation on ODNs and bacterial gDNA may enhance immune activity induced by CpG DNA.