Retargeting of pre-set regions on chromosome for high gene expression in mammalian cells

We have developed a system to hunt and reuse special gene integration sites that allow for high and stable gene expression. A vector, named pRGFP8, was constructed. The plasmid pRGFP8 contains a reporter gene, gfp2 and two extraneous DNA fragments. The gene gfp2 makes it possible to screen the high expression regions on the chromosome. The extraneous DNA fragments can help to create the unique loci on the chromosome and increase the gene targeting frequency by increasing the homology. After transfection into Chinese hamster ovary cells (CHO) cells, the linearized pRGFP8 can integrate into the chromosome of the host cells and form the unique sites. With FACS, 90 millions transfected cells were sorted and the cells with strongest GFP expression were isolated, and then 8 stable high expression GFP CHO cell lines were selected as candidates for the new host cell. Taking the unique site created by pRGFP8 on the chromosome in the new host cells as a targeting locus, the gfp2 gene was replaced with the gene of interest, human ifngamma, by transfecting the targeting plasmid pRIH-IFN. Then using FACS, the cells with the dimmest GFP fluorescence were selected. These cells showed they had strong abilities to produce the protein of interest, IFN-gamma. During the gene targeting experiment, we found there is positive correlation between the fluorescence density of the GFP CHO host cells and the specific production rate of IFN-gamma. This result shows that the strategy in our expression system is correct: the production of the interesting protein increases with the increase fluorescence of the GFP host cells. This system, the new host cell lines and the targeting vector, can be utilized for highly expressing the gene of interest. More importantly, by using FACS, we can fully screen all the transfected cells, which can reduce the chances of losing the best cells.

Supporting Reuse and Evolution in Software Design

Program design is an area of programming that can benefit significantly from machine-mediated assistance. A proposed tool, called the Design Apprentice (DA), can assist a programmer in the detailed design of programs. The DA supports software reuse through a library of commonly-used algorithmic fragments, or cliches, that codifies standard programming. The cliche library enables the programmer to describe the design of a program concisely. The DA can detect some kinds of inconsistencies and incompleteness in program descriptions. It automates detailed design by automatically selecting appropriate algorithms and data structures. It supports the evolution of program designs by keeping explicit dependencies between the design decisions made. These capabilities of the DA are underlaid bya model of programming, called programming by successive elaboration, which mimics the way programmers interact. Programming by successive elaboration is characterized by the use of breadth-first exposition of layered program descriptions and the successive modifications of descriptions. A scenario is presented to illustrate the concept of the DA. Technques for automating the detailed design process are described. A framework is given in which designs are incrementally augmented and modified by a succession of design steps. A library of cliches and a suite of design steps needed to support the scenario are presented.