1.4.1 Genetics in mammalian cells
The use of genetic manipulation aiming at the specific delivery and expression of ectopic genes in a genetically defined organism is a powerful tool for the discovery and characterisation of biological pathways. In particular, over the years, this
approach has been extensively applied to relatively simple unicellular organisms such as bacteria and, most importantly, yeast. The use o f lower eukaryotes is still the preferred way to study basic biological pathways and it is indeed a very useful approach. There are two main reasons for this. Firstly, almost invariably genes that are found to be important in yeast have a homologue with a similar function in
mammalian cells and therefore their characterisation in the lower eukaryote is relevant to the biology of more complex organisms. Secondly, genetics in yeast is technically relatively simple and the experimental procedures in use have been optimised over the years and are very reliable (Botstein, 1988).
Having said that, it is clear that a similar approach directly on mammalian cells could prove even more informative, especially when applied to the characterisation of more cell specific pathways that are not shared by all cell types. For example, the discovery of the cell cycle genes and their characterisation in yeast was extremely useful and the information gathered in this system could very effectively be applied to mammalian cells (Nasmyth, 1996). This is because the cell cycle mechanism is such a
from yeast to humans. Unfortunately this does not apply for those specialised functions that characterise cells o f more complex multicellular organisms. For example the biological pathway leading to melanin production is such a specific activity o f pigment cells that it can only be studied and characterised in melanocytes.
When facing the challenge of developing a tool that allows genetic manipulation of mammalian cells in a similar way to what is commonly done in bacteria and yeast, the following requirements need to be fulfilled:
a. Efficient delivery o f the genomic information b. Stable delivery of the genomic information c. Recovery of the genomic information
Over the years many vectors have been devised for the successful delivery o f genomic information in mammalian cells. Normally DNA in mammalian cells is introduced using chemical methods that allow to bypass the physical barrier represented by the cellular membrane. The most commonly used protocols consist in precipitating the DNA in calcium phosphate salts which are in turn internalised by the cells (Graham,
1973), or in enveloping the DNA in liposomal vesicles that can subsequently fuse with the plasma membrane delivering the DNA into the cells (Mannino, 1988). Although these methodologies can be quite efficient, depending on the cell line used, they have numerous limitations. For example the number of DNA molecules
molecules being introduced in one cell. This is not a problem if the DNA molecules introduced in a population are all the same, but it is not acceptable in the case, for example, of cDNA library screening where one has millions o f different clones that need to be examined according to the biological properties they confer on the cell. For such an application, one would ideally be able to introduce only one DNA molecule per cell, so that the phenotype observed can be related to a specific transcript.
Another major limitation o f common transfection methods is their inherent instability. Unlike vectors used for bacteria or yeast transformation, those employed for
mammalian transfections are not recognised by the cell as endogenous genomic information. This means that the plasmids are not duplicated when the cell divides and, over time and cell duplications, the vector DNA is lost resulting in a decrease in the population of tranfected cells still carrying the information originally delivered. A way to get around this problem is to introduce into the vector DNA a drug selection marker so that, in the presence o f the appropriate drug, cells that lose the vector can be selected against and cannot overtake the transfected population. If the selection is carried out long enough, the result will be to select for a clonal population that have integrated the vector DNA in their genome thereby becoming stably transfected. Unfortunately this is a relatively rare event and it can take up to three months before a stably transfected cell line is produced from a transient transfection.
Finally, in order to do genetics in mammalian cells it is fundamental to be able to recover the genetic information originally delivered. This is particularly important in
the case o f functional screenings of complex cDNA libraries where nothing is known o f the cDNA introduced in the cells and the selection is based on phenotypic changes.
1.4.2 Retroviral vectors
Retroviral vectors, especially those based on Murine Leukemia Virus (MLV), have been successfully used in the last few years for genetically modifying mammalian cells and they present a number of advantages over more conventional transfection methods (Hu and Pathak, 2000). First, being based on retroviruses, the delivery o f the genetic material goes through an infection step and most cell lines, provided that they are actively dividing, can be efficiently infected by MLV. Second, the number of viruses used on the target cells can be titrated so that statistically there is not more then one infectious event per cell, eliminating the issue o f more then one cDNA molecule being introduced in the same cell. Finally, and perhaps most importantly, every single infection event automatically becomes a stable transfection. This is an intrinsic characteristic of the system and is a consequence o f the retroviruses life cycle. Retroviruses are so called because, unlike most living organisms, their genome is made of two identical molecules of RNA. Upon infection of the target cell, the viral RNA genome is reverse-transcribed by a retroviral specific protein (the reverse
transcriptase) into a double strand molecule o f DNA (Fig 1.5). This in turn actively integrates into the target cell DNA becoming part of the host genomic information and as such is transmitted to all the cell progeny. This means that a cell infected by a
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