- ADJUNCT ASSISTANT PROFESSOR Genetics and Genomic Sciences
Ph.D., University of Melbourne
E. coli vector for delivery of high molecular weight DNA into mammalian cells
Intracellular bacteria such as Shigella, Yersinia, and Salmonella, have evolved the capability to invade mammalian cells to establish pathogenicity. In collaboration with Catherine Grillot-Courvalin (Pasteur Institut), my lab has adapted an E. coli vector expressing the Y. pseudotuberculosis invasin (inv) gene to enter mammalian cells and deliver high molecular weight BAC clones up to 200 kb in size. The internalized bacterium is engineered with a cell-wall defect to release its DNA in the cytoplasm, where some of them escape into the nucleus for expression. Using this invasive E. coli vector, intact human genomic loci recovered from BAC libraries can be easily modified using recombineering and delivered, without DNA purification, directly into mammalian cells for functional studies. Efforts are ongoing to improve the efficiency of this vector to deliver high-molecular weight DNA into various mammalian cells.
Recombineering: recombinogenic engineering of DNA in vivo
My lab previously developed a recombineering system that permits in vivo modification of DNA substrates using homologous recombination in E. coli. Because homologous recombination does not require presence of suitably placed restriction sites, recombineering technology permits facile manipulation of very large DNA including BAC clones and the E. coli chromosome. Recombineering enables various manipulations of full-length genomic inserts within BAC clones and the E. coli chromosomal loci, including i) point mutations, ii) deletions, iii) insertions, and iv) gene fusions. This system has also been optimized for stable manipulation of highly repetitive DNA where large stretches of the repeats have been known to potentially trigger instability by uncontrolled self-recombination. Current work in the lab is directed to better understand the action of the recombineering enzymes on various DNA substrates and to advance improved applications using this system.
Artificial chromosome vectors
Current research is focused to develop artificial chromosome vectors for long-term gene delivery to cells. Artificial chromosome will overcome many of the limitations of previous gene therapy vectors because artificial chromosomes 1) will segregate accurately during cell division to provide long-term gene expression, 2) will avoid problems associated with integration into the genome, such as insertional mutagenesis and transgene silencing caused by position effect, and 3) can deliver therapeutic genes along with surrounding regulatory sequences that will provide correct spatial and temporal control of expression pattern.
Chen Q, Narayanan K. Crude protein extraction protocol for phage N15 protelomerase in vitro enzymatic assays. Analytical biochemistry 2011 Jul; 414(1).
Narayanan K, Chen Q. Bacterial artificial chromosome mutagenesis using recombineering. Journal of biomedicine & biotechnology 2011; 2011.
Lee CW, Ng AY, Bong CW, Narayanan K, Sim EU, Ng CC. Investigating the decay rates of Escherichia coli relative to Vibrio parahemolyticus and Salmonella Typhi in tropical coastal waters. Water research 2011 Feb; 45(4).
Sim EU, Ang CH, Ng CC, Lee CW, Narayanan K. Differential expression of a subset of ribosomal protein genes in cell lines derived from human nasopharyngeal epithelium. Journal of human genetics 2010 Feb; 55(2).
Narayanan K, Sim EU, Ravin NV, Lee CW. Recombination between linear double-stranded DNA substrates in vivo. Analytical biochemistry 2009 Apr; 387(1).
Lee CW, Ng AY, Narayanan K, Sim EU, Ng CC. Isolation and characterization of culturable bacteria from tropical coastal waters.. Ciencias Marinas 2009; 35.
Narayanan K. Intact recombineering of highly repetitive DNA requires reduced induction of recombination enzymes and improved host viability. Analytical Biochemistry 2008; 375.
Ooi YS, Warburton PE, Ravin NV, Narayanan K. Recombineering linear DNA that replicate stably in E. coli. Plasmid 2008; 59.
Narayanan k, warburton pe. DNA modification and functional delivery into human cells using Escherichia coli DH10B. Nucleic Acids Res 2003; 31.
Jamsai d, Nefedov n, Narayanan k, Ioannou pa, Fucharoen s, Williamson r, Orford m. Insertion of common mutations into the B-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette. J. Biotechnol 2003; 101.
Narayanan k, Stewart af, Zhang y, Williamson r. Efficient and precise engineering of a 200 kb B-globin human/bacterial artificial chromosome in E. coli DH10B using an inducible homologous recombination system. Gene Ther 1999; 6.
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