Transgenic plants and animals

• Transgenic plants are plants that have

been genetically engineered, a breeding
approach that uses recombinant DNA
techniques to create plants with new
characteristics.
• A transgenic animal is one that carries a
foreign gene that has been inserted into its
genome. The foreign gene is constructed
using recombinant DNA methodology.
 Advantages
• In plants:
• Increased and improved nutrients
• Longer shelf life, less waste
• Enhanced taste and quality
• Reduced maturation time
• Higher yielding crops, more efficient use of land
• Improved resistance to disease or illness
• Increased food security for growing populations and growth
challenges
 Advantages
• In animals:
• Used in biomedical science--cancer research; immunology;
developmental biology; gene expression and regulation; and
models for human genetic diseases such as muscular
dystrophy, and sickle cell anemia.
• Potential applications for transgenic animals include
manipulation of milk composition, growth, disease resistance,
reproductive performance, and production of pharmaceutical
proteins by livestock.
 Risk associated with Genetic
Modification
• Safety
– Potential human health implications.
– Potential environmental impact.
• Ethics
– “Playing God”
– Tampering with nature by mixing genes among species.
• Labeling
– Not mandatory in some countries (e.g., Canada and the United
States).
– Mixing GM crops with non-GM confounds labeling attempts.
 Risk associated with Genetic Modification
• Biodiversity
• Addition of Bt gene into plants including corn, potatoes and
cotton to increase resistance to plants
• Bt gene obtained from Bacillus thuringiensis (a soil bacterium
that produces a natural insecticide)
• Problem: plants producing Bt toxin are releasing toxin in
pollen
• Pollen from a Bt plant was dusted on to milkweed:
• - only 56% of young monarch butterfly larvae lived
• - whereas pollen from organic plants dusted on the milkweed
produced a survival rate of 100%.
• Approximately half of the monarch butterfly population live in
the “corn belt” of the USA
Who Uses this technology?
 Researches on GMOs
• Transgenic animals are used as experimental models to
perform phenotypic and for testing in biomedical research.
• Genetically animals are becoming more vital to the discovery
and development of cures and treatments for many serious
diseases.
• By altering the DNA or transferring DNA to an animal, we can
develop certain proteins that may be used in medical
treatment.
• Stable expressions of human proteins have been developed in
many animals, including sheep, pigs, and rats.
• Human-alpha-1-antitrypsin,which has been tested in sheep
and is used in treating humans with this deficiency and
transgenic pigs with human-histo-compatibility have been
studied in the hopes that the organs will be suitable for
transplant with less chances of rejection.
 Controversy
• There are controversies around GMOs on several levels,
including whether making them is ethical, whether food
produced with them is safe, whether such food should be
labeled and if so how, whether agricultural biotech is needed
to address world hunger now or in the future, and more
specifically to GM crops—intellectual property and market
dynamics; environmental effects of GM crops; and GM crops'
role in industrial agricultural more generally.
 Animal Genetic Engineering:
Methodology and Applications
• Transgenic animals created by two methods
1. DNA microinjection
2. Embryonic stem cells & gene knock-outs/ also Cre-loxP
• Transgenic animals: some examples
 GH-mice
 Marathon mice
 ATyrn goats
• Transgenic fish: some examples
 GloFish (GFP-zebra fish)
 GH-salmon
• Mammalian Cloning by nuclear transfer
 Dolly the sheep
 Pet cloning
Establishing transgenic mice
by DNA microinjection

• Most commonly used method

• Only 5% or less of the treated
eggs become transgenic progeny
• Need to check mouse pups for
DNA (by PCR or Southerns), RNA
(by northerns or RT-PCR), and
protein (by western or by some
specific assay method)
• Expression will vary in transgenic
offspring: due to position effect
and copy number
 Creating a transgenic mouse using the
DNA microinjection method
• See https://www.youtube.com/watch?v=HiF9RDZJ0Pg

• See https://www.youtube.com/watch?v=ysq-lqp1-Ho
 Establishing
transgenic
animals using
engineered
embryonic stem
(ES) cells
But what are ES
cells?
Transgenic animals-Engineered embyronic
stem cell method (used for gene knockouts)
Step 1: Get the ES cells
Step 2: Genetically engineer the ES cells
 Step 3: Place
engineered ES cells
into an early embryo
(Fig. 19.4)

see the Chapter 5 video

 Transgenic
animals-Using
Cre-loxP for
tissue or time-
specific gene
knockouts
 Transgenic mice: applications
• Transgenic models for Alzheimer disease, amyotrophic lateral
sclerosis, Huntington disease, arthritis, muscular dystrophy,
tumorigenesis, hypertension, neurodegenerative disorders,
endocrinological dysfunction, coronary disease, etc.
• Using transgenic mice as test systems (e.g., protein [CFTR]
secretion into milk, protection against mastitis caused by
Staphylococcus aureus using a modified lysostaphin gene)
• Conditional regulation of gene expression (tetracycline-
inducible system)
• Conditional control of cell death (used to model and study
organ failure; involves the organ-specific engineering of a toxin
receptor into the mice and then addition of the toxin to kill that
organ)
 Another Transgenic mouse application:
Marathon Mice
Instead of improving times by fractions of a second,
the genetically enhanced “marathon” mice (above,
on the treadmill in San Diego) ran twice as far and
nearly twice as long as ordinary rodents. The
peroxisome proliferator-activated receptor (PPAR-
delta) gene was overexpressed in these transgenic
mice. For details, see
http://www.salk.edu/otm/Articles/PLoSBiology_Octo
ber2004.pdf
Dr. Ron Evans and one of his genetically
engineered “marathon” mice. The enhanced
PPAR-delta activity not only increased fat
burning, but transformed skeletal muscle fibers,
boosting so-called "slow-twitch" muscle fibers,
which are fatigue resistant, and reducing 'fast-
twitch' fibers, which generate rapid, powerful
contractions but fatigue easily.
 Transgenic cattle, sheep,
goats, and pigs
• Using the mammary gland as a
bioreactor (see adjacent figure)
• Increase casein content in milk
• Express lactase in milk (to
remove lactose)
• Resistance to bacterial, viral,
and parasitic diseases
• Reduce phosphorous excretion
 Exogenous proteins expressed in the
mammary glands of transgenic animals
• Erythropoietin
• Factor IX
• Factor VIII
• Fibrinogen
• Growth hormone
• Hemoglobin
• Insulin
• Monoclonal antibodies
• Tissue plasminogen activator (TPA)
• a1-antitrypsin
• Antithrombin (ATyrn)-prevents clotting; 1st approved
recombinant drug produced in an animal (goat);
approved by the FDA in 2009
• See https://www.youtube.com/watch?v=eJCReJbGZKs
 “Enviropigs”
• Transgenic pigs expressing the
phytase gene in their salivary
glands
• The phytase gene is introduced
via DNA microinjection and uses
the parotid secretory protein
promoter to specifically drive
expression in the salivary glands
• Phytate is the predominant
storage form of phosphorus in
plant-based animal feeds (e.g.,
soybean meal)
• Pigs and poultry cannot digest
phytate and thus excrete large
amounts of phosphorus
• “Enviro-pigs” excrete 75% less
phosphorus
• See EnviropigTM an environmentally
https://www.youtube.com/watch?v=mAf friendly breed of pigs that utilizes
CauLF-14 plant phosphorus efficiently.
And then there is “transgenic art” with
GFP…
 Transgenic fish
• Genes are introduced into fertilized eggs by DNA
microinjection or electroporation
• No need to implant the embryo; development is
external
• Genetically engineered for more rapid growth
using the growth hormone gene (salmon, trout,
catfish, tuna, etc.)
• Genetically engineered for greater disease
resistance
• Genetically engineered to serve as a biosensor
for water pollution
 GloFish: http://www.glofish.com/

Where do GloFish® fluorescent zebra fish come from?

GloFish® fluorescent zebra fish were originally bred to help detect
environmental pollutants. By adding a natural fluorescence gene to the
fish, scientists hope to one day quickly and easily determine when our
waterways are contaminated. The first step in developing these
pollution detecting fish was to create fish that would be fluorescent all
the time. It was only recently that scientists realized the public's
interest in sharing the benefits of this research. We call this the
GloFish® fluorescent fish.
 Transgenic salmon over-expressing GH
• This picture shows the respective
growths of a GM salmon and a
non-GM one at the same age
(Credit: Aqua Bounty).

• The FDA approved this GM

salmon in November 2015!
• https://www.youtube.com/watch?
v=ibkhBTC3Wl4

But why is this GM fish growing so fast?

These GM salmon grow so fast because of a change made to one of the roughly
40,000 genes in their DNA. In normal salmon, the gene that controls the production of
growth hormone (GH) is activated by light, so the fish generally grow only during the
sunny summer months. But by attaching a constitutive "promoter sequence", Aqua
Bounty ended up with salmon that make growth hormone all year round.
Gene construct: Ocean Pout AFP promoter-salmon GH cDNA-3’ Ocean Pout AFP gene
Note AFP=antifreeze protein
Cloning livestock by
nuclear transfer
(e.g., sheep)-“Hello Dolly”
https://www.youtube.com/watch?v=AG
xdWG6GO3k
And now there is pet cloning for a “small” fee…

Nine-week-old "Little Nicky" peers out from
her carrying case in Texas. Little Nicky,
a cloned cat, was sold to its new owner
by Genetic Savings and Clone for $50,000
in December 2004.
August 07, 2008 | Bernann McKinney with one of
the 5 puppies cloned from Booger, her late pet pit
bull. It cost her $50,000. When Booger was
diagnosed with cancer, a grief-stricken McKinney
sought to have him cloned -- first by the now-
defunct Genetic Savings and Clone, and then by
South Korean company RNL Bio.