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Archive for the ‘Genetic Engineering’ Category

First cow to produce human insulin in its milk created in Brazil – Cosmos

Monday, March 18th, 2024

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Engineering the Microbiome: CRISPR Leads the Way – The Scientist

Monday, March 18th, 2024

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The de-extinction process to bring woolly mammoths back to life – The Week

Monday, March 18th, 2024

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CRISPR gene editing applications are expanding dramatically in agriculture. Here are the latest advances – Genetic Literacy Project

Monday, March 18th, 2024

CRISPR gene editing applications are expanding dramatically in agriculture. Here are the latest advances  Genetic Literacy Project

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Polymerase Chain Reaction (PCR) – National Human Genome Research Institute

Monday, March 18th, 2024

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Colossal Genetic Breakthrough Could Be the Key to Reviving Wooly Mammoths – Syfy

Monday, March 18th, 2024

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Breaking: Crop gene editing greenlighted by European Parliament. Fight over labeling looms as measure moves to the … – Genetic Literacy Project

Saturday, February 10th, 2024

Breaking: Crop gene editing greenlighted by European Parliament. Fight over labeling looms as measure moves to the ...  Genetic Literacy Project

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GMOs on the spotlight in East Africa again – The Citizen

Saturday, February 10th, 2024

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Blog: Let’s Not Ignore the Science in the Fight Against Malaria – chimpreports.com

Saturday, February 10th, 2024

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Genetic engineering techniques – Wikipedia

Tuesday, January 9th, 2024

Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

The ability to genetically engineer organisms is built on years of research and discovery on gene function and manipulation. Important advances included the discovery of restriction enzymes, DNA ligases, and the development of polymerase chain reaction and sequencing.

Added genes are often accompanied by promoter and terminator regions as well as a selectable marker gene. The added gene may itself be modified to make it express more efficiently. This vector is then inserted into the host organism's genome. For animals, the gene is typically inserted into embryonic stem cells, while in plants it can be inserted into any tissue that can be cultured into a fully developed plant.

Tests are carried out on the modified organism to ensure stable integration, inheritance and expression. 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.

Early techniques randomly inserted the genes into the genome. Advances allow targeting specific locations, which reduces unintended side effects. Early techniques relied on meganucleases and zinc finger nucleases. Since 2009 more accurate and easier systems to implement have been developed. Transcription activator-like effector nucleases (TALENs) and the Cas9-guideRNA system (adapted from CRISPR) are the two most common.

Many different discoveries and advancements led to the development of genetic engineering. Human-directed genetic manipulation began with the domestication of plants and animals through artificial selection in about 12,000 BC.[1]:1 Various techniques were developed to aid in breeding and selection. Hybridization was one way rapid changes in an organism's genetic makeup could be introduced. Crop hybridization most likely first occurred when humans began growing genetically distinct individuals of related species in close proximity.[2]:32 Some plants were able to be propagated by vegetative cloning.[2]:31

Genetic inheritance was first discovered by Gregor Mendel in 1865, following experiments crossing peas.[3] In 1928 Frederick Griffith proved the existence of a "transforming principle" involved in inheritance, which was identified as DNA in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty. Frederick Sanger developed a method for sequencing DNA in 1977, greatly increasing the genetic information available to researchers.

After discovering the existence and properties of DNA, 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.[4] DNA ligases, which join broken DNA together, were discovered earlier in 1967.[5] By combining the two enzymes it became possible to "cut and paste" DNA sequences to create recombinant DNA. Plasmids, discovered in 1952,[6] 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 (replicated) and aided identification and isolation of genetic material.

As well as manipulating DNA, techniques had to be developed for its insertion into an organism's genome. Griffith's 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).[7] Transformation using electroporation was developed in the late 1980s, increasing the efficiency and bacterial range.[8] In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, had been discovered. In the early 1970s it was found that this bacteria inserted its DNA into plants using a Ti plasmid.[9] 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.[10]

The first step is to identify the target gene or genes to insert into the host organism. This is driven by the goal for the resultant organism. In some cases only one or two genes are affected. For more complex objectives entire biosynthetic pathways involving multiple genes may be involved. Once found genes and other genetic information from a wide range of organisms can be inserted into bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal, multiply quickly, relatively easy to transform and can be stored at -80C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research.[11]

Genetic screens can be carried out to determine potential genes followed by other tests that identify the best candidates. A simple screen involves randomly mutating DNA with chemicals or radiation and then selecting those that display the desired trait. For organisms where mutation is not practical, scientists instead look for individuals among the population who present the characteristic through naturally-occurring mutations. Processes that look at a phenotype and then try and identify the gene responsible are called forward genetics. The gene then needs to be mapped by comparing the inheritance of the phenotype with known genetic markers. Genes that are close together are likely to be inherited together.[12]

Another option is reverse genetics. This approach involves targeting a specific gene with a mutation and then observing what phenotype develops.[12] The mutation can be designed to inactivate the gene or only allow it to become active under certain conditions. Conditional mutations are useful for identifying genes that are normally lethal if non-functional.[13] As genes with similar functions share similar sequences (homologous) it is possible to predict the likely function of a gene by comparing its sequence to that of well-studied genes from model organisms.[12] The development of microarrays, transcriptomes and genome sequencing has made it much easier to find desirable genes.[14]

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 a biological insecticide, developed commercially in 1938. The cry proteins were discovered to provide the insecticidal activity in 1956, and by the 1980s, scientists had successfully cloned the gene that encodes this protein and expressed it in plants.[15] The gene that provides resistance to the herbicide glyphosate was found after seven years of searching in bacteria living in the outflow pipe of a Monsanto RoundUp manufacturing facility.[16] In animals, the majority of genes used are growth hormone genes.[17]

All genetic engineering processes involve the modification of DNA. Traditionally DNA was isolated from the cells of organisms. Later, genes came to be cloned from a DNA segment after the creation of a DNA library or artificially synthesised. Once isolated, additional genetic elements are added to the gene to allow it to be expressed in the host organism and to aid selection.

First the cell must be gently opened, exposing the DNA without causing too much damage to it. The methods used vary depending on the type of cell. Once it is open, the DNA must be separated from the other cellular components. A ruptured cell contains proteins and other cell debris. By mixing with phenol and/or chloroform, followed by centrifuging, the nucleic acids can be separated from this debris into an upper aqueous phase. This aqueous phase can be removed and further purified if necessary by repeating the phenol-chloroform steps. The nucleic acids can then be precipitated from the aqueous solution using ethanol or isopropanol. Any RNA can be removed by adding a ribonuclease that will degrade it. Many companies now sell kits that simplify the process.[18]

The gene researchers are looking to modify (known as the gene of interest) must be separated from the extracted DNA. If the sequence is not known then a common method is to break the DNA up with a random digestion method. This is usually accomplished using restriction enzymes (enzymes that cut DNA). A partial restriction digest cuts only some of the restriction sites, resulting in overlapping DNA fragment segments. 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 in a particular fragment, the DNA 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 must 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.[19]

For known DNA sequences, restriction enzymes that cut the DNA on either side of the gene can be used. Gel electrophoresis then sorts the fragments according to length.[20] Some gels can separate sequences that differ by a single base-pair. The DNA can be visualised by staining it with ethidium bromide and photographing under UV light. A marker with fragments of known lengths can be laid alongside the DNA to estimate the size of each band. The DNA band at the correct size should contain the gene, where it can be excised from the gel.[18]:4041 Another technique to isolate genes of known sequences involves polymerase chain reaction (PCR).[21] PCR is a powerful tool that can amplify a given sequence, which can then be isolated through gel electrophoresis. Its effectiveness drops with larger genes and it has the potential to introduce errors into the sequence.

It is possible to artificially synthesise genes.[22] Some synthetic sequences are available commercially, forgoing many of these early steps.[23]

The gene to be inserted must be combined with other genetic elements in order for it to work properly. The gene can 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. A selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is used 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.[24]

Once the gene is constructed it must be stably integrated into the genome of the target organism or exist as extrachromosomal DNA. There are a number of techniques available for inserting the gene into the host genome and they vary depending on the type of organism targeted. In multicellular eukaryotes, if the transgene is incorporated into the host's germline cells, the resulting host cell can pass the transgene to its progeny. If the transgene is incorporated into somatic cells, the transgene can not be inherited.[25]

Transformation is the direct alteration of a cell's genetic components by passing the genetic material through the cell membrane. About 1% of bacteria are naturally able to take up foreign DNA, but this ability can be induced in other bacteria.[26] Stressing the bacteria with a heat shock or electroporation can make the cell membrane permeable to DNA that may then be incorporated into the genome or exist as extrachromosomal DNA. Typically the cells are incubated in a solution containing divalent cations (often calcium chloride) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. It is suggested that exposing the cells to divalent cations in cold condition may change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall. Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms. Up-taken DNA can either integrate with the bacterials genome or, more commonly, exist as extrachromosomal DNA.

In plants the DNA is often inserted using Agrobacterium-mediated recombination,[27] taking advantage of the Agrobacteriums T-DNA sequence that allows natural insertion of genetic material into plant cells.[28] Plant tissue are cut into small pieces and soaked in a fluid containing suspended Agrobacterium. The bacteria will attach to many of the plant cells exposed by the cuts. The bacteria uses conjugation to transfer a DNA segment called T-DNA from its plasmid into the plant. The transferred DNA is piloted to the plant cell nucleus and integrated into the host plants genomic DNA.The plasmid T-DNA is integrated semi-randomly into the genome of the host cell.[29]

By modifying the plasmid to express the gene of interest, researchers can insert their chosen gene stably into the plants genome. The only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation.[30][31] The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the plasmid. An alternative method is agroinfiltration.[32][33]

Another method used to transform plant cells is biolistics, where particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos.[34] 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. Plants cells can also be transformed using electroporation, which uses an electric shock to make the cell membrane permeable to plasmid DNA. Due to the damage caused to the cells and DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial transformation.[citation needed]

Transformation has a different meaning in relation to animals, indicating progression to a cancerous state, so the process used to insert foreign DNA into animal cells is usually called transfection.[35] There are many ways to directly introduce DNA into animal cells in vitro. Often these cells are stem cells that are used for gene therapy. Chemical based methods uses natural or synthetic compounds to form particles that facilitate the transfer of genes into cells.[36] These synthetic vectors have the ability to bind DNA and accommodate large genetic transfers.[37] One of the simplest methods involves using calcium phosphate to bind the DNA and then exposing it to cultured cells. The solution, along with the DNA, is encapsulated by the cells.[38] Liposomes and polymers can be used as vectors to deliver DNA into cultured animal cells. Positively charged liposomes bind with DNA, while polymers can designed that interact with DNA.[36] They form lipoplexes and polyplexes respectively, which are then up-taken by the cells. Other techniques include using electroporation and biolistics.[39] In some cases, transfected cells may stably integrate external DNA into their own genome, this process is known as stable transfection.[40]

To create transgenic animals the DNA must be inserted into viable embryos or eggs. This is usually accomplished using microinjection, where DNA is injected through the cell's nuclear envelope directly into the nucleus.[26] Superovulated fertilised eggs are collected at the single cell stage and cultured in vitro. When the pronuclei from the sperm head and egg are visible through the protoplasm the genetic material is injected into one of them. The oocyte is then implanted in the oviduct of a pseudopregnant animal.[41] Another method is Embryonic Stem Cell-Mediated Gene Transfer. The gene is transfected into embryonic stem cells and then they are inserted into mouse blastocysts that are then implanted into foster mothers. The resulting offspring are chimeric, and further mating can produce mice fully transgenic with the gene of interest.[42]

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector.[43] Genetically modified viruses can be used as viral vectors to transfer target genes to another organism in gene therapy.[44] First the virulent genes are removed from the virus and the target genes are inserted instead. The sequences that allow the virus to insert the genes into the host organism must be left intact. Popular virus vectors are developed from retroviruses or adenoviruses. Other viruses used as vectors include, lentiviruses, pox viruses and herpes viruses. The type of virus used will depend on the cells targeted and whether the DNA is to be altered permanently or temporarily.

As often 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.[45][46] Each plant species has different requirements for successful regeneration. If successful, the technique produces an adult plant that contains the transgene in every cell.[47] In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells.[27] Offspring can be screened for the gene. All offspring from the first generation are heterozygous for the inserted gene and must be inbred to produce a homozygous specimen.[citation needed] 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.

Cells that have been successfully transformed with the DNA contain the marker gene, while those not transformed will not. 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. 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.[48]

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. Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene.[49] These tests can also confirm the chromosomal location and copy number of the inserted gene. Once confirmed methods that look for and measure the gene products (RNA and protein) are also used to assess gene expression, transcription, RNA processing patterns and expression and localization of protein product(s). These include northern hybridisation, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis.[50] When appropriate, the organism's offspring are studied to confirm that the transgene and associated phenotype are stably inherited.

Traditional methods of genetic engineering generally insert the new genetic material randomly within the host genome. This can impair or alter other genes within the organism. Methods were developed that inserted the new genetic material into specific sites within an organism genome. Early methods that targeted genes at certain sites within a genome relied on homologous recombination (HR).[51] By creating DNA constructs that contain a template that matches the targeted genome sequence, it is possible that the HR processes within the cell will insert the construct at the desired location. Using this method on embryonic stem cells led to the development of transgenic mice with targeted knocked out. It has also been possible to knock in genes or alter gene expression patterns.[52]

If a vital gene is knocked out it can prove lethal to the organism. In order to study the function of these genes, site specific recombinases (SSR) were used. The two most common types are the Cre-LoxP and Flp-FRT systems. Cre recombinase is an enzyme that removes DNA by homologous recombination between binding sequences known as Lox-P sites. The Flip-FRT system operates in a similar way, with the Flip recombinase recognizing FRT sequences. By crossing an organism containing the recombinase sites flanking the gene of interest with an organism that expresses the SSR under control of tissue specific promoters, it is possible to knock out or switch on genes only in certain cells. This has also been used to remove marker genes from transgenic animals. Further modifications of these systems allowed researchers to induce recombination only under certain conditions, allowing genes to be knocked out or expressed at desired times or stages of development.[52]

Genome editing uses artificially engineered nucleases that create specific double-stranded breaks at desired locations in the genome. The breaks are subject to cellular DNA repair processes that can be exploited for targeted gene knock-out, correction or insertion at high frequencies. If a donor DNA containing the appropriate sequence (homologies) is present, then new genetic material containing the transgene will be integrated at the targeted site with high efficiency by homologous recombination.[53] There are four families of engineered nucleases: meganucleases,[54][55] ZFNs,[56][57] transcription activator-like effector nucleases (TALEN),[58][59] the CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPRassociated protein (e.g. CRISPR/Cas9).[60][61] Among the four types, TALEN and CRISPR/Cas are the two most commonly used.[62] Recent advances have looked at combining multiple systems to exploit the best features of both (e.g. megaTAL that are a fusion of a TALE DNA binding domain and a meganuclease).[63] Recent research has also focused on developing strategies to create gene knock-out or corrections without creating double stranded breaks (base editors).[62]

Meganucleases were first used in 1988 in mammalian cells.[64] Meganucleases are endodeoxyribonucleases that function as restriction enzymes with long recognition sites, making them more specific to their target site than other restriction enzymes. This increases their specificity and reduces their toxicity as they will not target as many sites within a genome. The most studied meganucleases are the LAGLIDADG family. While meganucleases are still quite susceptible to off-target binding, which makes them less attractive than other gene editing tools, their smaller size still makes them attractive particularly for viral vectorization perspectives.[65][53]

Zinc-finger nucleases (ZFNs), used for the first time in 1996, are typically created through the fusion of Zinc-finger domains and the FokI nuclease domain. ZFNs have thus the ability to cleave DNA at target sites.[53] By engineering the zinc finger domain to target a specific site within the genome, it is possible to edit the genomic sequence at the desired location.[65][66][53] ZFNs have a greater specificity, but still hold the potential to bind to non-specific sequences.. While a certain amount of off-target cleavage is acceptable for creating transgenic model organisms, they might not be optimal for all human gene therapy treatments.[65]

Access to the code governing the DNA recognition by transcription activator-like effectors (TALE) in 2009 opened the way to the development of a new class of efficient TAL-based gene editing tools. TALE, proteins secreted by the Xanthomonas plant pathogen, bind with great specificity to genes within the plant host and initiate transcription of the genes helping infection. Engineering TALE by fusing the DNA binding core to the FokI nuclease catalytic domain allowed creation of a new tool of designer nucleases, the TALE nuclease (TALEN).[67] They have one of the greatest specificities of all the current engineered nucleases. Due to the presence of repeat sequences, they are difficult to construct through standard molecular biology procedure and rely on more complicated method of such as Golden gate cloning.[62]

In 2011, another major breakthrough technology was developed based on CRISPR/Cas (clustered regularly interspaced short palindromic repeat / CRISPR associated protein) systems that function as an adaptive immune system in bacteria and archaea. The CRISPR/Cas system allows bacteria and archaea to fight against invading viruses by cleaving viral DNA and inserting pieces of that DNA into their own genome. The organism then transcribes this DNA into RNA and combines this RNA with Cas9 proteins to make double-stranded breaks in the invading viral DNA. The RNA serves as a guide RNA to direct the Cas9 enzyme to the correct spot in the virus DNA. By pairing Cas proteins with a designed guide RNA CRISPR/Cas9 can be used to induce double-stranded breaks at specific points within DNA sequences. The break gets repaired by cellular DNA repair enzymes, creating a small insertion/deletion type mutation in most cases. Targeted DNA repair is possible by providing a donor DNA template that represents the desired change and that is (sometimes) used for double-strand break repair by homologous recombination. It was later demonstrated that CRISPR/Cas9 can edit human cells in a dish. Although the early generation lacks the specificity of TALEN, the major advantage of this technology is the simplicity of the design. It also allows multiple sites to be targeted simultaneously, allowing the editing of multiple genes at once. CRISPR/Cpf1 is a more recently discovered system that requires a different guide RNA to create particular double-stranded breaks (leaves overhangs when cleaving the DNA) when compared to CRISPR/Cas9.[62]

CRISPR/Cas9 is efficient at gene disruption. The creation of HIV-resistant babies by Chinese researcher He Jiankui is perhaps the most famous example of gene disruption using this method.[68] It is far less effective at gene correction. Methods of base editing are under development in which a nuclease-dead Cas 9 endonuclease or a related enzyme is used for gene targeting while a linked deaminase enzyme makes a targeted base change in the DNA.[69] The most recent refinement of CRISPR-Cas9 is called Prime Editing. This method links a reverse transcriptase to an RNA-guided engineered nuclease that only makes single-strand cuts but no double-strand breaks. It replaces the portion of DNA next to the cut by the successive action of nuclease and reverse transcriptase, introducing the desired change from an RNA template.[70]

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20.3: Genetic Engineering – Biology LibreTexts

Tuesday, January 9th, 2024

Genetic engineering is the alteration of an organisms genotype using recombinant DNA technology to modify an organisms DNA to achieve desirable traits. The addition of foreign DNA in the form of recombinant DNA vectors generated by molecular cloning is the most common method of genetic engineering. The organism that receives the recombinant DNA is called a genetically modified organism (GMO). If the foreign DNA that is introduced comes from a different species, the host organism is called transgenic. Bacteria, plants, and animals have been genetically modified since the early 1970s for academic, medical, agricultural, and industrial purposes. In the US, GMOs such as Roundup-ready soybeans and borer-resistant corn are part of many common processed foods.

Although classical methods of studying the function of genes began with a given phenotype and determined the genetic basis of that phenotype, modern techniques allow researchers to start at the DNA sequence level and ask: What does this gene or DNA element do? This technique, called reverse genetics, has resulted in reversing the classic genetic methodology. This method would be similar to damaging a body part to determine its function. An insect that loses a wing cannot fly, which means that the function of the wing is flight. The classical genetic method would compare insects that cannot fly with insects that can fly, and observe that the non-flying insects have lost wings. Similarly, mutating or deleting genes provides researchers with clues about gene function. The methods used to disable gene function are collectively called gene targeting. Gene targeting is the use of recombinant DNA vectors to alter the expression of a particular gene, either by introducing mutations in a gene, or by eliminating the expression of a certain gene by deleting a part or all of the gene sequence from the genome of an organism.

The process of testing for suspected genetic defects before administering treatment is called genetic diagnosis by genetic testing. Depending on the inheritance patterns of a disease-causing gene, family members are advised to undergo genetic testing. For example, women diagnosed with breast cancer are usually advised to have a biopsy so that the medical team can determine the genetic basis of cancer development. Treatment plans are based on the findings of genetic tests that determine the type of cancer. If the cancer is caused by inherited gene mutations, other female relatives are also advised to undergo genetic testing and periodic screening for breast cancer. Genetic testing is also offered for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases.

Gene therapy is a genetic engineering technique used to cure disease. In its simplest form, it involves the introduction of a good gene at a random location in the genome to aid the cure of a disease that is caused by a mutated gene. The good gene is usually introduced into diseased cells as part of a vector transmitted by a virus that can infect the host cell and deliver the foreign DNA (Figure (PageIndex{1})). More advanced forms of gene therapy try to correct the mutation at the original site in the genome, such as is the case with treatment of severe combined immunodeficiency (SCID).

Traditional vaccination strategies use weakened or inactive forms of microorganisms to mount the initial immune response. Modern techniques use the genes of microorganisms cloned into vectors to mass produce the desired antigen. The antigen is then introduced into the body to stimulate the primary immune response and trigger immune memory. Genes cloned from the influenza virus have been used to combat the constantly changing strains of this virus.

Antibiotics are a biotechnological product. They are naturally produced by microorganisms, such as fungi, to attain an advantage over bacterial populations. Antibiotics are produced on a large scale by cultivating and manipulating fungal cells.

Recombinant DNA technology was used to produce large-scale quantities of human insulin in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in humans because of differences in the gene product. Currently, the vast majority of diabetes sufferers who inject insulin do so with insulin produced by bacteria.

Human growth hormone (HGH) is used to treat growth disorders in children. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector. Bacterial HGH can be used in humans to reduce symptoms of various growth disorders.

Although several recombinant proteins used in medicine are successfully produced in bacteria, some proteins require a eukaryotic animal host for proper processing. For this reason, the desired genes are cloned and expressed in animals, such as sheep, goats, chickens, and mice. Animals that have been modified to express recombinant DNA are called transgenic animals. Several human proteins are expressed in the milk of transgenic sheep and goats, and some are expressed in the eggs of chickens. Mice have been used extensively for expressing and studying the effects of recombinant genes and mutations.

Manipulating the DNA of plants (i.e., creating GMOs) has helped to create desirable traits, such as disease resistance, herbicide and pesticide resistance, better nutritional value, and better shelf-life (Figure (PageIndex{3})). Plants are the most important source of food for the human population. Farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established.

Plants that have received recombinant DNA from other species are called transgenic plants. Because they are not natural, transgenic plants and other GMOs are closely monitored by government agencies to ensure that they are fit for human consumption and do not endanger other plant and animal life. Because foreign genes can spread to other species in the environment, extensive testing is required to ensure ecological stability. Staples like corn, potatoes, and tomatoes were the first crop plants to be genetically engineered.

Gene transfer occurs naturally between species in microbial populations. Many viruses that cause human diseases, such as cancer, act by incorporating their DNA into the human genome. In plants, tumors caused by the bacterium Agrobacterium tumefaciens occur by transfer of DNA from the bacterium to the plant. Although the tumors do not kill the plants, they make the plants stunted and more susceptible to harsh environmental conditions. Many plants, such as walnuts, grapes, nut trees, and beets, are affected by A. tumefaciens. The artificial introduction of DNA into plant cells is more challenging than in animal cells because of the thick plant cell wall.

Researchers used the natural transfer of DNA from Agrobacterium to a plant host to introduce DNA fragments of their choice into plant hosts. In nature, the disease-causing A. tumefaciens have a set of plasmids, called the Ti plasmids (tumor-inducing plasmids), that contain genes for the production of tumors in plants. DNA from the Ti plasmid integrates into the infected plant cells genome. Researchers manipulate the Ti plasmids to remove the tumor-causing genes and insert the desired DNA fragment for transfer into the plant genome. The Ti plasmids carry antibiotic resistance genes to aid selection and can be propagated in E. coli cells as well.

Bacillus thuringiensis (Bt) is a bacterium that produces protein crystals during sporulation that are toxic to many insect species that affect plants. Bt toxin has to be ingested by insects for the toxin to be activated. Insects that have eaten Bt toxin stop feeding on the plants within a few hours. After the toxin is activated in the intestines of the insects, death occurs within a couple of days. Modern biotechnology has allowed plants to encode their own crystal Bt toxin that acts against insects. The crystal toxin genes have been cloned from Bt and introduced into plants. Bt toxin has been found to be safe for the environment, non-toxic to humans and other mammals, and is approved for use by organic farmers as a natural insecticide.

The first GM crop to be introduced into the market was the Flavr Savr Tomato produced in 1994. Antisense RNA technology was used to slow down the process of softening and rotting caused by fungal infections, which led to increased shelf life of the GM tomatoes. Additional genetic modification improved the flavor of this tomato. The Flavr Savr tomato did not successfully stay in the market because of problems maintaining and shipping the crop.

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OpenStax, Biology. OpenStax CNX. May 27, 2016 http://cnx.org/contents/s8Hh0oOc@9.10:8CA_YwJq@3/Cloning-and-Genetic-Engineerin

Moen I, Jevne C, Kalland K-H, Chekenya M, Akslen LA, Sleire L, Enger P, Reed RK, Oyan AM, Stuhr LEB. 2012.Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation.BMC Cancer. 12:21. doi:10.1186/1471-2407-12-21 PDF

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20.3: Genetic Engineering - Biology LibreTexts

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Genetic Modification Techniques and Applications – Technology Networks

Tuesday, January 9th, 2024

Genetic Modification Techniques and Applications  Technology Networks

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Genetic Modification Techniques and Applications - Technology Networks

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Transformation-associated recombination (TAR) cloning and its applications – EurekAlert

Tuesday, January 9th, 2024

Transformation-associated recombination (TAR) cloning and its applications  EurekAlert

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Transformation-associated recombination (TAR) cloning and its applications - EurekAlert

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Are GMOs Safe? The Benefits of Science-Enhanced Foods | TIME – TIME

Tuesday, January 9th, 2024

Are GMOs Safe? The Benefits of Science-Enhanced Foods | TIME  TIME

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Are GMOs Safe? The Benefits of Science-Enhanced Foods | TIME - TIME

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Here’s the skinny on fast-emerging genetic engineering tools that are rapidly improving global farming – Genetic Literacy Project

Tuesday, January 9th, 2024

Here's the skinny on fast-emerging genetic engineering tools that are rapidly improving global farming  Genetic Literacy Project

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Here's the skinny on fast-emerging genetic engineering tools that are rapidly improving global farming - Genetic Literacy Project

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What Fruit Looked Like 100 Years Ago, Before Genetic Modification – Green Matters

Tuesday, January 9th, 2024

What Fruit Looked Like 100 Years Ago, Before Genetic Modification  Green Matters

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What Fruit Looked Like 100 Years Ago, Before Genetic Modification - Green Matters

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APHIS Reopens Comment Period on Proposed Additional Exemptions for Plants Modified or Produced Through Genetic Engineering – The National Law Review

Tuesday, January 9th, 2024

APHIS Reopens Comment Period on Proposed Additional Exemptions for Plants Modified or Produced Through Genetic Engineering  The National Law Review

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APHIS Reopens Comment Period on Proposed Additional Exemptions for Plants Modified or Produced Through Genetic Engineering - The National Law Review

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Restriction Enzyme Market is set to Reach US$ 647.41 million at a CAGR of 5.7% from the forecast period 2023 to … – GlobeNewswire

Tuesday, January 9th, 2024

Restriction Enzyme Market is set to Reach US$ 647.41 million at a CAGR of 5.7% from the forecast period 2023 to ...  GlobeNewswire

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Restriction Enzyme Market is set to Reach US$ 647.41 million at a CAGR of 5.7% from the forecast period 2023 to ... - GlobeNewswire

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Genetic engineering – DNA Modification, Cloning, Gene Splicing

Wednesday, December 13th, 2023

Most recombinant DNA technology involves the insertion of foreign genes into the plasmids of common laboratory strains of bacteria. Plasmids are small rings of DNA; they are not part of the bacteriums chromosome (the main repository of the organisms genetic information). Nonetheless, they are capable of directing protein synthesis, and, like chromosomal DNA, they are reproduced and passed on to the bacteriums progeny. Thus, by incorporating foreign DNA (for example, a mammalian gene) into a bacterium, researchers can obtain an almost limitless number of copies of the inserted gene. Furthermore, if the inserted gene is operative (i.e., if it directs protein synthesis), the modified bacterium will produce the protein specified by the foreign DNA.

A subsequent generation of genetic engineering techniques that emerged in the early 21st century centred on gene editing. Gene editing, based on a technology known as CRISPR-Cas9, allows researchers to customize a living organisms genetic sequence by making very specific changes to its DNA. Gene editing has a wide array of applications, being used for the genetic modification of crop plants and livestock and of laboratory model organisms (e.g., mice).

The correction of genetic errors associated with disease in animals suggests that gene editing has potential applications in gene therapy for humans. Gene therapy is the introduction of a normal gene into an individuals genome in order to repair a mutation that causes a genetic disease. When a normal gene is inserted into a mutant nucleus, it most likely will integrate into a chromosomal site different from the defective allele; although this may repair the mutation, a new mutation may result if the normal gene integrates into another functional gene. If the normal gene replaces the mutant allele, there is a chance that the transformed cells will proliferate and produce enough normal gene product for the entire body to be restored to the undiseased phenotype.

Genetic engineering has advanced the understanding of many theoretical and practical aspects of gene function and organization. Through recombinant DNA techniques, bacteria have been created that are capable of synthesizing human insulin, human growth hormone, alpha interferon, a hepatitis B vaccine, and other medically useful substances. Plants may be genetically adjusted to enable them to fix nitrogen, and genetic diseases can possibly be corrected by replacing dysfunctional genes with normally functioning genes.

Genes for toxins that kill insects have been introduced in several species of plants, including corn and cotton. Bacterial genes that confer resistance to herbicides also have been introduced into crop plants. Other attempts at the genetic engineering of plants have aimed at improving the nutritional value of the plant.

In 1980 the new microorganisms created by recombinant DNA research were deemed patentable, and in 1986 the U.S. Department of Agriculture approved the sale of the first living genetically altered organisma virus, used as a pseudorabies vaccine, from which a single gene had been cut. Since then several hundred patents have been awarded for genetically altered bacteria and plants. Patents on genetically engineered and genetically modified organisms, particularly crops and other foods, however, were a contentious issue, and they remained so into the first part of the 21st century.

Grains of golden rice, a genetically modified rice (Oryza sativa) that contains beta-carotene.(more)

Special concern has been focused on genetic engineering for fear that it might result in the introduction of unfavourable and possibly dangerous traits into microorganisms that were previously free of theme.g., resistance to antibiotics, production of toxins, or a tendency to cause disease. Indeed, possibilities for misuse of genetic engineering were vast. In particular, there was significant concern about genetically modified organisms, especially modified crops, and their impacts on human and environmental health. For example, genetic manipulation may potentially alter the allergenic properties of crops. In addition, whether some genetically modified crops, such as golden rice, deliver on the promise of improved health benefits was also unclear. The release of genetically modified mosquitoes and other modified organisms into the environment also raised concerns.

In the 21st century, significant progress in the development of gene-editing tools brought new urgency to long-standing discussions about the ethical and social implications surrounding the genetic engineering of humans. The application of gene editing in humans raised significant ethical concerns, particularly regarding its potential use to alter traits such as intelligence and beauty. More practically, some researchers attempted to use gene editing to alter genes in human sperm, which would enable the edited genes to be passed on to subsequent generations, while others sought to alter genes that increase the risk of certain types of cancer, with the aim of reducing cancer risk in offspring. The impacts of gene editing on human genetics, however, were unknown, and regulations to guide its use were largely lacking.

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Genetic engineering - DNA Modification, Cloning, Gene Splicing

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Star Trek: Julian Bashir’s Tale of Genetic Engineering and the Complexity of Parental Acceptance – GameRant

Wednesday, December 13th, 2023

Star Trek: Julian Bashir's Tale of Genetic Engineering and the Complexity of Parental Acceptance  GameRant

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Star Trek: Julian Bashir's Tale of Genetic Engineering and the Complexity of Parental Acceptance - GameRant

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