FINALS LEC 2: GENETIC TECHNOLOGIES Flashcards
focuses specifically on the STUDY, MANIPULATION, & ANALYSIS OF GENES & GENETIC MATERIAL, involving the techniques & tools used to understand, modify and utilize genetic information
GENETIC TECHNOLOGY
- technology that UTILIZES BIOLOGICAL SYSTEMS, living organisms/parts of this to develop/create different products
- incorporates genetic technology as oe of its componnents
- AIM: deals with the manipulation of the genes of organisms to ALTER their behavior, characteristics, or value
- use or alteration of cells or biological molecules for specific applications, including products and processes.
- is an ancient art as well as a modern science, and is familiar as well as futuristic.
BIOTECHNOLOGY
TECHNIQUES IN BIOTECHNOLOGY
(popular terms that refer broadly to any biotechnology that MANIPULATES DNA)
- GENETIC ENGINEERING
- GENETIC MODIFICATION
- Organisms that HARBOR DNA FROM OTHER SPECIES
- organisms that has been genetically modified by the introduction of 1 OR MORE GENES FROM ANOTHER SPECIES and their DNA is called __________________
EX: ENVIROPIG
- Genetically modified to secrete bacterial phytase in its saliva, which enables the animal to excrete low - phosphorus manure
TRANSGENIC ORGANISM
- DNA is called RECOMBINANT
WHY IS CREATING A TRANSGENIC POSSIBLE?
- ALL LIFE USES THE SAME GENETIC CODE
- DNA MOVES & MIXES BETWEEN SPECIES IN NATURE
- HUMAN-DIRECTED GENETIC MODIFICATION USUALLY GIVES ORGANISMS TRAITS THEY WOULD NOT HAVE NATURALLY
WHY IS CREATING A TRANSGENIC POSSIBLE?
- same codons encode the same amino acid
- ex: green mice
- they contain gene that encodes for a jellyfish’s green fluorescent protein (GFP, used by researchers to mark genes of interest
ALL LIFE USES THE SAME GENETIC CODE
WHY IS CREATING A TRANSGENIC POSSIBLE?
- fish that can tolerate very cold water
- tomatoes that grow in saltwater
- bacteria that synthesize human insulin
HUMAN-DIRECTED GENETIC MODIFICATION USUALLY GIVES ORGANISMS TRAITS THEY WOULD NOT HAVE NATURALLY
- official right to be the ONLY PERSON OR COMPANY ALLOWED TO MAKE OR SELL A NEW PRODUCT FOR A CERTAIN PERIOD (20 years from the date of filing)
- invention of transgenic organisms may be CONSIDERED AN INTELLECTUAL PROPERTY and therefore PATENTABLE
PATENT
QUALIFICATIONS FOR PATENT PROTECTION
- NOVELTY
- USEFUL
- NOT OBVIOUS TO AN EXPERT IN THE FIELD
QUALIFICATIONS FOR PATENT PROTECTION
- invention must be NEW and NOT DISCLOSED TO THE PUBLIC before the filing of the patent application
NOVELTY
QUALIFICATIONS FOR PATENT PROTECTION
- invention must have a SPECIFIC UTILITY or PRACTICAL APPLICATION
USEFUL
QUALIFICATIONS FOR PATENT PROTECTION
- invention should not be an OBVIOUS/STRAIGHTFORWARD/COMBINATION/MODIFICATION of EXISTING KNOWLEDGE/technologies
NOT OBVIOUS TO AN EXPERT IN THE FIELD
DNA SEQUENCE MIGHT BE A PATENTABLE if it is a part of a medical device used to diagnose an inherited/infectious disease or a research tool
T or F?
TRUEEE
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- U.S. patent act enacted. A patented invention must be new, useful, and not obvious.
1790
1873
1930
1980
1790
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- Louis Pasteur is awarded first patent on a life form, for yeast used in industrial processes..
1790
1873
1930
1980
1873
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- New plant variants can be patented.
1790
1873
1930
1980
1930
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- First patent awarded on a genetically modified organism, a bacterium given four DNA rings that enable it to metabolize components of crude oil.
1790
1873
1930
1980
1980
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- First patent awarded for a transgenic organism, a mouse that manufactures human protein in its milk. Harvard University granted a patent for “OncoMouse,” transgenic for human cancer..
1988
1992
1996-1999
2000
1988
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- Biotechnology company awarded patent for all forms of transgenic cotton. Groups concerned that this will limit the rights of subsistence farmers contest the patent several times.
1988
1992
1996-1999
2000
1992
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- Companies patent partial gene sequences and certain disease-causing genes for developing specific medical tests.
1988
1992
1996-1999
2000
1996-1999
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- With gene and genome discoveries pouring into the Patent and Trademark Office, requirements for showing utility of a DNA sequence are tightened.
1988
1992
1996-1999
2000
2000
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- Attempts to enforce patents on non-protein-encoding parts of the human genome anger researchers who support open access to the information.
2003
2007
2009
2003
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- Patent requirements must embrace a new, more complex definition of a gene.
2003
2007
2009
2007
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- Patents on breast cancer genes challenged.
2003
2007
2009
2009
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- Direct-to-consumer genetic testing companies struggle to license DNA patents for multigene and SNP association tests.
- Patents on breast cancer genes invalidated.
2010
2011
2013
2010
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- U.S. government considers changes to gene patent laws.
2010
2011
2013
2011
TECHNOLOGY TIMELINE:
PATENTING LIFE AND GENES
- U.S. Supreme Court declares genes unpatentable.
2010
2011
2013
2013
- 1st gene modification biotechnology and was initially done with bacteria to make them produce peptides and proteins useful as drugs.
- adds genes from one type of organism to the genome of another.
- also known as “GENE CLONING”
- 1ST drug manufactured using this technology was INSULIN, which was produced in bacterial cells (Escherichia coli)
RECOMBINANT DNA
Drugs Produced Using Recombinant DNA Technology
- Dilates BLOOD VESSELS, promotes urination
Atrial natriuretic peptide
Colony-stimulating factors
Deoxyribonuclease (DNase)
Epidermal growth factor
Atrial natriuretic peptide
Drugs Produced Using Recombinant DNA Technology
- Help restore bone marrow after marrow transplant; restore blood cells following cancer chemotherapy
Atrial natriuretic peptide
Colony-stimulating factors
Deoxyribonuclease (DNase)
Epidermal growth factor
Colony-stimulating factors
Drugs Produced Using Recombinant DNA Technology
- Thins secretions in lungs of people with cystic fibrosis
Atrial natriuretic peptide
Colony-stimulating factors
Deoxyribonuclease (DNase)
Epidermal growth factor
Deoxyribonuclease (DNase)
Drugs Produced Using Recombinant DNA Technology
- Accelerates healing of wounds and burns; treats gastric ulcers
Atrial natriuretic peptide
Colony-stimulating factors
Deoxyribonuclease (DNase)
Epidermal growth factor
Epidermal growth factor
Drugs Produced Using Recombinant DNA Technology
- Stimulates production of red blood cells in cancer patients
Erythropoietin (EPO)
Factor VIII
Glucocerebrosidase
Human growth hormone
Erythropoietin (EPO)
Drugs Produced Using Recombinant DNA Technology
- Promotes blood clotting in treatment of hemophilia A
Erythropoietin (EPO)
Factor VIII
Glucocerebrosidase
Human growth hormone
Factor VIII
Drugs Produced Using Recombinant DNA Technology
- Corrects enzyme deficiency in Gaucher disease
Erythropoietin (EPO)
Factor VIII
Glucocerebrosidase
Human growth hormone
Glucocerebrosidase
Drugs Produced Using Recombinant DNA Technology
- Promotes growth of muscle and bone in people with very short stature due to hormone deficiency
Erythropoietin (EPO)
Factor VIII
Glucocerebrosidase
Human growth hormone
Human growth hormone
Drugs Produced Using Recombinant DNA Technology
- Allows cells to take up glucose in treatment of type 1 diabetes
Insulin
Interferons
Interleukin-2
Lung surfactant protein
Insulin
Drugs Produced Using Recombinant DNA Technology
- Treat genital warts, hairy cell leukemia, hepatitis B and C, Kaposi sarcoma, multiple sclerosis
Insulin
Interferons
Interleukin-2
Lung surfactant protein
Interferons
Drugs Produced Using Recombinant DNA Technology
- Treats kidney cancer recurrence
Insulin
Interferons
Interleukin-2
Lung surfactant protein
Interleukin-2
Drugs Produced Using Recombinant DNA Technology
- Helps lung alveoli to inflate in infants with respiratory distress syndrome
Insulin
Interferons
Interleukin-2
Lung surfactant protein
Lung surfactant protein
Drugs Produced Using Recombinant DNA Technology
- Lowers blood pressure
Renin inhibitor
Somatostatin
Superoxide dismutase
Thrombin
Tissue plasminogen activator
Renin inhibitor
Drugs Produced Using Recombinant DNA Technology
- Decreases growth in muscle and bone in pituitary gigantism
Renin inhibitor
Somatostatin
Superoxide dismutase
Thrombin
Tissue plasminogen activator
Somatostatin
Drugs Produced Using Recombinant DNA Technology
- Prevents further damage to heart muscle after heart attack
Renin inhibitor
Somatostatin
Superoxide dismutase
Thrombin
Tissue plasminogen activator
Superoxide dismutase
Drugs Produced Using Recombinant DNA Technology
- Stops post surgical bleeding
Renin inhibitor
Somatostatin
Superoxide dismutase
Thrombin
Tissue plasminogen activator
Thrombin
Drugs Produced Using Recombinant DNA Technology
- Dissolves blood clots in treatment of heart attack, stroke, and pulmonary embolism
Renin inhibitor
Somatostatin
Superoxide dismutase
Thrombin
Tissue plasminogen activator
Tissue plasminogen activator
REQUIREMENT FOR THE MANUFACTURING OF RECOMBINANT DNA:
- RESTRICTION ENZYMES
- CLONING VECTORS
- RECIPIENT CELLS
REQUIREMENT FOR THE MANUFACTURING OF RECOMBINANT DNA:
- enzyme that cut DNA at specific sequences
- also called RESTRICTION ENDONUCLEASES because they cut DNA within the molecule [“endo”] rather than from the ends [“exo”]
RESTRICTION ENZYMES
CLONING VECTORS
RECIPIENT CELLS
RESTRICTION ENZYMES
REQUIREMENT FOR THE MANUFACTURING OF RECOMBINANT DNA:
- plasmids, viruses, artificial chromosomes
- pieces of DNA used to deliver specific DNA sequences to cells
RESTRICTION ENZYMES
CLONING VECTORS
RECIPIENT CELLS
CLONING VECTORS
REQUIREMENT FOR THE MANUFACTURING OF RECOMBINANT DNA:
- bacteria/cultured single cells
RESTRICTION ENZYMES
CLONING VECTORS
RECIPIENT CELLS
RECIPIENT CELLS
To identify if the cell has successfully taken up the plasmid with the DNA of interest inserted __________ will be used.
LacZ Gene
- encodes for beta-galactosidase
- by inserting foreign DNA into the gene, the ability of cell to produce beta-galactosidase is disrupted
- Procedure: plate the transformed bacteria on a selective growth medium that contains the necessary antibiotic for plasmid selection
- BLUE-WHITE SCREENING: to identify bacteria that have the plasmid with the DNA of interests inserted into the ___ using a chromogenic substrate
LacZ Gene
SAFER VACCINES ARE CREATED
- Flu vaccine consisting genes encoding the hemagglutinin proteins from __________ & _________________
2 INFLUENZA A STRAINS, 1 INFLUENZA B STRAIN
SAFER VACCINES ARE CREATED
- New vaccine that protects against malaria, based on altering _________________ (bacterium) that normally inhabits the mosquito gut by inserting genes from Escherichia coli
Pantoea agglomerans
- organisms having the foreign genes inserted into their genetic material
- Eukaryotic cells growing in culture are generally better at producing human proteins
- more efficient way to express some recombinant genes is in a body fluid of a transgenic animal
ex: transgenic cows, sheep, and goats - have expressed human genes in their milk
TRANSGENIC ORGANISMS
PROCESS OF CREATING A TRANSGENIC ORGANISMS
- GENE ISOLATION
- GENE CLONING
- GENE INSERTION
- SELECTION AND BREEDING
PROCESS OF CREATING A TRANSGENIC ORGANISMS:
- the gene (transgene) responsible for the desired trait is identified and isolated from the DNA of the donor organism
- GENE ISOLATION
- GENE CLONING
- GENE INSERTION
- SELECTION AND BREEDING
GENE ISOLATION
PROCESS OF CREATING A TRANSGENIC ORGANISMS:
- the isolated gene is then REPLICATED many times using the PCR or other cloning techniques
- GENE ISOLATION
- GENE CLONING
- GENE INSERTION
- SELECTION AND BREEDING
GENE CLONING
PROCESS OF CREATING A TRANSGENIC ORGANISMS:
- the replicated gene is INSERTED into the genome of the target organism using VARIOUS METHODS
- GENE ISOLATION
- GENE CLONING
- GENE INSERTION
- SELECTION AND BREEDING
GENE INSERTION
PROCESS OF CREATING A TRANSGENIC ORGANISMS:
- the transgenic organism is identified through markers or other selection methods and is then bred to maintain and pass on the introduced gene to subsequent generation
- GENE ISOLATION
- GENE CLONING
- GENE INSERTION
- SELECTION AND BREEDING
SELECTION AND BREEDING
GENE INSERTION TECHNIQUES
- CHEMICALS & BRIEF JOLTS OF ELECTRICITY
- GUNLIKE DEVICE (GENE GUN)
- LIPOSOMES
- VIRUSES
- BACTERIA
- USEFUL MODELS OF HUMAN DISEASES
- inserting mutant gene that causes disease into mice results in a mouse model of the disease
- drug candidates can be tested on them and abandoned before being tested in humans if they caused significant side effects
TRANSGENIC ANIMAL MODELS
LIMITATIONS ON TRANSGENIC ANIMAL MODELS
- located where a transgene will be inserted in a genome
- number of copies to insert
- level of gene expression necessary for a phenotype to emerge may also differ in the model and humans
- animal models might not mimic the human condition exactly
GENETICALLY MODIFIED FOODS
- controlled breeding of plants and animals to select individuals with certain combination of inherited traits that are useful
ex: seedless fruits and lean meat, form of genetic modifications based on phenotype (taste or appearance) affecting many genes
TRADITIONAL AGRICULTURE
GENETICALLY MODIFIED FOODS
- manipulate 1 or few specific genes at a time
- organisms altered to have genes from other species or to over- or under express their own genes are termed ___________________
EX: GOLDEN RICE
- genetically modified variety of rice (Oryza sativa) that has been engineered to produce beta-carotene, a precursor of Vitamin A
- developed by the not-for-profit “International Rice Research Institute in the Philippines”
- Phytoene synthase (psy) gene from daffodil (Narcissus pseudonarcissus) & beta-carotene common soil bacterium Erwinia uredova
DNA- BASED TECHNIQUES
- GENETICALLY MODIFIED ORGANISMS (GMOs)
SOME GENETICALLY MODIFIED FOODS
- less sugar
GRAPES
CASSAVA, PAPAYA, PLUM
CATTLE
SUGAR BEETS, CORN, SOYBEAN
GRAPES
SOME GENETICALLY MODIFIED FOODS
- resist viral infection
GRAPES
CASSAVA, PAPAYA, PLUM
CATTLE
SUGAR BEETS, CORN, SOYBEAN
CASSAVA, PAPAYA, PLUM
SOME GENETICALLY MODIFIED FOODS
- resist mad cow disease
GRAPES
CASSAVA, PAPAYA, PLUM
CATTLE
SUGAR BEETS, CORN, SOYBEAN
CATTLE
SOME GENETICALLY MODIFIED FOODS
- tolerate an herbicide
GRAPES
CASSAVA, PAPAYA, PLUM
CATTLE
SUGAR BEETS, CORN, SOYBEAN
SUGAR BEETS, CORN, SOYBEAN
SOME GENETICALLY MODIFIED FOODS
- more iron and Vitamin A
BANANA, RICE
CANOLA
SALMON
BANANA, RICE
SOME GENETICALLY MODIFIED FOODS
- altered fatty composition
BANANA, RICE
CANOLA
SALMON
CANOLA
SOME GENETICALLY MODIFIED FOODS
- faster growth
BANANA, RICE
CANOLA
SALMON
SALMON
POTENTIAL RISKS OF GMO FOODS
- There are inadequate studies on their effects to humans and the environment
- The technology promotes mutation in organisms which the long - term effect is still unknown
- Human composition might have the following effects: allergic reactions, gene mutation, antibiotic resistance, nutritional value
- Genetic uniformity
- use of plants or microorganisms to DETOXIFY ENVIRONMENTAL POLLUTANTS
ex: Genes from trees that accumulate so much nickel from soil slashing
2. transgenic poplar trees can thrive in mercury-tainted soil
3. deploying transgenic bacteria that normally break down trinitrotoluene
4. Oil-eating microbes
BIOREMEDIATION
- requires detecting mRNAs in particular cells under particular conditions
- helps to understand how genes are regulated and how different factors influence the production of gene products in cells and organisms
MONITORING GENE EXPRESSION
- piece of glass/plastic that is about 1.5cm²
- used to measure that abundance of specific mRNA molecules in a sample and can provide a snapshot of gene expression patterns
ex: it can reveal gene expression in response to spinal cord injury
GENE EXPRESSION DNA MICROARRAYS (GENE CHIPS)
what color?
- gene expressed in CSF only when the spinal cord is INJURED
RED
GREEN
YELLOW
BLACK OR LACK OF FLUORESCENCE
RED
what color?
- gene expressed in CSF only when the spinal cord is INTACT
RED
GREEN
YELLOW
BLACK OR LACK OF FLUORESCENCE
GREEN
what color?
- gene expressed in CSF only whether or not the spinal cord has been injured
RED
GREEN
YELLOW
BLACK OR LACK OF FLUORESCENCE
YELLOW
what color?
- DNA sequences not expressed in CSF because they DO NOT EXPRESSED IN CSF because they do not show up either sample
RED
GREEN
YELLOW
BLACK OR LACK OF FLUORESCENCE
BLACK OR LACK OF FLUORESCENCE
MANIPULATION OF THE EXPRESSION OF SPECIFIC GENES:
- techniques that block synthesis of, or DEGRADE mRNA
- leads to DECREASE or CESSATION of the production of a specific gene product (RNA or protein)
GENE SILENCING
GENE SILENCING
- BLOCKS EXPRESSION OF A GENE by introducing RNA that is complementary to the gene’s mRNA transcript
- early application: tomato intended to stay fresh longer (the technology squelched activity of ripening enzyme)
- variations: using of antisense RNA, using of morpholis
ANTISENSE TECHNOLOGY
RIBOZYMES
RNA INTERFERENCE (RNAi)
ANTISENSE TECHNOLOGY
GENE SILENCING
- RNA molecules that are part of ribosomes that have catalytic activity like enzymes
- FIT THE SHAPE of certain RNA molecules
- ACTS AS GENE-SILENCING AGENTS by specifically CLEAVING & INACTIVATING target mRNA molecules
ANTISENSE TECHNOLOGY
RIBOZYMES
RNA INTERFERENCE (RNAi)
RIBOZYMES
GENE SILENCING
- technique based on that fact that RNA molecules can fold into short, double-stranded regions where the base is complementary
- ANDREW FIRE & CRAIG MELLO were awarded Nobel Prize in Physiology of Medicine for explaining how RNAi works
- introduction of small interfering RNA (siRNA) molecule that BINDS TO & PREVENTS TRANSLATION of a specific mRNA
ANTISENSE TECHNOLOGY
RIBOZYMES
RNA INTERFERENCE (RNAi)
RNA INTERFERENCE (RNAi)
- techniques that create double - stranded breaks in the DNA double helix, enabling insertion of a desired DNA sequence or removal of a sequence
2 DISTINCT APPROACHES
1. SOMATIC GENOME EDITING
2. GERMLINE GENOME EDITING
GENOME EDITING
GENOME EDITING DISTINCT APPROACHES
- change must AFFECT ALL CELL/ENOUGH CELLS to alter the phenotype, such as treating symptoms of a genetic disease
SOMATIC GENOME EDITING
GERMLINE GENOME EDITING
SOMATIC GENOME EDITING
GENOME EDITING DISTINCT APPROACHES
- introduces the genetic change to every cell in an organism because it is carried out at the beginning of the development
SOMATIC GENOME EDITING
GERMLINE GENOME EDITING
GERMLINE GENOME EDITING
GENOME EDITING TECHNIQUES:
- uses PROTEIN MOTIFS (parts of proteins that have characteristic shapes) called ZINC FINGERS consisting of a b-pleated sheet linked to an a-helix by a zinc atom
- different zinc fingers bind different 3 - base DNA sequences
ZINC FINGER NUCLEASES (ZFNs)
TRANSCRIPTION-ACTIVATOR-LIKE- EFFECTOR NUCLEASE (TALEN) TECHNOLOGY
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS-CRISPR-ASSOCIATED PROTEIN 9 (CRISPR-Cas9)
ZINC FINGER NUCLEASES (ZFNs)
GENOME EDITING TECHNIQUES:
- restriction enzyme from Xanthomonas (bacterium) that infects plants cuts DNA on both strands
- transcription activator-like (TAL) effector repeats guide targeting to the DNA
ZINC FINGER NUCLEASES (ZFNs)
TRANSCRIPTION-ACTIVATOR-LIKE- EFFECTOR NUCLEASE (TALEN) TECHNOLOGY
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS-CRISPR-ASSOCIATED PROTEIN 9 (CRISPR-Cas9)
TRANSCRIPTION-ACTIVATOR-LIKE- EFFECTOR NUCLEASE (TALEN) TECHNOLOGY
- application of genome editing to kill, alter, or render infertile a pathogen
- based on HOMING (natural form of DNA repair) , which removes 1 of a pair of alleles of a selected gene and replaces the it with another copy of the remaining allele
- COUNTERS MENDEL’S LAW OF SEGREGATION
- could be used to MODIFY DISEASE-CARRYING ORGANISMS, to reduce or prevent the transmission of certain diseases
GENE DRIVE
GENOME EDITING TECHNIQUES:
- uses a bacterial enzyme and RNA to make double
ZINC FINGER NUCLEASES (ZFNs)
TRANSCRIPTION-ACTIVATOR-LIKE- EFFECTOR NUCLEASE (TALEN) TECHNOLOGY
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS-CRISPR-ASSOCIATED PROTEIN 9 (CRISPR-Cas9)
CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS-CRISPR-ASSOCIATED PROTEIN 9 (CRISPR-Cas9)
GERMLINE EDITING OF THE HUMAN GENOME IS CONTROVERSIAL
- raises profound ethical question about the ethical boundaries of human genetic modification and the potential implications for future generations (designer babies or genetic enhancements)
ETHICAL CONCERNS
SAFETY & INTENDED CONSEQUENCES
IRREVERSIBILITY
ETHICAL CONCERNS
GERMLINE EDITING OF THE HUMAN GENOME IS CONTROVERSIAL
- uncertain and there is a risk of unintended genetic changes/off-target effects that could have unforeseen and potentially harmful consequences for the edited individuals and future generations
ETHICAL CONCERNS
SAFETY & INTENDED CONSEQUENCES
IRREVERSIBILITY
SAFETY & INTENDED CONSEQUENCES
GERMLINE EDITING OF THE HUMAN GENOME IS CONTROVERSIAL
- once a heritable genetic change is introduced into the germline, it will be passed on to all subsequent generations
- potential for making mistakes that cannot be corrected in the future
ETHICAL CONCERNS
SAFETY & INTENDED CONSEQUENCES
IRREVERSIBILITY
IRREVERSIBILITY
