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Lecture 8 genetic engineering of animal cells
- 1. Genetic engineering of animal cells in culture To study individual genes, it may be necessary to transfer the gene into the cells under study. In this section we will discuss some of the common methods that are used to introduce genes into mammalian cells. This is essential so that one can genetically manipulate cells so that they are able to produce large levels of the desired protein (overexpression). In the diagram, a gene is spliced into a plasmid, called shuttle vectors, and then introduced into the mammalian cell.
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- To study individual genes, it may be necessary to transfer the gene into the cells under study. In this section we will discuss some of the common methods that are used to introduce genes into mammalian cells. This is essential so that one can genetically manipulate cells so that they are able to produce large levels of the desired protein (overexpression). In the diagram, a gene is spliced into a plasmid, called shuttle vectors, and then introduced into the mammalian cell.
- CHO cells are very popular for the expression of human recombinant glycoproteins because the glycosylation enzymes resemble those found in human cell lines. They can grow as anchorage dependent or as cell suspensions.
- Introduction of DNA into cells with commercially available cationic lipids. Charged head groups (positive) are drawn to the phosphate backbone of DNA.
- Electroporation Exposure of suspended cells to a pulsed electric field that causes the transient formation of pores in the cell membrane, allowing for exchange of macromolecules between the extracellular environment and the cytoplasm. Removal of the electrical field results in spontaneous sealing of the pores. Microinjection DNA injected with microneedles or pipettes directly into the nucleus, or into the cytoplasm. Requires highly trained and skilled personnel. Typically used when there are limited numbers of recipient cells.
- Bacteria are used to produce plasmids containing the human gene of interest. The cell walls are removed using lysozyme, resulting in the formation of protoplasts. The protoplasts are brought in contact with the mammalian cells, leading to protoplast fusion to mammalian cells, using Polyethylene glycol (PEG).
- Overview of methods discussed, in handout.
- Non-dominant genes: target cells must have endogenous genes mutated or removed, only works with mutant cells. If target genes are successfully transfected with shuttle vector containing wild-type marker gene, this will allow cells to survive in specially treated media. Dominant genes: do not require mutant cells, can be used with any cell type. Dominant marker genes do not have to be mammalian, may be bacterial in origin, allows the cell to survive in special medium. Antibiotic resistance genes: shuttle vector contains antibiotic, allows for successful transfectants to survive.