History of Genetic Engineering

• The assignment requires the listing of at least 5 products of genetic engineering across fields such as medicine, agriculture, industry, and environment.
• One product must be chosen to explain the "why" and "how" of its engineering, including its pros and cons.

Glow-in-the-dark cats

• Scientists utilize a virus to insert DNA from jellyfish.
• The inserted gene allows the cat to produce a fluorescent protein in its fur.

Genetically Modified Salmon

• Genes from two other fish cause the salmon to continually produce growth hormones.
• An 18-month-old GM salmon is 24 inches long and weighs 6.6 lbs.
• An 18-month-old Farm salmon is 13 inches long and weighs 2.8 lbs.

Seedless Grapes

• Grapes have been genetically modified to be seedless.

Introduction to Genetic Engineering

• Genetic engineering, also known as recombinant DNA technology and biotechnology, was coined in 1919 by Karl Ereky, a Hungarian engineer.
• Humans have used artificial selection to manipulate organisms for thousands of years, as early as 8000 and 1000 B.C., domesticating animals such as horses, camels, and oxen
• Around 6000 B.C., yeast was used to make beer.
• Plants such as maize, wheat, and rice were bred around 5000 B.C.
• Socrates speculated on why children may not resemble their parents around 420 B.C.
• Hippocrates proposed around 400 B.C. that males contribute character through semen, establishing the idea of heredity.
• Hindu philosophers in 100-300 A.D. considered reproduction and inheritance, noting familial diseases and the inheritance of parental characteristics.
• The 19th century saw an increase in biochemical studies that led to new direction in biology because of research on nucleic and amino acids and speed increase in fermentation
• Mendel presented his work on peas in 1864-1865, which was neglected for some time, and the term gene/genetics wasn't yet coined.
• The first biotechnology products, including the use of agar, came about in 1882, described by the Koch lab.
• An autoclave was developed in 1884 by a French company, Chamberland's Autoclaves.
• X-rays were discovered in 1895 by W. Roentgen.
• X-ray crystallography was applied in 1913 by physicist Sir William Henry Bragg and his son William Lawrence Bragg, among others.
• In 1945, William Astbury created the term molecular biology.
• In the early 1950s, Rosalind Franklin and Maurice Wilkins obtained the X-ray diffraction data for DNA that would aid Watson and Crick.
• Hershey showed that DNA is the hereditary material, with help from Martha Chase
• During the late 1960s, Werner Arber identified restriction enzymes in bacteria that cleave DNA.
• In 1970, Temin and Baltimore each discovered reverse transcriptase, leading to recombinant DNA technology.
• In 1971, Paul Berg (Stanford) succeeded in splicing and recombining genetic material.
• The first recombinant DNA was produced in 1972 at Boyer Laboratory.
• The first biotechnology company, Genentech, was founded in 1976.

Genetic Engeneering in The 1980s

• Polymerase chain reaction (PCR) method was invented in 1983 by Karen Mullis.
• 1982 saw the appearance of Genentech's recombinant interferon gamma and Eli Lilly's recombinant human insulin on the market.
• 1986 saw the launch of The Human Genome Initiative, later the Human Genome Project, which was completed around two decades later.
• In the 1990s, GenPharm International created the first transgenic dairy cow to produce human milk proteins for infant formula; authorized gene therapy also began for a 4-year-old girl with adenosine deaminase deficiency (ADA).
• In 1997, flash news reported the cloning of Dolly the sheep from Scotland's Roslin Institute, via somatic cell nuclear transfer.

Recombinant DNA Technology

• Involves using enzymes and lab techniques to manipulate and isolate DNA segments.
• It can combine (splice) DNA from different species or create new genes with novel functions.
• Resulting copies are referred to as recombinant DNA. Typically involves propagating recombinant DNA in a bacterial or yeast cell.
• Encyclopedia Britannica defines as "the joining together of DNA molecules from different organisms and inserting it into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture and industry.
• Is a biotech approach with multidisciplinary applications that may address important aspects of life, from health issues(recombinant antibodies) to food resources, and resistance to environmental effects (Eberle C. 2022)
• Recombinant DNA is made when combining components from different DNA sources.
• Between 1972 and 1974, Stanley Cohen, Herbert Boyer, and their colleagues developed recombinant DNA technology techniques, spurring the biotechnology industry.
• In 1959, scientists found that bacteria had extra loops of DNA called "plasmids" in addition to their chromosomes.
• In nature, bacteria swap plasmids, transferring beneficial genes such as antibiotic resistance.
• By the early 1970s, investigators isolated plasmids and discovered restriction endonucleases that act like scissors.
• Herbert Boyer had expertise with restriction endonucleases and Stanley Cohen studied plasmids, and after meeting at a conference in 1972, the two decided to combine their research efforts. 
• The Cohen-Boyer team cut a plasmid loop from bacteria, inserted a gene from a different bacterial species, and closed the plasmid.
• This yielded the plasmid containing recombined DNA from two sources.
• They created the first genetically modified organisms by inserting the plasmid into bacteria.
• It was proven possible to transfer genes between two very different organisms when a frog gene was inserted into bacteria. -The technology for creating these “molecular chimeras” was patented on December 2, 1980 (US Patent 4,237,224.)
• Isolate Bacterial DNA
• Use enzymes to cut open DNA
• Use enzymes to connect insulin gene and bacterial DNA
• Insert Recombinant DNA into Bacteria
• DNA of Interest and Plasmid digested with restriction enzyme

Tools of Recombinant DNA Technology

• Plasmids are physically separate from chromosomal DNA and replicate independently.
• They have a few genes, including antibiotic resistance, which can be passed from cell to cell.
• Methods are used to splice genes for study into plasmids.
• When a plasmid copies itself, it also copies the inserted gene.
• The bacterial cell contains various microscopic organisms (e.g. Escherichia coli, Plasmid, Pili) for replication
• Restriction enzymes serve as molecular scissors for cutting DNA sequences at specific spots.

Different types of restriction enzymes

• Useful restriction enzymes for rDNA technology recognize and cleave specific DNA sequences to produce sticky or blunt-ended fragments. (e.g Smal and BamHI)
• Bacillus amyloliquefaciens is a recognition site for producing a sticky enzyme (BamHI). The sticky end is GATC
• Staphylococcus aureus is a recognition site for producing a sticky enzyme (Sau3A). The sticky end is GATC
• Escherichia coli is a recognition site for producing a sticky enzyme (EcoRI). The sticky end is GAATC
• Haemophilus influenzae is a recognition site for producing a sticky enzyme (HindIII). The sticky end is AAGCTI
• Serratia marcescens is a recognition site for producing a Blunt end enzyme (Smal). The sticky end is CCCGGG
• Nocardia otitidis-caviarum is a recognition site for producing a sticky enzyme (Not1). The sticky end is GCGGCCGC
• DNA Ligase attaches two pieces of DNA together at the sticky ends

Process of Recombinant DNA Technology

• Multiple steps maintained in sequence that generates the desired product.
• Isolate the desired DNA in pure form, free from macromolecules to isolate of genetic material.
• Restriction enzymes play a role in determining where the gene is inserted into the vector genome, this is refereed to as ‘restriction enzyme digestions’ to cut the gene at the recognition sites.
• Amplifying the gene copies through Polymerase Chain Reaction (PCR) to amplify a single copy of DNA into thousands to millions.
• Joining of two pieces of DNA ( a cut fragment and the vector) using DNA ligase is known as Ligation of DNA molecules.
• Insertion of Recombinant DNA Into Host, where the recombinant DNA is introduced into a recipient host cell and is referred to as transformation.
• The recombinant DNA gets multiplied and expressed like a manufactured protein.
• The production of an protein (insulin) uses human cells, bacteria cells(transgenic) to combine into plasmids to grow cultures.
• DNA can also be transferred through vectorless gene transfer methods such as transformation and transduction.

Vectorless Gene Transfer

• Uses electroporation, also called electropermeabilization.
• Aids genetic material (DNA and RNA), proteins, drugs, or other molecules to enter cells by using pulsed electrical current to create pores in the cell membrane for temporary access.
• It can be used on a wide variety of cells including mammalian, insect, yeast, plant and bacterial cells.
• Another method uses protoplast fusion, in which the protoplasm derived from somatic cells of plant cells produce a hybrid protoplast.
• This process can be obtained chemically with mixture (polyethylene glycol (PEG) and high calcium concentration @ pH 8), or by electrofusion.
• The pomato is a transgenic crop produced by protoplast fusion of a tomato plant and a potato plant.
• Microinjection includes a transformation method used to insert DNA into giant cells; introducing DNA into animal cells (eggs, oocytes, embryos) or plant protoplasts using a micropipette. A technique appropriate for producing transgenic mice.
• DNA is incorporated straight into cytoplasm or nucleus.
• Holding pipette holds the host cell and DNA is inserted using a microinjection needle

Transduction

• When genetically engineered bacteriophages-viruses that parasitize bacteria are introduced the cell to create desired recombinant DNA
• Phage injects DNA and break down host bacterial DNA
• Viral particles inject DNA into the cell via infection cycle
• A donor’s DNA is then inserted into a recipient (transduction)
• Cell creates new viral particles (viral RNA -> DNA -> mRNA -> proteins)
• New viral particles are transduced w/ donor DNA
• Cells lyse and release viral particles
• Donor DNA is incorporated into recipient gene

Applications of Recombinant DNA Technology

• Cyanobacteria used for the production of plastic (polyethylene) and fuel (butanol).
• E. coli bacteria can be modified to produce diesel fuel.

Health and Medicine

• Treatment of genetic diseases ( Gene Therapy), removal and replacement of defective genes w/ normal healthy functional genes.
• Treat Sickle cell anemia, or Severe Combined Immuno-Deficiency(SCID).
• Example, SCID is due to a defect in the gene which results in the enzyme adenosine deaminase (ADA) being present in only twenty five per cent of cases.
• Production of medically useful biologicals (e.g. Insulin and recombinant human growth hormone)
• Development of vaccines
• Pharmacogenomics: the study of genes effects on a person's response to drugs.

Environment

• The development and usage of alternative fuels that burn cleaner and improve air quality through reduced pollution
• Utilizing technology of bioremediation, micro-organisms can be used to decompose wastes and clean up contaminated sites.
• Utilize the creation of disease resistant cultivars to improve crop production which is less environmentally intrusive by reducing utilization of agrochemicals

Agriculture

• Reasons to Genetically Modify Crops,
  • Insect resistant
  • Herbicide resistant,
  • Drought/freeze resistant
  • disease resistant,
  • Higher yield,
  • Faster growth,
  • Improved nutrition &
  • Longer shelf life

Explore the milestones in genetic engineering history. This quiz covers key figures, experiments, and discoveries. Test your knowledge of molecular biology.