Genetic engineering techniques

Genetic engineering techniques enable modification of the DNA of living organisms. A variety of editing techniques have been developed since DNA's structure was first discovered.

Targets

Bacteria are commonly engineered for research purposes. Typically this is through transformation to add a plasmid containing a gene of interest, but editing of the chromosome is also used. Plants and animals have both been genetically modified for research, agricultural and medical applications. In plants, the most widely inserted genes provide herbicide resistance or insecticidal properties.^[1] In animals, the most widely used are growth hormone genes. Finally, genetically modified viruses are also used as viral vectors to transfer target genes to another organism in gene therapy.

Procedure

The first step involves choosing and isolating the gene that will be inserted into/removed from the genetically modified organism.

The gene must generally be combined with a promoter and terminator region as well as a selectable marker gene.

Then the genes must be spliced into the target's DNA. For animals, the gene must be inserted into embryonic stem cells.

The resulting organism must have the presence of the target gene confirmed.

First generation offspring are heterozygous, requiring them to be inbred to create the homozygous pattern necessary for stable inheritance. Homozygosity must be confirmed in second generation specimens, which then become the final product.

History

Human directed genetic manipulation began with the domestication of plants and animals through artificial selection in about 12,000 BC.^[2]:1 Various techniques were developed to aid in breeding and selection. Hybridization was one way rapid changes in an organisms makeup could be introduced. Hybridization most likely first occurred when humans first grew similar, yet slightly different plants in close proximity.^[3]:32 Some plants were able to be propagated by vegetative cloning.^[3]:31 X-rays were first used to deliberately mutate plants in 1927. Between 1927 and 2007, more than 2,540 genetically mutated plant varieties had been produced using x-rays.^[4]

It wasn't until the mid 1800s that DNA and genes were discovered, which would form the basis of modern genetic manipulation. Genetic inheritance was first discovered by Gregor Mendel in 1865 following experiments crossing peas.^[5] In 1928 Frederick Griffith proved the existence of a "transforming principle" involved in inheritance, which was identified as DNA in 19. Frederick Sanger developed a method for sequencing DNA in 1977, greatly increasing the genetic information available to researchers.

As well as discovering how DNA works, tools had to be developed that allowed it to be manipulated. In 1970 Hamilton Smiths lab discovered restriction enzymes, enabling scientists to isolate genes from an organism's genome.^[6] DNA ligases, that join broken DNA together, had been discovered earlier in 1967^[7] and by combining the two enzymes it was possible to "cut and paste" DNA sequences to create recombinant DNA. Plasmids, discovered in 1952,^[8] became important tools for transferring information between cells and replicating DNA sequences. Polymerase chain reaction (PCR), developed by Kary Mullis in 1983, allowed small sections of DNA to be amplified and aided identification and isolation of genetic material.

As well as manipulating the DNA, techniques had to be developed for its insertion (known as transformation) into an organism's genome. Griffiths experiment had already shown that some bacteria had the ability to naturally uptake and express foreign DNA. Artificial competence was induced in Escherichia coli in 1970 by treating them with calcium chloride solution (CaCl₂).^[9] Transformation using electroporation was developed in the late 1980s, increasing the efficiency and bacterial range.^[10] In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, had been discovered and in the early 1970s it was found that the bacteria inserted its DNA into plants using a Ti plasmid.^[11] By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants.^[12]

An important part of genetic engineering is to identify useful genes to transform into the genetically modified organism. The bacteria Bacillus thuringiensis was first discovered in 1901 as the causative agent in the death of silkworms. Due to these insecticidal properties the bacteria was used as an biological insecticide, commercially developed in 1938. The cry proteins were discovered to provide the insecticidal activity in 1956 and by the 1980s scientists had successfully cloned the gene coding for this protein and expressed it in plants.^[13] The gene that provides resistance to the glyphosate herbicide was found, after seven years searching, in the outflow pipe of a Monsanto roundup manufacturing facility.^[14] In animals the majority of genes used are growth hormone genes.^[15]

Libraries

Target genes can be cloned from a DNA segment after the creation of a DNA library. The libraries generally cover the organism's genome multiple times and its size will depend on how large the genome is.

Techniques

Gene isolation

The DNA is first digested with a random digestion method, commonly by cutting the DNA with restriction enzymes (enzymes that cut DNA). A partial restriction digest cuts only some of the restriction sites, resulting in overlapping DNA fragment lengths. The DNA fragments are put into individual plasmid vectors and grown inside bacteria. Once in the bacteria the plasmid is copied as the bacteria divides. To determine if a useful gene is present on a particular fragment the bacterial library is screened for the desired phenotype. If the phenotype is detected then it is possible that the bacteria contains the target gene. If the gene does not have a detectable phenotype or a DNA library does not contain the correct gene, other methods can be used to isolate it. If the position of the gene can be determined using molecular markers then chromosome walking is one way to isolate the correct DNA fragment. If the gene expresses close homology to a known gene in another species, then it could be isolated by searching for genes in the library that closely match the known gene.^[16]

If the DNA sequence of the gene and the organism is known, restriction enzymes can cut the DNA on either side of the gene and gel electrophoresis can sort the fragments according to length.^[17] The DNA band at the correct size should contain the gene, where it can be excised from the gel. Polymerase chain reaction (PCR) can be used to amplify the gene, which can then be isolated through gel electrophoresis.^[18] It is also possible to synthesize the gene.^[19]

Additional material

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.^[20]

Gene targeting

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 success of gene targeting can be enhanced with the use of engineered nucleases such as zinc finger nucleases,^[21][22] engineered homing endonucleases,^[23][24] transcription activator-like effector nuclease.^[25][26] or CRISPR. Engineered nucleases can also introduce mutations at endogenous genes that generate a gene knockout.^[27][28]

Transformation

A. tumefaciens attaching itself to a carrot cell

About 1% of bacteria are naturally able to take up foreign DNA but it can be induced in other bacteria.^[29] Stressing the bacteria with a heat shock or an electric shock can make the cell membrane permeable to DNA that may then incorporate into the genome or exist as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus or through the use of viral vectors. In plants the DNA is generally inserted using Agrobacterium-mediated recombination or biolistics.^[30]

In Agrobacterium-mediated recombination the plasmid construct must also contain T-DNA. Agrobacterium naturally inserts DNA from a tumor inducing plasmid into any susceptible plant that it infects, causing crown gall disease. The T-DNA region of this plasmid is responsible for insertion of the DNA. The DNA to be inserted is cloned into a binary vector that contains T-DNA and can be grown in both E. coli and Agrobacterium. Once the binary vector is constructed the plasmid is transformed into Agrobacterium containing no plasmids and plant cells are infected. The Agrobacterium naturally inserts the genetic material into the plant cells.^[31]

In biolistic particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material enters the cells and transforms them. This method can be used on plants that are not susceptible to Agrobacterium infection and also allows transformation of plant plastids.

Another transformation method for plant and animal cells is electroporation, which involves subjecting cells to an electric shock, which can make the cell membrane permeable to plasmid DNA. In some cases the electroporated cells will incorporate the DNA. Due to the associated cell and DNA damage, the transformation efficiency of biolistics and electroporation is lower than with agrobacteria and microinjection.^[32]

Selection

Not all the organism's cells will be transformed with the new genetic material; typically a selectable marker is used to differentiate transformed from untransformed cells. Cells that have been successfully transformed with the DNA it will also contain the marker gene. By growing the cells in the presence of an antibiotic or chemical that selects or marks the cells expressing that gene, it is possible to separate modified from unmodified cells. Another screening method involves a DNA probe that sticks only to the inserted gene. Multiple strategies can remove the marker from the mature plant.^[33]

Regeneration

As often only a single cell is transformed with genetic material the modified organism must be grown from that single cell. Bacteria consist of a single cell and reproduce clonally, so regeneration is not necessary for them. In plants this is accomplished through the use of tissue culture. Each plant species has different requirements for successful regeneration. If successful, the technique produces an adult plant that contains the transgene in every cell.

In animals it is necessary to ensure that the inserted DNA is present in embryonic stem cells. Offspring can be screened for the gene. All offspring from the first generation will be heterozygous for the inserted gene and must be inbred to produce a homozygous specimen.

Confirmation

The finding that a recombinant organism contains the inserted genes is not usually sufficient to ensure that they will be appropriately expressed in the intended tissues. To confirm the presence of the gene, PCR, Southern hybridization and DNA sequencing are employed to determine the chromosomal location and number of gene copies.

To assess gene expression, transcription, RNA processing patterns and expression and localization of protein product(s) must usually be assessed, using methods including northern hybridization, quantitative RT-PCR, Western blot, immunofluorescence and phenotypic analysis. When appropriate, the organism's offspring are studied to confirm that the trans-gene and associated phenotype are stably inherited.

In some cases further generations must be produced and confirmed, to ensure the absence of undesirable traits in the modified organism. For hybrid products such as maize, the modified organism is crossbred with other cultivars that possess required traits.

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