Technology and innovation
Innovations that are required to deliver safe and efficient gene therapy demand a detailed knowledge of many aspect of nuclear function. But in particular require that we learn what happens when we try to mimic the natural behaviour of a gene by expressing it from an extra-chromosomal DNA molecule. There are many problems in doing this. The first crucial one to understand is seen when we look at what happens following introduction of the gene delivery vector into a population of cells.
This figure shows the wide range of expression that is seen when standard transfection protocols are used. Cells have a wide range of reporter gene expression shown by the intensity of green fluorescence - and this reflects the range of episomal genes within the different cells. The vector also contains a gene that confers resistance to a cytotoxic drug, so that by growing cells under selection it is possible to clone populations in which the vector gene is efficiently expressed. In fact, while many hundreds of cells have the vector immediately following transfection, only 1-2% of these will usually grow to make clones in which the reporter gene is efficient expressed and retained. A typical clone is shown right column and it is clear that all cells now have similar levels of reporter gene expression.
A crucial part of the project is to understand what happens in the cells when the stable clones are established. We know that the changes are not genetic the DNA sequence of the vector is the same after stable clones have formed so they must reflect how the DNA interacts with protein and how this chromatin.