Genetic Engineering - Process

Process

The first step is to choose and isolate the gene that will be inserted into the genetically modified organism. Presently, most genes transferred into plants provide protection against insects or tolerance to herbicides. In animals the majority of genes used are growth hormone genes. The gene can be isolated using restriction enzymes to cut DNA into fragments and gel electrophoresis to separate them out according to length. Polymerase chain reaction (PCR) can also be used to amplify up a gene segment, which can then be isolated through gel electrophoresis. If the chosen gene or the donor organism's genome has been well studied it may be present in a genetic library. If the DNA sequence is known, but no copies of the gene are available, it can be artificially synthesized.

The gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly. The gene can also be modified at this stage for better expression or effectiveness. As well as the gene to be inserted most constructs contain a promoter and terminator region as well as a selectable marker gene. The promoter region initiates transcription of the gene and can be used to control the location and level of gene expression, while the terminator region ends transcription. The selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is needed to determine which cells are transformed with the new gene. The constructs are made using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning. The manipulation of the DNA generally occurs within a plasmid.

The most common form of genetic engineering involves inserting new genetic material randomly within the host genome. Other techniques allow new genetic material to be inserted at a specific location in the host genome or generate mutations at desired genomic loci capable of knocking out endogenous genes. The technique of gene targeting uses homologous recombination to target desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of gene targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger nucleases, engineered homing endonucleases, or nucleases created from TAL effectors. In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout .