Vectors and hosts

To carry a gene into a host and have it copied, we need a vector: a DNA molecule that can replicate inside the host. The most common are plasmids and bacteriophages (viruses of bacteria), because they replicate in bacterial cells independently of the main chromosome. Some plasmids exist in only one or two copies per cell; others in 15-100; some phages, in very high numbers.

A good cloning vector needs three features:

• An origin of replication (ori), where copying starts. It also controls the copy number: how many copies of the vector, and so of the gene, the cell will make.
• A selectable marker: a gene that lets us tell transformed cells (which took up the vector) from the rest. Usually it gives resistance to an antibiotic such as ampicillin, tetracycline or kanamycin: only cells carrying the vector survive on a plate with that antibiotic.
• Cloning sites: a place where the foreign DNA can be inserted. It should be cut by a given restriction enzyme at only one site; several sites would break the vector into pieces.

pBR322, a plasmid of E. coli, is a classic example. It has an ori and two resistance genes, ampR and tetR, with restriction sites inside them. If the foreign DNA is inserted at the BamH I site in the tetR gene, that gene is broken. Recombinant cells then grow on ampicillin but not on tetracycline; cells without the plasmid grow on neither.

Testing every colony on two plates is slow, so a simpler method was developed: blue-white selection. The foreign DNA is inserted into a gene for the enzyme β-galactosidase, breaking it: insertional inactivation. The cells are grown with a chromogenic substrate. Colonies whose enzyme still works turn it blue; recombinant colonies, whose gene has been broken, stay colourless (white).

Vectors for plants and animals take advantage of natural 'genetic engineers'. The bacterium Agrobacterium tumifaciens naturally carries a piece of DNA into plant cells using its Ti plasmid, causing tumours. The Ti plasmid has been disarmed so it no longer causes disease, but still delivers genes. Similarly, disarmed retroviruses are used to carry genes into animal cells.

Getting DNA into host cells, which normally do not take it up easily:

• Bacteria are made competent by treating them with calcium ions, then given a heat shock (ice, then 42 °C, then ice) so the DNA enters.
• Micro-injection: DNA is injected straight into the nucleus of an animal cell.
• Biolistics, or the gene gun: tiny particles of gold or tungsten coated with DNA are shot into plant cells at high speed.
• Disarmed pathogens carry the DNA in naturally.