9 - pac Gene as Efficient Dominant Marker and Reporter Gene in Mammalian Cells

Abstract

Publisher Summary DNA-mediated gene transfer has become a standard technique to introduce genes into animal cells. It has enabled the functional studies of alterations produced in vitro, as well as the overproduction of the corresponding gene products. However, because the vast majority of the genes are not selectable, the development of selectable markers for cotransformation experiments has been a crucial step in the generalization of this approach. Since the recovery of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity mediated by DNA, biochemical markers have been extensively used. Some of them are selectable but not dominant markers. Antibiotic resistance markers constitute the paradigm among the dominant ones, which include those encoding hygromycin phosphotransferase and puromycin acetyltransferase (pac). The latter encodes puromycin acetyltransferase from Streptomyces alboniger. Its development as a dominant marker and its practical use are the subjects of this chapter. Puromycin is an aminonucleoside antibiotic that blocks protein synthesis by interacting with the A site of the large ribosomal subunit of both prokaryotic and eukaryotic ribosomes because it resembles an amino acid attached to the terminal adenosine of tRNA. This interaction is followed by peptide bond formation, which releases the nascent polypeptide in the form of polypeptidylpuromyein. The antibiotic inhibits the growth of gram-positive bacteria and various animal and insect cells. Puromycin acetyltransferase (PAC) has many advantages. It can be used both as a selectable marker and as a reporter gene. It is possible to predict the efficiency of a given construct for transformation by a fast transient expression assay and the determination of PAC activity.