Genetic Engineering: History and Applications

• Genetic engineering definition: recombinant DNA technology and biotechnology coined in 1919 by Karl Ereky.
• Humans have used artificial selection to manipulate organisms for thousands of years, with domesticated species as early as 8000 to 1000 B.C.
• Yeast has been used to make beer as early as 6000 B.C..
• Plants such as maize, wheat and rice have been bred since 5000 B.C..
• In 420 B.C., Socrates speculated on why children do not necessarily resemble their parents.
• Hippocrates proposed in 400 B.C. that males contribute to a child's character through semen, establishing the idea of heredity.
• Hindu philosophers in 100-300 A.D. observed familial diseases, and believed children inherited all characteristics from their parents.
• The 19th century saw an exponential increase in biochemical studies and the speeding up of the fermentation industry.
• Mendel presented his work on peas in 1864-1865, but it was largely neglected for some time and the terms gene or genetics was not yet coined.
• In 1882, the first biotechnology products which included the use of agar as described by the Koch lab.
• The autoclave was developed in 1884 by the French company Chamberland's Autoclaves.
• X-rays where discovered in 1895 by W. Roentgen.
• X-ray crystallography was invented in 1913 by Sir William Henry Bragg and his son William Lawrence Bragg.
• In 1945, William Astbury coined the term molecular biology while working in X-ray diffraction analysis of biological macromolecules.
• Rosalind Franklin and Maurice Wilkins obtained X-ray diffraction data for DNA in the early 1950s, which was critical to Watson and Crick's model.
• Hershey showed the hereditary material is DNA, not protein, while working with Martha Chase in 1951.
• Luria and Hershey showed bacteriophages mutate and introduced criteria for distinguishing mutations.
• Werner Arber identified restriction enzymes in bacteria that cleave DNA in the late 1960s.
• Temin and Baltimore independently identified reverse transcriptase in 1970, leading to recombinant DNA technology.
• Paul Berg proved the possibility to splice and recombine genetic material in 1971.
• The first recombinant DNA was produced in Boyer Laboratory in 1972
• The first biotechnology company, Genentech, was born in 1976.
• The polymerase chain reaction(PCR) was invented in 1983 by Karen Mullis
• Genentech and Eli Lilly marketed recombinant interferon gamma and human insulin respectively around 1982.
• The Human Genome Project was launched in 1986 and completed nearly two decades later.
• GenPharm International created the first transgenic dairy cow for human milk proteins for infant formula in the 1990s.
• First authorized gene therapy began on child with immune disorder in the 1990s as well.
• In 1997, Dolly the sheep, was cloned at Scotland's Roslin Institute by somatic cell nuclear transfer.

Recombinant DNA Technology

• Enzymes and laboratory techniques are leveraged to manipulate and isolate DNA segments of interest.
• Method used to combine or splice DNA from different species or create new functions.
• Resulting copies are known as recombinant DNA.
• Work involves propagating this recombinant DNA in bacteria or yeast.
• Cellular machinery copies the engineered DNA along with its own.
• It joins DNA molecules from different organisms and inserts it into a host organism to produce new genetic combinations.
• Recombinant DNA is a biotechnology approach with multidisciplinary applications that addresses important aspects of life.
• Applications range from health issues like recombinant antibodies, to food resources, and environmental effects .
• Recombinant DNA is made from combining DNA from different sources.

Recombinant DNA in the Lab

• From 1974 Stanford University and University of California's Stanley Cohen and Herbert Boyer built on DNA pioneers such as Paul Berg.
• Their work developed that form the basis of recombinant DNA and helped spur biotechnology .
• Since 1959, bacteria contain extra loops of DNA called plasmids.
• Bacteria naturally swap plasmids with one another.
• This allows codes for beneficial genes like antibiotic resistance.
• By the early 1970s, investigators had isolated several plasmids.
• Special enzymes known as restriction endonucleases function as scissors to open loops of plasmids.
• Herbert Boyer had expertise with restriction endonucleases.
• Stanley Cohen studied plasmids.
• After meeting at a meeting in 1972, the pair combined their research efforts to cut open a plasmid loop, insert gene and close plasmid.
• This created a recombinant DNA molecule, a plasmid containing recombined DNA from two different sources.
• The plasmid was then inserted in bacteria.
• The team demonstrated recombinant DNA could be used by bacteria
• The team therefore created the first genetically modified organisms.
• A year later, the technique was used insert gene between two different organisms, an example being a frog inserted into bacteria.
• The technology for creating "molecular chimeras” was patented on December 2, 1980 (US Patent 4,237,224.)

Tools of Recombinant DNA Technology

• Plasmids are physically separate from chromosomal DNA.
• Plasmids replicate independently.
• Typically, plasmids have a small number of genes, including antibiotic resistance, which can be passed between cells.
• Scientists use recombinant DNA methods to slice genes that they want to study into a plasmid.
• When the plasmid copies itself, it copies the inserted gene.
• Restriction enzymes are molecular scissors that cut DNA sequences at a specific site and plays a role in gene manipulation.
• Restriction enzymes recognize specific DNA sequences and cleave either sticky or blunt-ended fragments.
• DNA Ligase attaches 2 pieces of DNA together.

Process of Recombinant DNA Technology

• Recombinant DNA technology proceeds through multiple steps, maintained to generate the desired product.
• Step-1, Isolation of Genetic Material is isolating the desired DNA in its pure form (free from other molecules).
• Step-2, Cutting the gene at the recognition site.
• Restriction enzymes determine the location at which the desired gene is inserted into the vector genome in 'restriction enzyme digestions’.
• Step-3, Amplifying the gene copies through Polymerase chain reaction (PCR).
• A single copy of DNA is amplified into thousands to millions of copies using restriction enzymes.
• Step-4, Ligation of DNA Molecules joins the DNA fragment and a vector with the use of enzyme DNA ligase.
• Step-5, Insertion of Recombinant DNA Into Host.
• Recombinant DNA (rDNA) is introduced after ligation.
• The process of inserting rDNA is called transformation.
• Recombinant DNA is inserted into the host cell and multiplied.
• Recombinant DNA gets expressed in the form of the manufactured protein under optimal conditions.
• Transformation, Vectorless Gene Transfer, and Transduction are ways of inserting Recombinant DNA.

Vectorless Gene Transfer

• Electroporation, also called electropermeabilization, is a non-viral delivery system that allows genetic material.
• This material can be DNA, RNA, proteins, drugs or other molecules to enter cells.
• Temporary pores in the cell's membrane are created with use of an accurately pulsed electrical current.
• The process can be used on many types of cells including mammalian, insect, yeast, plant, and bacterial cells.
• In Protoplast fusion the protoplasm of living plant cell fuse.
• Somatic cells of the plant cells fuse to create hybrid protoplast.
• The chemicals Polyethylene glycol (PEG and a high concentration of calcium a pH of 8 can facilitate the process.
• The process can be obtained physically by electrofusion.
• An example of protoplast fusion is the pomato, this is a transgenic crop of a tomato and potato plant.
• Microinjection is where the transformation method inserts DNA into giant cells.
• DNA is introduced into animal cells or plant protoplasts from micropipette
• The process is more appropriate for producing transgenic mice by incorporating DNA into the cytoplasm.

Transduction

• In Transduction, genetically engineered bacteriophages - viruses that parasitize bacteria are introduced into the cells.
• Process is intended to create a desired recombinant DNA.

Applications of Recombinant DNA Technology

• Cyanobacteria have been modified to produce plastic and fuel byproducts of photosynthesis.
• E. Coli bacteria have been modified to produce diesel fuel.

Health and Medicine

• Gene therapy is the treatment of genetic diseases and it works by removing and replacing defective genes.
• This replacement is done with normal healthy functional genes using gene therapy.
• Examples include Sickle cell anemia, Immune-Deficiency (SCID), a defect for adenosine deaminase (ADA) in 25 per cent of the cases.
• Production of medically useful biologicals, such as insulin and producing Vaccines can be done through Recombinant DNA Technology.
• Pharmacogenomics studies how genes affect a person's response to drugs.
• Pharmacology is the study of drugs.
• Genomics is the study of genes.
• Pharmacogenomics combines pharmacology and genomics to devlop effective, safe medications.
• These medications can be prescribed based on a person's genetic makeup.

Environment

• Alternative cleaner fuels, air air quality via pollution reduction is developed through genetic engineering.
• Micro-organisms decompose wastes or remediate contaminated sites.
• Disease resistant cultivars, crops production via environmentally intrusive process reduces agrochemical use.

Agriculture

• Genetic modification can make crops:
  • Insect Resistant
  • Herbicide resistant
  • Drought/freeze resistant
  • Disease resistant
  • Higher yield
  • Faster growth
  • Improved nutrition
  • Longer shelf life

Glow-in-the-dark cats

• Scientists used a virus to insert DNA from jellyfish.
• The gene made the resulting cat produce a fluorescent protein in its fur.

GM Salmon

• Genes from two other fish were used to cause the GM salmon to continually produce growth hormones
• GM salmon length: 24 inches.
• GM salmon weight: 6.6 lbs.
• Farm salmon length: 13 inches.
• Farm salmon Weight: 2.8 lbs.
• Both fish are 18 months old at time of measure.

Plants/Animals with Desirable or Enhanced Traits:

• Seedless Grapes.

Explore the history of genetic engineering, from its roots in artificial selection to modern recombinant DNA technology. Test your knowledge on key figures, milestones like Dolly the sheep, and applications in medicine and biotechnology.