cloning vectors contain a site that allows DNA fragments to be inserted, for example a multiple cloning site or polylinker which has several commonly used restriction sites to which DNA fragments may be ligated. After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called transformation. These plasmids contain a selectable marker, usually an antibiotic resistance gene, which confers on the bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics. The cells after transformation are exposed to the selective media, and only cells containing the plasmid may survive. In this way, the antibiotics act as a filter to select only the bacteria containing the plasmid DNA. The vector may also contain other marker genes or reporter genes to facilitate selection of plasmids with cloned inserts. Bacteria containing the plasmid can then be grown in large amounts, harvested, and the plasmid of interest may then be isolated using various methods of plasmid preparation. A plasmid cloning vector is typically used to clone DNA fragments of up to 15 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are used. Protein production Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing the protein the gene codes for, for example, insulin. Gene therapy Plasmids may also be used for gene transfer as a potential treatment in gene therapy so that it may express the protein that is lacking in the cells. Some forms of gene therapy require the insertion of therapeutic genes at pre-selected chromosomal target sites within the human genome. Plasmid vectors are one of many approaches that could be used for this purpose. Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double-strand break to the DNA genome and cause homologous recombination. Plasmids encoding ZFN could help deliver a therapeutic gene to a specific site so that cell damage, cancer-causing mutations, or an immune response is avoided. Disease models Plasmids were historically used to genetically engineer the embryonic stem cells of rats