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Trends Biotechnology

150429 TB13 Transgenic animals

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Methods of creating transgenic animals or cloning animals.

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A transgenic, or genetically modified, organism is one that has been altered through recombinant DNA technology, which involves either the combining of DNA from different genomes or the insertion of foreign DNA into a genome.

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Key Developments in Recombinant DNA Technology Four key developments helped lead to construction of the first recombinant DNA organism. • Two developments involved cutting and joining

pieces of DNA from different genomes using enzymes.

• Two other developments involved techniques used to transfer foreign DNA into new host cells.

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1. Joining enzymes Martin Gellert and his colleagues purified and characterized an enzyme in Escherichia coli responsible for the actual joining, or recombining, of separate pieces of DNA. This enzyme is now known as DNA ligase. All living cells use some version of DNA ligase to join short strands of DNA during replication.

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Using E. coli extract, the researchers next showed that only in the presence of ligase was it possible to repair single-stranded breaks in λ phage DNA. λ phage is a virus particle that infects E. coli.The enzyme was able to form a 3'-5'-phosphodiester bond between the 5'-phosphate end of the last nucleotide on one DNA fragment and the 3'-OH end of the last nucleotide on an adjacent fragment.

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2. Cutting enzymes Restriction enzymes, which cut DNA at specific sequences. Werner Arber and his colleagues were investigating host-controlled restriction of bacteriophages. Bacteriophages are viruses that invade and often destroy their bacterial host cells; host-controlled restriction is the defense mechanisms that bacterial cells have evolved to protect themselves from these invading viruses. It is enzymatic activity provided by the host cell.

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The enzymes were called "restriction enzymes" because of the way they restrict the growth of bacteriophages. Restriction enzymes damage invading bacteriophages by cutting the phage DNA at very specific nucleotide sequences (now known as restriction sites). The identification and characterization of restriction enzymes gave biologists the means to cut specific pieces of DNA for subsequent recombination.

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3. Inserting Foreign DNA into a New Host Cell In the 1970s scientists began to use vectors to efficiently transfer genes into bacterial cells. The first vectors were plasmids, or small DNA molecules that live naturally inside bacterial cells and replicate separately from a bacterium's chromosomal DNA.

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Plasmids' use as a vector was discovered by Stanley Cohen. Some bacteria had antibiotic resistance factors, or R factor-plasmids that replicated independently inside the bacterial cell. Cohen showed that calcium chloride-treated E. coli can be genetically transformed into antibiotic-resistant cells by the addition of purified plasmid DNA (in this case, purified R-factor DNA) to the bacteria during transformation.

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Recombinant Plasmids in Bacteria Cohen and his colleagues were also the first to make a novel plasmid DNA from two separate plasmid species which, when introduced into E. coli, possessed all the nucleotide base sequences and functions of both parent plasmids. Restriction endonuclease enzymes cut the double-stranded DNA molecules of the two parent plasmids.

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DNA ligase rejoined, or recombined, the DNA fragments from the two different plasmids. The newly recombined plasmid DNA was put into E. coli. "the nucleotide sequences cleaved are unique and self-complementary so that DNA fragments produced by one of these enzymes can associate by hydrogen-bonding with other fragments produced by the same enzyme" (Cohen et al., 1973).So the researchers were able to join two DNA fragments from completely different plasmids.

13© 2005 W. H. Freeman Pierce, Benjamin. Genetics: A Conceptual Approach, 2nd ed. (New York: W. H. Freeman and Company), 519. All rights reserved

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Any DNA—not just plasmids—from two different species could be joined. DNA has the same structure and function in all species and restriction and ligase enzymes cut and paste the same ways in different genomes - so recombinant DNA biology is possible.

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E. coli λ bacteriophage is now one of the most widely used vectors used to carry recombinant DNA into bacterial cells. About one-third of virus genome is considered nonessential, meaning that it can be removed and replaced by foreign DNA (i.e., the DNA being inserted). The nonessential genes are removed by restriction enzymes, the foreign DNA inserted in their place, and then the final recombinant DNA molecule is packaged into the virus's protein coat and prepared for introduction into its host cell.

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Phage λ is an effective cloning vector.

© 2005 W. H. Freeman Pierce, Benjamin. Genetics: A Conceptual Approach, 3rd ed. (New York: W. H. Freeman and Company), 521. Used with permission. All rights reserved.

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4. Vectors Used in Mammalian Cells Discovery of a vector for efficiently introducing genes into mammalian cells. Recombinant DNA could be introduced into the SV40 virus, a pathogen that infects both monkeys and humans.

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1972, Paul Berg and colleagues integrated segments of λ phage DNA, as well as a segment of E. coli DNA containing the galactose operon, into the SV40 genome. Recombinant DNA technologies could be applied to any DNA sequences, no matter how distantly related their species of origin.

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Recombinant DNA Technology Creates Recombinant Animals 1981, for example, Franklin Costantini and Elizabeth Lacy introduced rabbit DNA fragments containing the adult beta globin gene into murine (mouse) germ-line cells.(The beta globins are a family of polypeptides that serve as the subunits of hemoglobin molecules.)

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Another group had shown that foreign genes could be successfully integrated into murine somatic cells, but this was the first demonstration of their integration into germ cells. So Costantini and Lacy were the first to engineer an entire recombinant animal.

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Recombinant DNA technologies have been used to create many different types of recombinant animals, both for scientific study and for the manufacturing of human proteins.

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For instance, mice, goats, and cows have all been engineered to create medically valuable proteins in their milk.Hormones that were once isolated only in small amounts from dead humans can now be mass-produced by genetically engineered cells.

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Gene Therapy

A single base change - specifically, the appearance of a cytosine instead of a thymine - leads to a rare congenital blindness known as Leber congenital amaurosis.

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To fix disorders that are caused by mutations in a single gene insert the correct form of the mutant gene into the cells that need the functioning gene. But, the field of gene therapy, in which scientists work to treat disorders at the level of the sequence of mutated genes, is much more complicated.

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How to Get Therapeutic DNA Inside CellsA big problem for effective gene therapy involves finding the best way to deliver therapeutic genes into target cells within the body. Scientists have tested a number of methods, some of which use liposomes or cell surface receptors. But the most common method of DNA delivery uses something that is perfectly evolved to enter cells: the virus.

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Viruses are obligate intracellular parasites, designed to infect cells, often with great specificity to particular cell types. Viruses efficiently transfect their own DNA into a host cell, then use the host's cellular machinery to replicate their DNA and synthesize certain viral proteins, thereby producing more viral particles.

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We can use this process by removing the disease-causing portions of the viral genome and adding a foreign therapeutic gene. The engineered genome is then repackaged into the viral protein coat and allowed to infect the proper cell target. The cell infected by this virus is able to produce the protein or enzyme it lacks.

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A common virus to use as a vector is an adenovirus. Recombinant DNA technology can introduce a wild-type gene into an adenoviral vector. The adenovirus infects the target cells, homologous recombination occurs, the native, mutant copy of the gene is replace by the wild-type version from the vector.

29Gene targeting with recombinant adenoviral vectors.

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A nonfunctional mutant of a reporter gene (W in the figure) is introduced or is present in a cell (a). In the recombinant targeting vector (b), the viral genes are replaced by a sequence that is homologous to the chromosomal locus targeted for modification. Usually, the modification is introduced between stretches of homology called 5' and 3' homology arms. Z designates an inactivating mutation in the viral repair template. This recombinant adeno-associated virus (rAAV) is used to transduce the cells. Recombination with the chromosomal target (c) can result in repair of the defect and recovery of a healthy cell (d). The frequency of gene targeting is determined as the fraction of infected cells that expresses a functional reporter. Stable integration is confirmed by antibiotic selection and Southern analysis. (In the figure, crosses [X] mark regions of homology between the chromosomal and viral DNA, and “ITR” indicates an inverted terminal repeat.)

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LCA: A Disease Candidate for Gene Therapy Leber congenital amaurosis (LCA) arises from a single-base mutation. This mutation stops the biological conversion of a photon of light to a chemical signal in the retina that is then sent to the visual cortex of the brain.

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Within this cycle, 11-cis-retinal protein, when struck by a photon of light, undergoes a conformational change that induces signal cascades that stimulate the visual cortex. The RPE65 gene codes for a protein that is critical for the regeneration of 11-cis-retinal protein into its starting form. Without RPE65, there is no 11-cis-retinal protein present to change light signals into chemical signals, and the result is blindness.

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About 10% of cases of LCA bear a mutation in the RPE65 gene. Because this form of LCA is the result of a single-gene defect and there are no other treatments available, gene therapy was considered as a possibility for this disorder.

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Work began using dogs as an animal model of LCA. In dogs, a four-base-pair deletion in the RPE65 gene (denoted RPE65 -/-) alters that gene such that functional protein cannot be not made.

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Investigators infected cells extracted from RPE65 -/- dogs with a recombinant adeno-associated virus (rAAV) that contained wild-type RPE65. Expression was recovered in the cells, suggesting that it was possible to rescue expression of the RPE65 protein.

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RPE65 immunocytochemistry in canine RPE cells and retinal sections.

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This image depicts cultured canine RPE cells immunolabeled with anti-RPE65 antibody, with nuclei stained with propidium iodide. (a) Wild-type cells label uniformly and intensely with anti-RPE65 antibody. (b) RPE65 -/- cells do not label before infection with AAV-RPE65. (c) Most RPE65 -/- cells label positively (indicated by arrowheads) following infection with AAV-RPE65, indicating the presence of wild-type RPE65 protein. One cell (indicated by the arrow) does not seem to have been transduced.

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To see if expression of wild-type RPE65 resulted in a physiological effect, researchers injected the virus directly into the dogs’ retinas or the surrounding tissue. They tested to see if the pathway from the retina to the visual cortex in the dogs' brains regained the ability to respond properly to light stimulation.

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It is generally thought that a lack of visual stimulation leads to a concomitant lack of development in the visual cortex, resulting in blindness. The researchers therefore worried that without visual stimulation, proper development of the visual cortex had not occurred in these LCA animals, so even if the function of RPE65 could be restored, the dogs' vision might not improve.

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Using electroretinography (ERG), the investigators compared the retinal response of wild-type animals with the retinal response of RPE65 mutant animals both before and after gene therapy. The results showed a rescue of the mutant animals' retinal response to light, with post-treatment dogs exhibiting a response to light similar to that of wild-type animals.

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Restoration of retinal and visual function in RPE65 mutant dogs by subretinal AAV-RPE65 therapy.

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Dark-adapted ERGs evoked by increasing intensities of blue light stimuli in a control dog (left) are compared with those evoked using the same stimuli in an RPE65 mutant dog (middle). After subretinal AAV-RPE65 therapy (right), the mutant dog shows an improved response, although not to normal levels, as well as an ERG waveform shape that is now similar to that of the control dog.

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Functional magnetic resonance imaging (fMRI) measured activity in the dogs' brains in response to light stimulation both before and after gene therapy. Following gene therapy, there was almost-normal response in the dogs' visual cortices to light stimulation; before treatment, there had been no activity in this region of the dogs' brains. Also there was normal pupil function and normal electrical activity in the retinas of the treated dogs, both of which were abnormal before gene therapy.

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The cure was tested in adult human patients with LCA. Gene therapy seemed to restore some functional vision to people with LCA.

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The results of a phase 1 trial showed improvement in 12 subjects (ages 8 to 44).Younger patients were associated with better results. Patients were excluded based on the presence of particular antibodies to the vector AAV2 and treatment was only administered to one eye as a precaution.

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Moving from Animals to Humans: Important Considerations

There are significant risks in gene therapy. A virus may recover its ability to cause disease once inside the body. The retrovirus might cause insertional mutagenesis.

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The virus inserts its genome within a host cell's chromosomes, thereby potentially disrupting important genes. If the viral genome is inserted into a tumor suppressor gene, it might cause the development of cancer.

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There may be immune and inflammatory responses when any foreign particle, such as a virus, is introduced into the body.

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A Major Setback to the Field of Gene Therapy In 1999, just nine years after the first human trial for gene therapy was conducted, an 18-year-old gene therapy trial volunteer died.

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He suffered from an enzyme deficiency that causes an inability to break down ammonia. Four days after receiving an injection of adenovirus carrying a corrected version of the gene, the volunteer died due to multiple organ failure.

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His death is believed to have been triggered by a severe immune response to the adenovirus carrier. In response, the U.S. FDA temporarily halted all gene therapy trials while the National Institutes of Health conducted reviews of all adverse reactions and deaths associated with this type of research.