Purpose of Method:
The MaRX method is a strategy for applying genetic techniques to mammalian
cells. Use of this technique allows for efficient
genetic
manipulation of mammalian cells and subsequent phenotypic screening.
Background Information:
Mammalian cells have always posed a challenge to molecular biologists because
of their asexual diploid nature. Mammalian cells cannot be mated
with each other to produce more genetically complex mutants, nor can they
be manipulated by plasmids. They are inaccessible to most common
genetic methods. In short, the only genetic technique available for use
on these cells is mutagenesis or viral infection. The MaRX system
outlines a clever approach to using these available techniques to overcome
the obstacles that mammalian cells in tissue culture present.
How the design works:
| # | Multistep Process | Comments |
| 1 | A special retrovirus is used as the vector to insert cDNA’s from a library into normal mammalian cells. These retroviruses are specially designed not to replicate themselves, and thus serve as a good tool to simply insert cDNAs into the chromosomes of mamalian cells. | Cells are infected from a library of different cDNAs in this manner. |
| 2 | Then, these infected cells are phenotypically screened on the basis of some biological characteristic (the focus of the study). | r |
| 3 | From these phenotypically isolated cells the provirus (cDNA inserted into the genome by the retrovirus) is then excised by means of recombinase enzyme that cuts the insert out at specifically designed recombination sites. | This step, which is essentially a reversion test, allows the experimenter to determine whether the desired phenotype was actually the positive result of an inserted cDNA or merely the result of the disruption of a normal gene. |
| 4 | A library of recovered proviruses is made from the excision products of the recombinase enzyme. | "The ability to resuce a library of functional proviruses from a selected cell population allows a gene to be enriched from a complex mixture through multiple rounds of phenotypic selection." (1) |
| 5 | The new phenotypcally distinct cells are then phenotypically screened again through stricter selection criteria. | r |
Figure 1.
The MaRX screening cycle. A cDNA library is converted into a library
of retroviruses by suing a packaging cell line (LinX). Then,
these infected recipient cells are selected or enriched on the basis of
a specific biological property. Proviruses are recovered and used
for virus production and subsequent rounds of screening. (Figure
adpated from Science, Vol.283 Feb 19, 1999)
1. Hannon, G.J, Sun P, Carnero A, Ying Xie L,
Maestro R, Conklin DS, Beach D. MaRX: an
approach to genetics in mammalian
cells. Science 1999. Feb. (283): 1129-1130
2. Edelson, Stevens. Gentica: functional genomics in vitro. BioCentury 1998. September A9
3. Campbell, N. A. Biology, 4th ed. Menlo Park, CA: Benjamin/Cummings
Publishing Company, Inc., 1996.
p. 372-374.
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