EMD - Calbiochem: Antibiotics Resource

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Overview
Antibiotics can be classified into several groups by their mechanism of action. These include inhibition of bacterial wall, protein, RNA, and DNA synthesis. They can also interfere with essential cell processes such as microtubule function. Antibiotics are used to give the desired organism a selective advantage over the undesired organism. For example, antibiotics such as ampicillicin which inhibits cell wall synthesis, selectively destroy bacterial cells over mammalian cells since mammalian cells lack a cell wall. Other antibiotics such as bleomycin, which damages DNA and prevents repair, act on all cells that possess DNA. These types of antibiotics are more toxic to rapidly dividing cells, such as neoplasms. However, they also exert their toxic effects on other mammalian cells.

Mechanisms by which cells become antibiotic-resistant include inactivation of the antibiotic, mutation of the drug target, removal by efflux, and over expression of the drug target. Examples of these mechanisms include the amp^r gene that codes for b-lactamase and the ble gene that codes for bleomycin resistance. These genes encode proteins that inactivate the drug. Alternatively, in the case of paclitaxel resistances the multi-drug-resistance (mdr) gene is overexpressed; the expressed mdr protein pumps out the toxic drug. Although resistance is a growing problem in medical treatment, molecular biologists routinely exploit these resistance genes to select out populations of cells which express a gene of interest. In general, a plasmid is created which contains both the gene of interest, and the resistance gene. Cells are transfected with the plasmid, allowed to recover, and treated with the antibiotic corresponding to the expressed resistance gene. The sensitivity of the cell population to antibiotics depends on several factors. These include the rate of division of the cell, the baseline level of toxicity and whether or not the parent (untransfected) cell line has become resistant. Many commonly-used cell lines have become resistant to multiple antibiotics usually by acquiring a resistance plasmid. In addition, they are very susceptible to mutations that may lead to resistance.

The length of time required to select cells is an important factor. The longer cells are exposed to antibiotics, the more likely that resistant populations will emerge that, may not contain the gene of interest. For example selection with BLEOCIN™, a highly toxic compound, will be faster and more specific than selection with other types of drugs. Due to the number of variables, it is best to perform a kill-curve on your cell line of interest with the antibiotic that is to be used. A rough kill-curve can be generated by plating identical numbers of cells in dishes, or wells, of a cell culture plate and incubating with increasing concentrations of antibiotic for approximately two weeks.

Calbiochem^� offers a wide variety of antibiotics for cell selection as well as for other purposes. We strive to provide the highest quality antibiotics for the best value.

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Drugs for the Selection of Genetic Markers