Chapter 5. General conclusions and future prospective 5.1. General conclusions In order to monitor gene transfer between Agrobacterium tumefaciens and mammalian cells, a sensitive detection system was developed, which takes advantage of both the viral elements and the EGFP reporter system. This system is superior over the utilization of antibiotics-resistance marker because it does not depend on stable integration of the transferred DNA. Using this system, the direct transfer of both plasmid and chromosome DNA from Agrobacterium into mammalian cells has been demonstrated. This process is dependent upon Agrobacterium and independent of the virulence genes. The frequency of gene transfer is approximately 10-4-10-5 transformants per recipient. E. coli and R. meliloti cells could not mediate such horizontal gene transfer (HGT) into mammalian cells in similar experiment conditions. Studies on mutant strains of Agrobacterium revealed that some bacterial genes such as chvG, chvH and katA, were involved in efficient Agrobacterium-mediated gene transfer into mammalian cells, suggesting that Agrobacterium cells may be actively involved in the gene transfer and the gene transfer is not a simple uptake of DNA released by cell lysis. Moreover, the interaction between A.tumefaciens and mammalian cells were studied using GFP as an efficient reporter. The results showed that Agrobacterium could efficiently attach to and invade into mammalian cells. The polymerization of mammalian cell actin might play important roles in these processes, as the addition of polymerization inhibitor, Cytochalasin D, could decrease both the rate of internalization and DNA delivery efficiency significantly. 155 Although the occurrence of horizontal gene transfer among prokaryotes is well established, the gene transfer between bacteria and eukaryotic cells is less well elucidated. The finding that non-specific DNA transfer from Agrobacterium into the mammalian cells might provide direct evidence to support the HGT between bacteria and eukaryotes. As a two-component regulatory system, ChvG/ChvI play important roles in plant transformation. In this study, ChvG was found to be involved in DNA transfer into mammalian cells and the regulation of acid-inducible genes, including aopB and katA, residing on the circular and linear chromosomes respectively, and the Tiplasmid-harbored vir genes, virB and virE. Thus, ChvG might be a global sensor protein that can directly or indirectly sense extracellular acidity since it can regulate unlinked acid-inducible genes encoding different functions in different ways. 5.2. Future prospective Agrobacterium was demonstrated to be able to deliver its DNA into mammalian cells. Nonetheless, the precise mechanism of gene transfer is still unclear. It will be necessary to identify the specific endogenous factor(s) of A. tumefaciens required for the efficient mammalian cell gene transfer. By identification and characterization of these factors, more information about the horizontal gene transfer between bacteria and mammalian cells could be yielded. Moreover, the studies were conducted in artificial environment; whether such DNA transfer indeed occurs in nature need further studies. So far, bacteria that have been used as gene deliver vectors are all human pathogens (Salmonella and Shigella) or harbored virulence gene(s) from pathogens. Thus the safety issue of these bacteria should be concerned when used in vivo. As 156 Agrobacterium is nonpathogenic to human, it may be used as a DNA vector with less safety concerns. The high DNA transfer efficiency of AG6 strain might allow us to develop an efficient human cell gene delivery or a good vehicle for DNA vaccination. The feasibility of using this system for DNA delivery needs to be investigated. In addition, ChvG has been proved to be involved in the expression of acidic pH-inducible genes, but the mechanism is still unclear. Detailed studies is necessary in order to get more information for a better understanding of the signal transduction pathway between Agrobacterium and its host cells. 157 . efficient Agrobacterium- mediated gene transfer into mammalian cells, suggesting that Agrobacterium cells may be actively involved in the gene transfer and the gene transfer is not a simple uptake of. independent of the virulence genes. The frequency of gene transfer is approximately 10 -4 -10 -5 transformants per recipient. E. coli and R. meliloti cells could not mediate such horizontal gene transfer. Chapter 5. General conclusions and future prospective 5. 1. General conclusions In order to monitor gene transfer between Agrobacterium tumefaciens and mammalian cells, a sensitive