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Process

Main article: Genetic engineering techniques

Polymerase chain reaction is a powerful tool used in molecular cloning

Creating a GMO is a multi-step process. Genetic engineers must first choose what gene they wish to insert into the organism. This is driven by what the aim is for the resultant organism and is built on earlier research. Genetic screens can be carried out to determine potential genes and further tests then used to identify the best candidates. The development of microarrays, transcriptomics and genome sequencing has made it much easier to find suitable genes. Luck also plays its part; the Roundup Ready gene was discovered after scientists noticed a bacterium thriving in the presence of the herbicide. # ISO certification in India

Gene isolation and cloning

Main article: Molecular cloning

The next step is to isolate the candidate gene. The cell containing the gene is opened and the DNA is purified. The gene is separated by using restriction enzymes to cut the DNA into fragments or polymerase chain reaction (PCR) to amplify up the gene segment. These segments can then be extracted through gel electrophoresis. If the chosen gene or the donor organism’s genome has been well studied it may already be accessible from a genetic library. If the DNA sequence is known, but no copies of the gene are available, it can also be artificially synthesised. Once isolated the gene is ligated into a plasmid that is then inserted into a bacterium. The plasmid is replicated when the bacteria divide, ensuring unlimited copies of the gene are available. The RK2 plasmid is notable for its ability to replicate in a wide variety of single-celled organisms, which makes it suitable as a genetic engineering tool. # ISO certification in India

Before the gene is inserted into the target organism it must be combined with other genetic elements. These include a promoter and terminator region, which initiate and end transcription. A selectable marker gene is added, which in most cases confers antibiotic resistance, so researchers can easily determine which cells have been successfully transformed. The gene can also be modified at this stage for better expression or effectiveness. These manipulations are carried out using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning. # ISO certification in India

Inserting DNA into the host genome

Main article: Gene delivery

A gene gun uses biolistics to insert DNA into plant tissue

There are a number of techniques used to insert genetic material into the host genome. Some bacteria can naturally take up foreign DNA. This ability can be induced in other bacteria via stress (e.g. thermal or electric shock), which increases the cell membrane’s permeability to DNA; up-taken DNA can either integrate with the genome or exist as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cell’s nuclear envelope directly into the nucleus, or through the use of viral vectors. # ISO certification in India

Plant genomes can be engineered by physical methods or by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors. In plants the DNA is often inserted using Agrobacterium-mediated transformation, taking advantage of the Agrobacteriums T-DNA sequence that allows natural insertion of genetic material into plant cells. Other methods include biolistics, where particles of gold or tungsten are coated with DNA and then shot into young plant cells, and electroporation, which involves using an electric shock to make the cell membrane permeable to plasmid DNA.

As only a single cell is transformed with genetic material, the organism must be regenerated from that single cell. In plants this is accomplished through the use of tissue culture. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Bacteria consist of a single cell and reproduce clonally so regeneration is not necessary. Selectable markers are used to easily differentiate transformed from untransformed cells. These markers are usually present in the transgenic organism, although a number of strategies have been developed that can remove the selectable marker from the mature transgenic plant. # ISO certification in India

A. tumefaciens attaching itself to a carrot cell

Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene. These tests can also confirm the chromosomal location and copy number of the inserted gene. The presence of the gene does not guarantee it will be expressed at appropriate levels in the target tissue so methods that look for and measure the gene products (RNA and protein) are also used. These include northern hybridisation, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis. # ISO certification in India

The new genetic material can be inserted randomly within the host genome or targeted to a specific location. The technique of gene targeting uses homologous recombination to make 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 through genome editing. Genome editing uses artificially engineered nucleases that create specific double-stranded breaks at desired locations in the genome, and use the cell’s endogenous mechanisms to repair the induced break by the natural processes of homologous recombination and nonhomologous end-joining. There are four families of engineered nucleases: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the Cas9-guideRNA system (adapted from CRISPR). TALEN and CRISPR are the two most commonly used and each has its own advantages. TALENs have greater target specificity, while CRISPR is easier to design and more efficient. In addition to enhancing gene targeting, engineered nucleases can be used to introduce mutations at endogenous genes that generate a gene knockout. # ISO certification in India