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Modification of genes by homologous recombination

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Innovation

Series E: 2; 2.15

Mario Capecchi, Martin Evans, Oliver Smithies

2007 Nobel Prize for their work on targeted gene modification.

Born in Italy in 1937, scientist Mario R. Capecchi emigrated to the United States after World War II and later became a geneticist and professor. His groundbreaking work on targeted gene modification won him a Nobel Prize in 2007. He is Distinguished Professor of Human Genetics at the University of Utah School of Medicine. Mario Capecchi is interested in the molecular genetic analysis of mammalian development, with emphasis on neurogenesis, organogenesis, patterning of the vertebral column, and limb development. He also contributes to the modeling of human disease in the mouse, from cancer to neuropsychiatric disorders.

Capecchi MR. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet, Jun;6(6):507-12. Review.

https://youtu.be/WQr6ZeNe-vE

 

Sir Martin John Evans FRS FMedSci (b. 1 January 1941, StroudGloucestershire[1][5]) is a Welsh biologist who, with Matthew Kaufman, was the first to culture mice embryonic stem cells and cultivate them in a laboratory in 1981. He is also known, along with Mario Capecchi and Oliver Smithies, for his work in the development of the knockout mouse and the related technology of gene targeting, a method of using embryonic stem cells to create specific gene modifications in mice.[5][6] In 2007, the three shared the Nobel Prize in Physiology or Medicine in recognition of their discovery and contribution to the efforts to develop new treatments for illnesses in humans.[7][8][9][10][11]

He won a major scholarship to Christ’s College, Cambridge at a time when advances in genetics were occurring there and became interested in biology and biochemistry. He then went to University College London where he learned laboratory skills supervised by Elizabeth Deuchar. In 1978, he moved to the Department of Genetics, at the University of Cambridge, and in 1980 began his collaboration with Matthew Kaufman. They explored the method of using blastocysts for the isolation of embryonic stem cells. After Kaufman left, Evans continued his work, upgrading his laboratory skills to the newest technologies, isolated the embryonic stem cell of the early mouse embryo and established it in a cell culture. He genetically modified and implanted it into adult female mice with the intent of creating genetically modified offspring, work for which he was awarded the Nobel Prize in 2007.

In 1981, Evans and Kaufman published results for experiments in which they described how they isolated embryonic stem cells from mouse blastocysts and grew them in cell cultures.[23][24] This was also achieved by Gail R. Martin, independently, in the same year.[25] Eventually, Evans was able to isolate the embryonic stem cell of the early mouse embryo and establish it in a cell culture. He then genetically modified it and implanted it into adult female mice with the intent of creating genetically modified offspring, the forbearers of the laboratory mice that are considered so vital to medical research today.[23] The availability of these cultured stem cells eventually made possible the introduction of specific gene alterations into the germ line of mice and the creation of transgenic mice to use as experimental models for human illnesses.[23]

Evans and his collaborators showed that they could introduce a new gene into cultured embryonic stem cells and then use such genetically transformed cells to make chimeric embryos.[26] In some chimeric embryos, the genetically altered stem cells produced gametes, thus allowing transmission of the artificially induced mutation into future generations of mice.[27] In this way, transgenic mice with induced mutations in the enzyme Hypoxanthine-guanine phosphoribosyltransferase (HPRT) were created.[28] Today, genetically modified mice are considered vital for medical research.

In the 1990s, he was a fellow at St Edmund’s College, Cambridge. In 1999, he became Professor of Mammalian Genetics and Director of the School of Biosciences at Cardiff University,[5][17] where he worked until he retired at the end of 2007.[18] He became a Knight Bachelor in the 2004 New Year Honours in recognition of his work in stem cell research.[5][19] He received the accolade from Prince Charles at Buckingham Palace on 25 June 2004.[20] In 2007, he was awarded the Nobel Prize in Physiology or Medicine along with Mario Capecchi and Oliver Smithies for their work in discovering a method for introducing homologous recombination in mice employing embryonic stem cells.[7] Evans was appointed president of Cardiff University and was inaugurated into that position on 23 November 2009.[21] Subsequently Evans became Chancellor of Cardiff University in 2012. [22]

 

The Whole of a Scientific Career: An Interview with Oliver Smithies

Jane Gitschier*

PLoS Genet. 2015 May; 11(5): e1005224.

Published online 2015 May 28. doi:  10.1371/journal.pgen.1005224

Smithies, of course, is well worth any pilgrimage. Nearing 90 years of age, he still works at the bench, seven days a week. He is enthusiastic, curious, gentle, and fearless in attacking new problems, to which he applies his gifts both as a tinkerer and a thinker. He is generous with his ideas and advice and beloved by his colleagues, students, and postdocs, now numbering so many that he has lost count. His scientific journey began in the mid-late 1940s as an undergraduate at Balliol College, Oxford, where his tutor introduced him to a new field, now called “molecular biology.” Smithies embraced the young field, and after a brief postdoctoral stint at University of Wisconsin, took his first job in Toronto. There, in the early 1950s, he invented starch gel electrophoresis, which had the property of fractionating proteins on the basis of size and led him to discover inherited differences in haptoglobin, a serum protein that binds hemoglobin. One variant, the product of an abnormal genetic exchange, piqued his life-long interest in homologous recombination. Three decades later, after an arduous, three-year experiment, he was able to demonstrate homologous recombination between a plasmid and the human genome in the pursuit of correcting genetic defects, a discovery for which he, much later, won the Nobel Prize.

 

Genetic engineering, also called genetic modification, is the direct manipulation of an organism’s genome using biotechnology. It is therefore a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or “knocked out”, using a nuclease. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations.

An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). The first GMOs were bacteria generated in 1973 and GM mice in 1974. Insulin-producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994. Glofish, the first GMO designed as a pet, was first sold in the United States December in 2003.[1]

Genetic engineering techniques have been applied in numerous fields including research, agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and medicines such as insulin and human growth hormone are now manufactured in GM cells, experimental GM cell lines and GM animals such as mice or zebrafish are being used for research purposes, and genetically modified crops have been commercialized.

In 1972 Paul Berg created the first recombinant DNA molecules by combining DNA from the monkey virus SV40 with that of the lambda virus.[26] In 1973 Herbert Boyer andStanley Cohen created the first transgenic organism by inserting antibiotic resistance genes into the plasmid of an E. coli bacterium.[27][28] A year later Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal.[29] These achievements led to concerns in the scientific community about potential risks from genetic engineering, which were first discussed in depth at the Asilomar Conference in 1975. One of the main recommendations from this meeting was that government oversight of recombinant DNA research should be established until the technology was deemed safe.[30][31]

In 1976 Genentech, the first genetic engineering company, was founded by Herbert Boyer and Robert Swanson and a year later the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.[32] In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented.[33] The insulin produced by bacteria, branded humulin, was approved for release by the Food and Drug Administration in 1982.[34]

The most common form of genetic engineering involves inserting new genetic material randomly within the host genome.[citation needed] 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,[62][63] engineered homing endonucleases,[64][65] or nucleases created from TAL effectors.[66][67] In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout.[68][69]

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