? The overall objective of this proposal is to develop a novel E. coli-based vector system for the modification of large pieces of genomic DNA and their functional delivery into mammalian cells. Ordered libraries of large BAC clones that cover >90% of the human genome are readily available. These BAC clones contain human genes complete with surrounding genomic sequences including endogenous promoters, introns, and cis-acting regulatory elements that will ensure accurate spatio-temporal gene expression. However, expression and functional studies using these valuable BAC libraries has been limited by difficulties in both modifying such large DNA clones and delivering them into mammalian cells. Therefore, we propose to develop a novel E. coli based vector system in BAC host strain DH10B that includes 1) convenient modification of BAC DNA by transient inducible expression of E. coli homologous recombination machinery and 2) delivery of DNA into mammalian cells via bacterial invasion by expression of the Y. pseudotuberculosis invasin gene. This process takes place entirely in the host E. coli without a requirement to physically isolate large DNA molecules and will facilitate the use of large DNA constructs for expression and functional studies.
Three Specific Aims for the development of this system are proposed. 1) The invasive E. coli vector system will be optimized for delivery of pGFP-neo reporter plasmid in a variety of transformed and primary cultured mammalian cells. 2) Human genomic BACs will be modified and conditions determined for their intact delivery to mammalian cells for expression studies. 3) Human Artificial Chromosome (HAC) formation will be examined by construction of improved vectors and their controlled delivery into mammalian cells. This proposed research will permit the study of tissue and temporal specificity of gene expression in a wide variety of mammalian cells. It will provide novel gene delivery vectors, including HAC development, for gene expression, which will be valuable for development of gene therapy strategies for treatment of human genetic metabolic diseases and chronic diseases. ? ? ?
| Narayanan, Kumaran; Sim, Edmund Ui-Hang; Ravin, Nikolai V et al. (2009) Recombination between linear double-stranded DNA substrates in vivo. Anal Biochem 387:139-41 |
| Ooi, Yaw-Shin; Warburton, Peter E; Ravin, Nikolai V et al. (2008) Recombineering linear DNA that replicate stably in E. coli. Plasmid 59:63-71 |
