Genetic Manipulation of Mammalian Cells for Protein Expression

Abstract

This chapter describes basic methodology used in expressing heterologous proteins in mammalian cells and in engineering their characteristics. Expressing genes in mammalian cells has become increasingly important to understand their functional significance. However, other mammalian expression systems are also available, including human embryonic kidney (HEK-293) cells, African green monkey CV1 kidney (COS) cells, and Madin-Darby canine kidney (MDCK) cells. In this section, the basic steps of genetic manipulation for heterologous protein production are outlined. Genetic manipulation for heterologous protein production often requires high-level expression, so for this purpose, strong constitutive promoters are typically used. Gene amplification is commonly used to increase the number of copies of the transgene and its transcript levels, thus generating high levels of recombinant protein expression. The authors have described a number of methods that incorporate flow cytometry for sorting high-producing cells. Episomal systems, which allow extrachromosomal replication of the expression vector in mammalian cells, are commonly employed. Within the context of cell engineering applications, genetic manipulations are employed to modify cellular functions. The use of viral expression systems is also common in cell engineering applications and is described in the chapter. The development of new technologies for genetic manipulation of mammalian cells and their screening will continue to drive discovery and improve robustness of our protein production processes.