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Chapter 23 : Genetic Manipulation of Mammalian Cells for Protein Expression

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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.

Citation: Kantardjieff A, Hu W, Seth G, Scott McIvor R. 2010. Genetic Manipulation of Mammalian Cells for Protein Expression, p 330-344. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch23

Key Concept Ranking

Genetic Elements
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Murine leukemia virus
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Rous sarcoma virus
0.48785308
Simian virus 40
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Figures

Image of FIGURE 1
FIGURE 1

Typical vector for expression of antibody product in mammalian cells. This representative plasmid contains both the heavy-chain (VH, CH1, CH2, CH) and light-chain (VL, CKappa) genes of the IgG gene. Each is driven by its own promoter (P) and has a polyadenylation signal (PolyA) downstream. A leader sequence (L) which serves as a signal for secretion is present for each IgG gene. A mammalian selectable marker, in this case, is included with its own promoter (P) and polyadenylation signal (SV40 pA). Sequence elements for replication and selection in are also included, namely, an origin of replication (pUC ori) and selectable marker ().

Citation: Kantardjieff A, Hu W, Seth G, Scott McIvor R. 2010. Genetic Manipulation of Mammalian Cells for Protein Expression, p 330-344. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch23
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Image of FIGURE 2
FIGURE 2

Overview of selection of stable and amplified clones.

Citation: Kantardjieff A, Hu W, Seth G, Scott McIvor R. 2010. Genetic Manipulation of Mammalian Cells for Protein Expression, p 330-344. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch23
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Image of FIGURE 3
FIGURE 3

Overview of production of replication-defective retroviral particles using packaging cells and subsequent transduction of target cells. A replication-incompetent vector, which includes the 5′ and 3′ LTR regions, the packaging sequence (ψ), a gene of interest (Gene X), and a mammalian selectable marker () with its own promoter is introduced into a packaging cell line which supplies the required viral accessory proteins, namely Gag, Pol, and Env. This bacterial plasmid also contains the required genetic elements for replication (ori) and selection () in Upon entry into the packaging cell line (via any desired transfection methodology), the plasmid is transported into the nucleus, where it is transiently transcribed. The viral transcript initiates at the 5′ LTR and terminates at the 3′ LTR, generating a full-length viral transcript. It contains the packaging sequence (ψ), which allows packaging into viral particles. Fully assembled viral particles subsequently bud out of the packaging cell line. These viral particles are harvested from the supernatant 48 h after transfection. The virus-containing supernatant is subsequently incubated with the target cells, and entry of the viral particles occurs through interaction with host-cell receptors. Once the viral core is released into the cytoplasm, reverse transcription of the RNA viral genome begins using reverse transcriptase contained within the viral particle. The DNA copy of the viral genome then integrates into the host genome, and the gene of interest and selectable marker can be expressed. One infectious virion produces one integrated copy of the viral genome. Figure adapted from reference .

Citation: Kantardjieff A, Hu W, Seth G, Scott McIvor R. 2010. Genetic Manipulation of Mammalian Cells for Protein Expression, p 330-344. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch23
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Tables

Generic image for table
TABLE 1

Commonly used constitutive promoters for expression constructs

Citation: Kantardjieff A, Hu W, Seth G, Scott McIvor R. 2010. Genetic Manipulation of Mammalian Cells for Protein Expression, p 330-344. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch23
Generic image for table
TABLE 2

Commonly used selectable markers

Citation: Kantardjieff A, Hu W, Seth G, Scott McIvor R. 2010. Genetic Manipulation of Mammalian Cells for Protein Expression, p 330-344. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch23
Generic image for table
TABLE 3

Common viral expression systems

Citation: Kantardjieff A, Hu W, Seth G, Scott McIvor R. 2010. Genetic Manipulation of Mammalian Cells for Protein Expression, p 330-344. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch23
Generic image for table
TABLE 4

Commonly used reporter genes (adapted from reference )

Citation: Kantardjieff A, Hu W, Seth G, Scott McIvor R. 2010. Genetic Manipulation of Mammalian Cells for Protein Expression, p 330-344. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch23

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