The cells that form the multi-cellular organism control its responses to various extracellular signals by employing dynamic inter- and intra-cellular networks of biological molecular interactions. The recent development of fluorescence imaging technologies has made it possible to investigate how these molecular network systems function when a signal from the outer world is received. However, in most cases, researchers use cell extracts or membrane-permeable cells to introduce the fluorescence-labeled protein into the intracellular system in question. Moreover, even when the fluorescent proteins are expressed in living cells, this involves the introduction of vectors and the cell is observed under a condition where the protein is over-expressed. In contrast, in living cells, the expression level and cytological localization of each protein is precisely regulated, which facilitates their proper functions. Thus, the observations made by studies such as those described above may not be reflective of the protein in its intrinsic physiological condition. If we wish to analyze the real dynamics of multiple target proteins by introducing their genes (cDNAs) into living cells, we need to integrate the genes, which are regulated by their own promoter, into a definite site on the chromosome, as this is the native state of the intrinsic genes in the genome.

This department has developed further the multisite Gateway (msGW) technology, which was originally created and developed by Invitrogen Co. We are now applying this technology to the construction of multi-gene expression clones that permit the simultaneous introduction of the genes into culture cells. These clones are designed to regulate the expression of the genes at a physiological level. Our research subjects thus include:

1. Construction of msGW vectors for the high-throughput introduction of multiple genes labeled by fluorescence protein tags.
2. Development of proper promoters and IRES signals to ensure expression is at physiological levels, and development and improvement of fluorescence protein tags for bio-imaging.
3. Development of technology to control the expression levels of the transgenes, and development of methods to quantify the fluorescently-tagged products when they are expressed at low levels.
4. Introduction of multiple genes simultaneously and stably into definite sites on the chromosome to obtain superior transformed cell lines.

Fig: 1 Multisite Gateway cloning technology.

Fig. 2 Generation of msGW clones for multiple purposes.

Fig. 3 Introduction of an expression clone into a targeted site of the cellular chromosome.