Lecture 20 - Genetically Modified Organisms

Genetically modified organism (GMO)

An organism whose genetic material has been altered using genetic engineering techniques; GMOs include microorganisms (bacteria and yeast), plants, and animals

GMO foods and regulation

GMO foods (also called bioengineered foods) have been available to consumers since the early 1990s; the FDA, USDA, and EPA work together to ensure GMOs are safe for people, animals, and the environment

Traditional breeding methods

Cross

How genetic engineering differs from traditional breeding

Modern technology allows scientists to take just one beneficial gene, such as insect resistance or drought tolerance, and transfer it into a plant, rather than mixing entire genomes

Reasons for genetic modification

Higher crop yields, longer storage life, better appearance, better nutrition, or a combination — the same goals as traditional breeding thousands of years ago

Four application areas of GMOs

1) Agricultural crops modified to improve yield, growth rates, and nutritional value; 2) Genetically modified animals used to grow human transplant tissues and organs (xenotransplantation); 3) Microorganisms as future clean fuel producers and biodegraders; 4) Pharmaceutical companies introducing vaccines, antibodies, and medicines into foods

Pink pineapple

A GMO that became commercially available in 2020

Polyphenol oxidase (PPO)

The enzyme responsible for browning; silencing it is how non

Why herbicide resistance is engineered

To save labor

Why insect resistance is engineered

To reduce pesticide use

Why browning resistance is engineered

To reduce food waste

Why virus resistance and fast growth are engineered

To achieve higher yield

Herbicide

resistant GMO crops

GMO cotton and corn

Cotton carries the Bacillus thuringiensis (Bt) toxin for insect resistance; corn carries Bt toxin for insect resistance and is also herbicide resistant

GMO potato and apple

Potato is insect resistant and resists bruising and browning; apple resists browning after being cut

GMO squash and papaya

Summer squash is resistant to some plant viruses; papaya is resistant to some viruses

GMO salmon

Carries a growth hormone gene from Chinook salmon, and is disease resistant and cold resistant

Agrobacterium tumefaciens method (steps 1–3)

1) The Ti plasmid is removed from the bacterium and the T

Agrobacterium tumefaciens method (steps 4–7)

4) The plasmid is reinserted into a bacterium; 5) The bacterium inserts the T

Phenylketonuria (PKU)

An inherited disorder that increases blood levels of phenylalanine, an amino acid obtained through the diet that is found in all proteins and in some artificial sweeteners

Biochemistry of PKU

Phenylalanine hydroxylase (PAH) normally converts phenylalanine to tyrosine; when PAH activity is reduced, phenylalanine accumulates and is converted into phenylpyruvate by a minor transaminase pathway with glutamate, so sufferers build up toxic phenylpyruvate (plus phenylacetate and phenylethylamine, detectable in urine) and become tyrosine deficient

SYNB1618

A GM probiotic developed by Synlogic from the safe strain Escherichia coli Nissle 1917, carrying genes for phenylalanine ammonia lyase (PAL), which converts phenylalanine to the non

Transgenic animal

An animal that carries a foreign gene deliberately inserted into its genome, giving it properties different from non

Transgenesis

The process by which introduction of foreign genes takes place

Why foreign genes are inserted into the germ line

So that the transgene can be transmitted to the progeny; delivery uses plasmid vectors, viral vectors, pronuclear injection, or liposomes

The first transgenic animal

The "Super Mouse," created in 1982 by Ralph Brinster (University of Pennsylvania) and Richard Palmiter (University of Washington) by inserting a human growth hormone gene (PEPCK

Model transgenic animals besides mouse

Pig, goat, cow, sheep, and fish

Five ways to insert a foreign gene into an animal

(i) Injecting DNA into the nucleus of a fertilized egg; (ii) injecting DNA into embryonic stem cells; (iii) using viruses to transfer the gene by recombinant DNA; (iv) introducing genetic material into sperm that fertilize eggs; (v) electrically induced membrane fusion

Research uses of transgenic animals

Understanding regulation of gene expression and immune system function, treating genetic disease, overcoming viral diseases, cancer research, toxicological studies, development of cell factories for therapeutics production, pharmaceutical research, disease modeling, and promotion of reproductive performance

Timeline of transgenic animal milestones

First transgenic pigs/sheep (~1985), AquaAdvantage GH salmon founder (late 1980s), Herman the lactoferrin bovine (~1990), first SCNT cloned mammal (late 1990s), phytase

Six technologies for making transgenic animals

Microinjection of DNA into early

Pronuclear injection (PNI)

Among the first techniques used, between 1980 and the 1990s, to generate transgenic animals by introducing DNA constructs into the pronucleus of fertilized eggs and transferring the injected eggs to foster mothers

Pronucleus

The stage of a fertilized egg where the sperm head has not yet merged with the egg nucleus, resulting in two distinct pronuclei that subsequently fuse to create a diploid zygote nucleus

Efficiency of PNI

In both sheep and goats, reported efficiencies of conventional PNI were about 1% of injected zygotes

Why PNI efficiency is low

Random integration and variable copy number of the integrated DNA constructs, which make transgene expression unpredictable

Technical obstacle to PNI in livestock

Difficulty visualizing the pronucleus, obstructed by a large amount of lipid granules in livestock eggs that make the cytoplasm nontransparent

Sperm

mediated gene transfer (SMGT)

Magnetic nanoparticles (MagNPs) in SMGT

They offer high carrying efficiency, non

MagNP

SMGT experiment

Advantages of the MagNP

SMGT method

Somatic cell nuclear transfer (SCNT)

Somatic cell cloning, in which the nucleus of a somatic (body) cell is transferred to the cytoplasm of an enucleated egg; the somatic nucleus is then reprogrammed by egg cytoplasmic factors to become a zygote nucleus

How ESCs are obtained from SCNT

The egg is allowed to develop to the blastocyst stage, at which point a culture of embryonic stem cells can be created from the inner cell mass; mouse, monkey, and human ESCs have been made this way

Status of SCNT as a method

Despite relatively low efficiency and the potential for developmental anomalies, SCNT has — alongside PNI — become a basic and dominant methodology for generating transgenic and gene

Dolly the sheep

Successfully cloned in 1996 by fusing the nucleus from a mammary

Cloning by embryo splitting

Also known as twinning by separation of blastomeres; a set of primary reproductive techniques applied to produce identical twins, multiplets, and chimeras

Embryo splitting procedure

Collect cleaved embryos from super

Limitation of embryo splitting

Technical difficulties and suboptimal pregnancy rates lead to production of only a relatively small number of individuals

Four stages shown in the embryo splitting figure

Blastomere separation (embryo split into individual cells), creation of an empty zona pellucida (blastomeres removed and discarded from mouse embryos), microinjection of a single blastomere into a surrogate zona pellucida using a transfer and holding pipette, and in vitro culturing of the split embryo to a blastocyst

Virus

based transgenesis

Intra

mammary injection strategy

Viral vectors plus RNAi

Used to mediate loss of gene expression in order to investigate potential biological functions of genes, suppress the expression of disease

Steps of viral

vector RNAi transgenesis

Examples of shRNA targets in livestock

Inhibiting replication of foot

Recombinases

Enzymes that catalyze site

Two recombinase strategies

Using a unidirectional tyrosine recombinase (Cre or FLP) to remove a transcriptional stop signal so a downstream gene can be expressed, or using a unidirectional serine recombinase (TP901) to flip a gene from an off to an on orientation

Transposons

Segments of DNA that can move to different positions in the genome of a single cell, potentially causing mutations, increasing or decreasing the amount of DNA in the genome, and — if the cell is a gamete precursor — affecting the genomes of descendants

RNA interference (RNAi)

A biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules; historically also called co

Four major classes of site

specific endonucleases

Common delivery approaches for endonucleases

Direct microinjection into embryos (mainly cytoplasmic injection in sheep and goats) and somatic cell nuclear transfer (mainly using fibroblast cells)

Repair outcomes after an endonuclease

induced DSB

Why mice are used to model human disease

Animal models are selected for similarity to humans in genetics, anatomy, and physiology; the mouse is the leading organism for disease research and the only mammal providing such a rich resource of genetic diversity coupled with the potential for extensive genome manipulation

Five ways to obtain desired gene mutations in mice

(i) Targeted mutations that eliminate function in the whole organism or a specific tissue; (ii) forward genetic screens using chemicals or transposons; (iii) introduction of exogenous transgenes as DNAs; (iv) bacterial artificial chromosomes (BACs); (v) reporter constructs

Natural variation

A change in one or more nucleotides between individuals

Reverse genetics

The ability to introduce a mutation by design

Forward genetics

Using agents including chemicals, radiation, and viruses to disrupt genes in order to identify their functions and the diseases associated with them

Transgenics (definition from the mouse slide)

A gene that does not inherently belong to the organism but is introduced into its genome from an outside source

Why the mammary gland is the preferred production site

Large quantities of protein can be extracted and purified, and milk is a secreted body fluid normally produced in large quantities that can be collected without causing harm to the animals

Therapeutic proteins from transgenic sheep and goats

Antithrombin III (AT III), tissue plasminogen activator (TPA), and α

Glycosidase from transgenic rabbits

Produced in rabbit milk and used to treat Pompe disease, a rare genetic disease characterized by abnormal buildup of the sugar molecule glycogen inside cells

Therapeutic proteins from eggs

The therapeutic proteins are encoded in the chicken's DNA and produced as part of the egg white

How the transgenic chickens were made

Lentiviral vectors were packaged and injected into embryos from newly laid eggs to generate G0 transgenic birds producing human cytokine interferon α2a and two species

Breeding and screening of transgenic chickens

Birds with vector sequence in the germ line at a frequency greater than 1% (found by PCR) were bred with wild

Cost advantage of chicken

based production

Nuclear transfer (definition)

Removing the DNA from an oocyte (unfertilized egg) and injecting the nucleus that contains the DNA to be cloned; in rare instances the newly constructed cell divides normally, replicating the new DNA while remaining pluripotent

Spermatogonial stem cells

The germ stem cells of the seminiferous epithelium in the testis

Cytoplasmic injection

Injecting a single live sperm directly into the center of a human egg

Perivitelline space injection

Microinjecting DNA constructs directly into the perivitelline space of mouse embryos at embryonic day 0.5 to generate transgenic mice

Zona

free transduction

Transfection vs. transduction

Transfection is deliberately introducing naked or purified nucleic acids into eukaryotic cells; transduction is introducing foreign DNA into a cell by a virus or viral vector

Incubation for delivery of spermatozoa

Co

Intratesticular injection

An injection that assesses the potential of agents to directly affect the testis

Intramammary injection

One of the most effective and commonly used methods to treat and/or prevent mastitis

Intracytoplasmic sperm injection (ICSI)

An in vitro fertilization procedure in which a single sperm cell is injected directly into the cytoplasm of an egg

In vitro fertilization (IVF)

Fertilization achieved by extracting eggs, retrieving a sperm sample, and manually combining egg and sperm in a laboratory dish

Artificial insemination

A fertility treatment method used to deliver sperm directly to the cervix or uterus in hopes of achieving pregnancy

Natural mating

The free expression of sexual behavior in captive

Transplantation

A medical procedure in which an organ is removed from one body and placed in the body of a recipient to replace a damaged or missing organ

Embryo transfer

The final stage of the IVF process, where the fertilized egg — now an embryo — is placed in the woman's uterus