Context-dependent DNA methylation signatures in animal livestock.

DNA methylation is an important epigenetic modification that is widely conserved across animal genomes. It is widely accepted that DNA methylation patterns can change in a context-dependent manner, including in response to changing environmental parameters. However, this phenomenon has not been analyzed in animal livestock yet, where it holds major potential for biomarker development. Building on the previous identification of population-specific DNA methylation in clonal marbled crayfish, we have now generated numerous base-resolution methylomes to analyze location-specific DNA methylation patterns. We also describe the time-dependent conversion of epigenetic signatures upon transfer from one environment to another. We further demonstrate production system-specific methylation signatures in shrimp, river-specific signatures in salmon and farm-specific signatures in chicken. Together, our findings provide a detailed resource for epigenetic variation in animal livestock and suggest the possibility for origin tracing of animal products by epigenetic fingerprinting.

Versatile seamless DNA vector production in E. coli using enhanced phage lambda integrase.

Seamless DNA vectors derived from bacterial plasmids are devoid of bacterial genetic elements and represent attractive alternatives for biomedical applications including DNA vaccines. Larger scale production of seamless vectors employs engineered Escherichia coli strains in order to enable tightly regulated expression of site-specific DNA recombinases which precisely delete unwanted sequences from bacterial plasmids. As a novel component of a developing lambda integrase genome editing platform, we describe here strain MG1655-ISC as a means to easily produce different scales of seamless vectors, ranging in size from a few hundred base pairs to more than ten kilo base pairs. Since we employed an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence, the resulting seamless vectors will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences. Future inclusion of single cognate sites for other genome targeting systems could enable modularity. These features, together with the demonstrated simplicity of in vivo seamless vector production, add to their utility in the biomedical space.

A non-viral genome editing platform for site-specific insertion of large transgenes.

BACKGROUND: The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA-despite being successful in E. coli and animal models-still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. METHODS: Previously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a 'gene of interest'. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. RESULTS: The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs. CONCLUSION: We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.

A novel λ integrase-mediated seamless vector transgenesis platform for therapeutic protein expression.

Advances in stem cell engineering, gene therapy and molecular medicine often involve genome engineering at a cellular level. However, functionally large or multi transgene cassette insertion into the human genome still remains a challenge. Current practices such as random transgene integration or targeted endonuclease-based genome editing are suboptimal and might pose safety concerns. Taking this into consideration, we previously developed a transgenesis tool derived from phage λ integrase (Int) that precisely recombines large plasmid DNA into an endogenous sequence found in human Long INterspersed Elements-1 (LINE-1). Despite this advancement, biosafety concerns associated with bacterial components of plasmids, enhanced uptake and efficient transgene expression remained problematic. We therefore further improved and herein report a more superior Int-based transgenesis tool. This novel Int platform allows efficient and easy derivation of sufficient amounts of seamless supercoiled transgene vectors from conventional plasmids via intramolecular recombination as well as subsequent intermolecular site-specific genome integration into LINE-1. Furthermore, we identified certain LINE-1 as preferred insertion sites for Int-mediated seamless vector transgenesis, and showed that targeted anti-CD19 chimeric antigen receptor gene integration achieves high-level sustained transgene expression in human embryonic stem cell clones for potential downstream therapeutic applications.

Genome wide association study of uric acid in Indian population and interaction of identified variants with Type 2 diabetes.

Abnormal level of Serum Uric Acid (SUA) is an important marker and risk factor for complex diseases including Type 2 Diabetes. Since genetic determinant of uric acid in Indians is totally unexplored, we tried to identify common variants associated with SUA in Indians using Genome Wide Association Study (GWAS). Association of five known variants in SLC2A9 and SLC22A11 genes with SUA level in 4,834 normoglycemics (1,109 in discovery and 3,725 in validation phase) was revealed with different effect size in Indians compared to other major ethnic population of the world. Combined analysis of 1,077 T2DM subjects (772 in discovery and 305 in validation phase) and normoglycemics revealed additional GWAS signal in ABCG2 gene. Differences in effect sizes of ABCG2 and SLC2A9 gene variants were observed between normoglycemics and T2DM patients. We identified two novel variants near long non-coding RNA genes AL356739.1 and AC064865.1 with nearly genome wide significance level. Meta-analysis and in silico replication in 11,745 individuals from AUSTWIN consortium improved association for rs12206002 in AL356739.1 gene to sub-genome wide association level. Our results extends association of SLC2A9, SLC22A11 and ABCG2 genes with SUA level in Indians and enrich the assemblages of evidence for SUA level and T2DM interrelationship.

Aquatic model for engine oil degradation by rhamnolipid producing Nocardiopsis VITSISB.

The present study was focused on isolation, screening, characterization and application of biosurfactant producing marine actinobacteria. Twenty actinobacteria were isolated from marine water sample and were primarily screened for biosurfactant production using hemolytic activity method. Among the 20 isolates, six showed positive result for hemolytic activity and those were taken for further secondary screening tests such as oil collapse method, oil spreading method and emulsification method. From the results of secondary screening analysis, two isolates (SIS-3 and SIS-20) were selected and further used to carry out biosurfactant characterization test such as pH, density, surface tension and viscosity determination. Comparing biosurfactant characterization results, SIS-3 was chosen for further analysis and application. FT-IR and GC-MS were carried out for analysis of biosurfactant from isolate SIS-3 and the compound detected was rhamnolipid. The isolate (SIS-3) was identified as Nocardiopsis using 16S rRNA gene sequencing and named as 'Nocardiopsis VITSISB' (KC958579) which was further applied for immobilizing whole cells for engine oil degradation by constructing an aquatic model and using natural products such as soybean meal, sugarcane juice as nutrient source. The oil was efficiently degraded by rhamnolipid producing Nocardiopsis VITSISB (KC958579) within 25 days which indicated that the strain can act as a natural candidate for the bioremediation of oil spill in ocean.

Cytotoxicity, Genotoxicity, and Phytotoxicity of Tannery Effluent Discharged into Palar River Basin, Tamil Nadu, India.

Ambur, a town located on the banks of Palar River, is considered one of the most polluted areas in India and occupied by hundreds of tanneries and leather product units. The present study was designed to evaluate the toxic effect of discharged tannery effluent (TE) on model agricultural crops, ecofriendly microorganisms, and human blood cells. The phytotoxic effects of TE tested on Allium cepa and Lemna minor revealed inhibition of root growth and significant reduction in number of fronds, protein, and chlorophyll content. Moreover, TE induced chlorosis and tissue necrosis in Nostoc muscorum at low concentration (10%). TE has also negative impact on ecofriendly microorganisms, Bacillus thuringiensis, Rhizobium etli, and Aspergillus terreus which play an important role in the nutrition of plant growth. The genotoxicity of TE was investigated in human leukocytes which showed interference with normal mitotic division with subsequent cell lysis. It also intervened with the normal replication process and induced micronucleus formation in the healthy leukocyte. 5% concentration of TE has been revealed to be toxic to erythrocytes. From this study TE found in the Palar River of Ambur has adverse effects on all the three levels of organisms in ecosystem even at lower concentrations.