Getting genes into cells: vectors, physical methods and markers

1The membrane is in the way

A recombinant plasmid in a test tube does nothing. To be copied and expressed it has to get inside a living cell, and the cell surface membrane is a barrier built to keep large, charged molecules such as DNA out. Plant cells add a cellulose wall on top. Over fifty years, gene technologists have found two kinds of way through: borrowing the tricks of viruses and bacteria that already inject DNA into cells, and forcing the DNA in by physical or chemical means. Whatever the method, only a small fraction of the cells take up the gene, so the second half of this lesson is about finding them.

2Biological vectors

• Plasmids into bacteria. Bacteria such as E. coli are chilled in calcium chloride solution with the plasmids and then given a brief heat shock at about 42 °C. The calcium ions and the sudden temperature change make the membrane more permeable for a moment, and some cells take up a plasmid.
• Viruses. A virus naturally delivers its genome into a cell. Bacteriophages can carry genes into bacteria. For human gene therapy, viruses such as adenoviruses, adeno-associated viruses and lentiviruses are modified so that they cannot reproduce, and the therapeutic gene replaces some of their own genes.
• Agrobacterium tumefaciens for plants. This soil bacterium infects plants through wounds and transfers part of its Ti plasmid, the T-DNA, into the plant's chromosomes. Its disease-causing genes are removed and the wanted gene is placed in the T-DNA. Plant cells infected in tissue culture are grown into whole transgenic plants.

3Non-biological methods of gene transfer

Some cells cannot be infected by a suitable vector, and some uses need no viral or bacterial sequences at all. Physical and chemical methods then carry the DNA across the membrane:

Physical methods avoid some of the risks of viral vectors, such as an immune reaction against the virus, but they are usually less efficient, can damage cells, and insert the gene at random positions in the genome.

4Finding the transformed cells

After transformation, a culture contains three kinds of cell: cells with no plasmid, cells with a plasmid that closed up without the gene, and cells with the recombinant plasmid. Marker genes sort them out.

Antibiotic resistance and replica plating. Using the plasmid of lesson 1, the gene is inserted into the BamHI site inside the tetracycline resistance gene, tetR. This insertional inactivation means:

• cells with no plasmid are killed by ampicillin;
• cells with a plasmid that closed up without the gene resist both ampicillin and tetracycline;
• cells with the recombinant plasmid resist ampicillin but are killed by tetracycline, because tetR is broken.

The bacteria are first grown on ampicillin agar (the master plate). A sterile velvet pad is pressed onto it and then onto a tetracycline plate, transferring a few cells of every colony to the same position. Colonies that grow on the master plate but not on tetracycline carry the recombinant plasmid, and they are picked from the master plate.

Fluorescent and colour markers. Antibiotic resistance genes raise the worry that resistance could spread to bacteria that cause disease, so other markers are now preferred where possible:

• The gene for green fluorescent protein (GFP), from the jellyfish Aequorea victoria, makes cells glow green under blue or ultraviolet light. Transformed cells can be seen directly, even inside a living plant or animal, without killing anything.
• In blue-white screening, the gene is inserted into a bacterial gene, lacZ, for the enzyme β-galactosidase. Normal colonies turn a colourless substrate in the agar (X-gal) blue; colonies with the insert cannot, and stay white.

Finally, the gene must be expressed. It needs a promoter that the host's RNA polymerase recognises. A human protein that must be folded and modified after translation, for example by adding sugar groups, may have to be made in yeast or in cultured mammalian cells rather than in bacteria, which lack a Golgi apparatus.