Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

Applications of DNA clones
Plasmid vectors for animal cells

The Amplification of recombinant Proteins cannot always be accomplished using bacterial expression systems, since many eukaryotic proteins require post-translational modifications (such as glycosylation, hydroxylation, etc.) to achieve full functionality—processes for which bacterial Cells lack the corresponding Enzymes. To produce recombinant proteins that require post-translational modification (maturation), cloned genes are introduced into Animal Cell Cultures.

This process of introducing specially constructed vectors into an animal cell is called transfection.

The Introduction of DNA into bacterial and Yeast cells is referred to as transformation. In microbiology, this term is used to describe heritable changes resulting from the uptake (acquisition) of exogenous (foreign) DNA. When applied to animal cells, however, the term "transformation" traditionally denotes A change in their growth pattern in culture caused by The conversion of normal cells into Cancer cells. To avoid terminological confusion, the term transfection was chosen to designate heritable changes in animal cells following the introduction of exogenous DNA.

There are two Methods of transfection, depending on whether or not the recombinant vector integrates into The Cell's genomic DNA.

In both cases, animal Cells must be pre-conditioned (treated) to facilitate the penetration of the recombinant plasmid vector into the cell.

This is achieved either by using specific Surfactants that increase the permeability of The Plasma Membrane to DNA, or by electroporation—the application of an electric discharge with an amplitude of several thousand volts, which temporarily creates lipid Pores in the plasma membrane. Plasmid Vectors are typically added in excess to the culture medium to ensure that as many cells as possible undergo transfection.

Figures 108 and 109 illustrate the schemes of transient and stable transfection using a vector containing all the necessary elements (ORI, amp^R Selection marker, polylinker, etc.) that ensure the functioning of the plasmid vector, though these are omitted from the figures to avoid overcrowding.

12.2.1. Transient Transfection. Transient transfection methods utilize vectors similar to shuttle vectors (Figure 95). To enable the plasmid vector to replicate in mammalian cells, a Replication origin from a virus that infects these cells is inserted into it. Additionally, the vector must contain a complex consisting of a strong promoter recognized by mammalian RNA polymerase, located adjacent to the cloned cDNA encoding the target protein (Figure 108).

Once introduced into a mammalian cell, the viral origin of replication ensures efficient replication of the plasmid vector, generating successive generations of Plasmids that will express the target protein.

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Figure 108 - Transient transfection

However, during Cell Division, these plasmids are not necessarily distributed equally between daughter cells. As a result, after some time, a significant portion of the cells in the culture no longer contains any plasmid molecules. This is precisely why this technique is called transient transfection.

12.2.2. Stable Transfection (Transformation). If the vector introduced into the cell integrates into the cell's genome (a permanent genomic alteration) with the help of special DNA Repair enzymes, such a cell is termed transformed.

Since vector integration into The Genome is a relatively rare event, the vector must contain a selection marker to allow the identification and Isolation of the few cells successfully transformed by the vector. In this case, the selection marker is often a Gene encoding neomycin phosphotransferase (designated as nео^r), which confers cellular resistance to the toxic neomycin analog designated as G-418. The MAIN STAGES OF cloned cDNA expression via stable transfection are shown in Figure 109.

Figure 109 - Stable transfection (transformation)

Only transformed cells carrying the expression vector within their Chromosomes will survive and proliferate in a medium containing G-418.

Because integration occurs at random chromosomal sites, screening the survivors in the G-418 medium yields a collection of clones that differ in their METABOLISM/31.html">Transcription rates of the integrated cDNA.

Therefore, the transformed cells are subjected to screening to identify and select the clones that express the target protein with maximum efficiency.