Selectable Markers and Gene Amplification in Animal Cells

Like all of the other DNA transfer processes we have discussed, the incorporation of foreign DNA into animal cells needs to be accompanied by a phenotypic change to distinguish the transfected cells from those that have not taken up the foreign DNA (Figure 12.7). Some of the first experiments to identify transfected animal cells involved the complementation of a nutritional defect in a cell line. For example, human cells defective in the gene encoding the enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT; part of

Figure 12.7. The structure of the bleomycin-bleomycin binding protein complex (Maru-yama etal., 2001). Binding sites for the drug (shown in red) are located in deep clefts in the sides of the dimeric protein (green and blue)

the inosinate cycle for the salvage of purine bases and the production of inosine monophosphate) and therefore unable to grow in medium containing hypoxanthine, aminopterin and thymidine (HAT, which blocks de novo inosine monophosphate production (Lester etal., 1980)) were transfected with total genomic DNA from a wild-type cell line. Very rarely, cells could be isolated that were able to grow in this medium, indicating that they had acquired the ability to make HPRT from the wild-type cells (Szybalska and Szybalski, 1962). Similarly, mouse cells deficient in the enzyme thymidine kinase (TK, which is part of the nucleotide biosynthesis salvage pathway) are unable to grow on HAT medium, but can be transfected with the herpes simplex virus (HSV) Tk gene to allow growth on this medium (Wigler etal., 1977). A number of other such metabolic markers have also been used to monitor the transfec-tion process, but they all suffer from the requirement of a mutant cell line in order to detect transformed cells. This need has been overcome by using dominant selectable markers that confer a drug resistance phenotype to the transfected cells (Table 12.1). Antibiotics such as ampicillin have no effect on eukaryotic cells due to the lack of an animal cell wall. Some other antibiotics, particularly those that are protein synthesis inhibitors, are active against both prokaryotes and eukaryotes. For example, the E. coli transposon Tn5 encodes the kanamycin-resistance gene (Yamamoto and Yokota, 1980). Kanamycin is an aminoglycoside antibiotic that interferes with translation and induces bacterial cell death through site-specific targeting of ribosomal 16S RNA. At higher concentrations, aminoglycosides also inhibit protein synthesis in mammalian cells probably through non-specific binding to eukaryotic ribosomes and/or nucleic acids (Mingeot-Leclercq, Glupczynski and Tulkens, 1999). To achieve resistance as a method of selection, another gene encoded within the Tn5 transposon (producing neomycin phosphotransferase) is placed under the

Table 12.1. Markers for the selection of DNA fragments added to higher eukaryotic cells

Marker

Gene product

Selection method

Reference neo Aminoglycoside phosphotransferase; neo gene from the bacterial transposon Tn5 hyg Hygromycin-B-transferase; hyg gene from E. coli pac Puromycin-N-acetyl transferase; pac gene from Streptomyces alboniger ble Bleomycin binding protein; a ble gene is located on the bacterial transposon Tn5, or the Sa ble gene from Staphylococcus aureus gpt Xanthine-guanine phosphoribosyltransferase; gpt gene isolated from E. coli

Select cells in G418 (0.1-1.0 |xg/mL), an aminoglycoside that blocks protein synthesis and is similar to kanamycin Select cells in Hygromycin B (10-300 |ig/mL), an aminocyclitol that inhibits protein synthesis Select cells in puromycin (0.5-5 |ig/mL), an antibiotic that inhibits protein synthesis Select cells in bleomycin or Zeocin (50-500 |ig/mL), an antibiotic that binds DNA and blocks RNA synthesis

Select cells in guanine-deficient media that contains inhibitors of de novo GMP synthesis and xanthine; this selects for gpt+ cells that can synthesize guanine from xanthine

(Colbere-Garapin etal., 1981)

(Blochlinger and Diggelmann, 1984)

(Vara etal, 1986)

(Genilloud, Garrido and Moreno, 1984)

(Mulligan and Berg, 1981)

control of a mammalian promoter, e.g. the HSV Tk promoter or the SV40 early promoter (Berg, 1981). We will discuss promoters used to drive expression of genes in animal cells in more detail below.

The exposure of animal cells to high levels of toxic drugs for prolonged periods for the purposes of recombinant selection can give rise, at a very low frequency, to the formation of cells that have become spontaneously highly resistant to the drug. For example, mouse cells exposed to methotrexate (a folic acid analogue that is an inhibitor of the enzyme dihydrofolate reductase, DHFR) have been shown to undergo three types of mutation to generate resistance (Schimke et al., 1978):

• mutations within the DHFR enzyme to generate inhibitor resistance,

• mutations that prevent cellular uptake of the drug and

• amplification of the Dhfr locus to increase the copy number of the Dhfr gene to produce sufficient quantities of the enzyme to overcome the effects of the drug. The amplification process appears to be quite random, with large regions of flanking DNA surrounding the Dhfr locus also becoming amplified.

Mutations in this last class are particularly important for the high-level expression of foreign genes. The foreign DNA is cloned into a plasmid vector that also bears the Dhfr gene. This is then transfected into methotrexate-resistant cells and recombinants selected for in the presence of high levels of the drug. Cells that amplify the Dhfr locus should also contain large numbers of copies of the foreign DNA (Wigler et al., 1980).

Was this article helpful?

0 0
Overcome Shyness 101

Overcome Shyness 101

You can find out step by step what you need to do to overcome the feeling of being shy. There are a vast number of ways that you can stop feeling shy all of the time and start enjoying your life. You can take these options one step at a time so that you gradually stop feeling shy and start feeling more confident in yourself, enjoying every aspect of your life. You can learn how to not be shy and start to become much more confident and outgoing with this book.

Get My Free Ebook


Post a comment