Monday, 25 October 2010

What are the barriers to Efficient Gene Transfer?

DNA, the common carrier of the genetic information for all living entities on this planet, is omnipresent and we are daily exposed to large quantities of foreign DNA (e.g., by food or bacterial infections). Under these circumstances, nature had to provide powerful barriers against the spontaneous insertion of foreign DNA sequences into the genomic DNA of cells. Barriers are the plasma membrane of the cell, the envelope of the cell’s nucleus, but also the possibility for DNA degradation in lysosomes and the cytoplasm. These protective mechanisms work rather well and even under optimized conditions it is by no means easy to genetically modify an eukaryotic cell (the terminus usually employed for this modification is to “transfect” the cell). However, the necessity to transfect cells for research purposes, the discovery of new and efficient reporter systems to verify the success of a transfection experiment (luciferase, green fluorescent protein) as well as the availability of powerful transfection reagents have spurred research in the area for many years. Several methods to transfer genes into cells have been developed during the last 30 years. However, considerable efforts to develop new techniques or to improve the efficiency of old ones are still being made.

Transfection reagents help to overcome the natural barriers to gene transfer by various strategies.

The steps involved in the transfer of a “gene” from the outside into the genome of the cell comprise of the following:

1. Compaction of the DNA,

2. Attachment to the cell surface,

3. Transport into the cytoplasm,

4. Import into the nucleus and

5. Insertion into the chromosomal DNA.

The mechanism by which a certain barrier is overcome is an important feature of the respective transfection reagent. In order to elucidate the difficulties in optimizing the genetic engineering of mammalian cells, the major steps of transfection as well as putative agents for reaching this goal will be discussed in detail in the following sections. The mechanisms for many of the above-mentioned five steps of transfection are still under discussion. This is especially the case for the later steps taking place inside the cell, i.e., transport into the cell and most importantly into the nucleus. The earlier stages of compaction and interaction with the cell surface are better understood. This has important consequences for our current ability to engineer transfection agents and procedures. It should be noted that man-made transfection procedures are still orders of magnitude less efficient than nature’s transfection agents, the viruses are. One to five infectious particles, i.e., viruses, per cell are sufficient in that case, compared to the 105– 106 plasmid molecules needed in most nonviral transfection methods.