Overview Function transfer production of a protein from one organism to another synthesize large quantities of a plasmid or its product Process insertion of a DNA segment of interest into a vector vector is usually a plasmid first: digestion of vector with restriction endonuclease second: sealing of the segment into the vector with DNA ligase insertion of the vector into a bacteria via transformation selection of the bacteria via a mechanism to distinguish which cells received the vector and those that did not usually via an antibiotic resistance gene Applications recombinant proteins produced by introduction of a plasmid containing the protein product of interest into bacteria bacterial colony grown some plasmids contain regulatory sequences which can turn on/off expression of the plasmid bacteria lysed and produced protein is purified ex.) recombinant insulin, factor VIII, bacterial factors for vaccination genome sequencing/libraries contains the entire genome of an organism spread between bacterial colonies the genome of an organism is degraded with a restriction endonuclease at specific palindromic sites individual pieces are inserted into a bacterial colony and cloned individually to access the library for amplification of a particular segment a blot is taken of the library and radiolabeled segment of interest is hybridized the labeled colony (in which hybridization took place) can be harvested and grown cDNA libraries "expression libraries" contain all the expressed genes of an organism in a similar fashion as a genomic library because mRNA has had the non-coding introns spliced out it can express genes from the cloned host bacteria do not contain the enzymes to splice RNA requires reverse transcriptase to convert mRNA into DNA for the library similar mechanism of accessing the library as the genomic library; however the probe is an 125I-antibody against the protein of interest gene therapy allows introduction of a normal copy of a gene into a cell that was defective in that gene uses a delivery vector to introduce the gene usually modified viruses viral genome is replaced with plasmid virus inserts the gene into the host as part of its normal life cycle problems viruses can indiscriminately insert copies of gene into undesirable regions oncogenesis cells must be dividing for viral integration adenovirus vectors do not require active division, but do not actually insert into the genome can be performed ex vivo cells removed from the body, modified, and transplanted back into the body in vivo cells modified in the body without removal has been used to treat SCID caused by deficiency in the IL-receptor γ chain