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Genetic Engineering of Plants:

Methodology
Plant transformation with the Ti plasmid of
Agrobacterium tumefaciens
Ti plasmid derived vector systems
Physical methods of transferring genes to plants
(microprojectile bombardment)
Chloroplast engineering
Use of reporter genes in transformed plant cells
Manipulation of gene expression in plants
Production of marker-free transgenic plants

Why genetically engineer plants?


To improve the agricultural or horticultural value of
plants
To serve as living bioreactors for the production of
economically important proteins or metabolites
To provide a renewable source of energy (biofuels)
To provide a powerful means for studying the
biological action of genes and gene products

Plant transformation with the Ti plasmid of


Agrobacterium tumefaciens
A. tumefaciens is a gram-negative soil bacterium
which naturally transforms plant cells, resulting in
crown gall (cancer) tumors
Tumor formation is the result of the transfer,
integration and expression of genes on a specific
segment of A. tumefaciens plasmid DNA called the
T-DNA (transferred DNA)
The T-DNA resides on a large plasmid called the Ti
(tumor inducing) plasmid found in A. tumefaciens

The Ti plasmid of Agrobacterium tumafaciens and the


transfer of its T-DNA to the plant nuclear genome

Fig. 18.3 The Ti plasmid of Agrobacterium tumafaciens and


its T-DNA region containing eukaryotic genes for auxin,
cytokinin, and opine production.

Chapter 18
Genetic Engineering of Plants: Methodology

Figure 18.3

Ti plasmid structure

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 18
Genetic Engineering of Plants: Methodology

Figure 18.1

Infection of a plant with A. tumefaciens


and formation of a crown gall tumor.

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Fig. 28-27
Crown Gall on
Tobacco

Fig. 18.1 Infection of a


plant with A. tumefaciens and
formation of crown galls

Figure 18.2 and 18.3


Ti plasmid structure and
function. Note the woundinduced plant phenolics
induce the vir genes on
the Ti plasmid.

The infection process:


1. Wounded plant cell releases phenolics and nutrients.
2. Phenolics and nutrients cause chemotaxic response of A. tumefaciens
3. Attachment of the bacteria to the plant cell.
4. Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene
transcription and allow for T-DNA transfer and integration into plant chromosomal DNA.
5. Transcription and translation of the T-DNA in the plant cell to produce opines (food) and
tumors (housing) for the bacteria.
6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use
opines as a C, H, O, and N source.

Chapter 18
Genetic Engineering of Plants: Methodology

Figure 18.4
Conserved nucleotides at the right and left borders of the Ti plasmid are imperfect
direct repeats.

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 18
Genetic Engineering of Plants: Methodology

Figure 18.6
Chemical structures of three
opines produced by plants.

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Fig. 18.7 The binary Ti plasmid system involves using a small


T-DNA plasmid (shown below) and a disarmed (i.e., no TDNA) Ti plasmid in A. tumefaciens

Clone YFG (your favorite gene) or


the target gene in the small T-DNA
plasmid in E. coli, isolate the plasmid
and use it to transform the disarmed
A. tumefaciens as shown.

(disarmed)

Disarmed
Ti plasmid

Plant genetic engineering with


the binary Ti plasmid system

Transgenic
plant

Chapter 18
Genetic Engineering of Plants: Methodology

Table 18.1

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 18
Genetic Engineering of Plants: Methodology

Table 18.2

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Fig. 18.10
Microprojectile
bombardment or
biolistic-mediated
DNA transfection
equipment
(a) lab version
(b) portable version

When the helium pressure


builds to a certain point, the
plastic rupture disk bursts, and
the released gas accelerates the
flying disk* with the DNAcoated gold particles on its
lower side. The gold particles
pass the stopping screen, which
holds back the flying disk, and
penetrate the cells of the plant.

Chapter 18
Genetic Engineering of Plants: Methodology

Figure 18.10

Microprojectile bombardment
(biolistics) apparatus

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 18
Genetic Engineering of Plants: Methodology

Chloroplasts can be genetically engineered using microparticle bombardment.

Figure 18.12

Figure 18.13

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 18
Genetic Engineering of Plants: Methodology

Table 18.5

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Table 18.5 Some plant cell reporter and


selectable marker gene systems
Enzyme activity

Selectable
marker

Reporter
gene

Neomycin phosphotransferase (kanr)

Yes

Yes

Hygromycin phosphotransferase (hygr)

Yes

Yes

Nopaline synthase

No

Yes

Octopine synthase

No

Yes

b-glucuronidase (GUS)

No

Yes

Firefly luciferase

No

Yes

b-galactosidase

No

Yes

Bromoxynil nitrilase

Yes

No

Green fluorescent protein (GFP)

No

Yes

Reporter Genes
For how reporter genes work, see:
http://bcs.whfreeman.com/lodish7e/#800911__811966__
GFP Researchers Win Nobel Prize (October 8, 2008)
Osamu Shimomura, Martin Chalfie, and Roger Tsien won
the Nobel Prize in chemistry for their work on green
flourescent protein, a tool that has become ubiquitous in
modern biology as a tag and molecular highlighter, vastly
improving our ability to understand what goes on inside
cells.
Perhaps you may even want to see a 10 minute YouTube
video on GFP; if so please see
http://www.youtube.com/watch?v=Sl2PRHGpYuU

Manipulation of gene expression in plants


Strong, constitutive promoters (35S Cauliflower mosaic virus
promoter or 35S CaMV or 35S)
Organ and tissue specific promoter (e.g., the leaf-specific
promoter for the small subunit of the photosynthetic enzyme
ribulosebisphosphate carboxylase or rbc)
Promoterless reporter gene constructs to find new organ- and
tissue-specific promoter (see Fig. 18.15)
Inducible promoters
Secretion of transgene products by inclusion of a signal peptide
sequence between a root promoter and YFG and growing the
transgenic plant hydroponically (YFG product will be secreted)

Chapter 18
Genetic Engineering of Plants: Methodology

Figure 18.21
Rhizosecretion using a
plant promoter active in
roots and a signal peptide
sequence.

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 18
Genetic Engineering of Plants: Methodology

Figure 18.26

Marker genes may be a safety issue, so it is best to remove themhere is one strategy.

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Genetic Engineering of Plants:


Applications
Insect-, pathogen-, and herbicide-resistant plants
Stress
Genetic manipulation of flower pigmentation
Modification of plant nutritional content
Modification of plant food taste and appearance
Plant as bioreactors
Edible vaccines
Renewable energy crops
Plant yield

Genetically engineered crops/foods allowed in the US food supply

Insect-resistant plants

Bt toxin
Cowpea trypsin inhibitor
Proteinase inhibitor II
a-amylase inhibitor
Bacterial cholesterol oxidase
Combinations of the above (e.g., Bt toxin and
proteinase inhibitor II)

Genetic engineering of Bt-plants


Expression of truncated Bt genes encoding the Nterminal portion of Bt increase effectiveness
Effectiveness enhanced by site-directed mutagenesis
increasing transcription/translation
Effectiveness further enhanced by making codon bias
changes (bacterial to plant)
35S CaMV and rbcS promoters used
Integration and expression of the Bt gene directly in
chloroplasts
Note that Lepidopteran insects like corn rootworm,
cotton bollworm, tobacco budworm, etc., cause
combined damages of over $7 Billion dollars yearly in
the US

Fig. 18.7/19.3 A binary T-DNA plasmid for delivering


the Bt gene to plants (not a cointegrate vector)
(NPT or kanr)
(35S-Bt gene-tNOS)

(Spcr)

Effectiveness of insecticide and Bt-tomato plants


in resisting insect damage
% of plants or fruits damaged
Insect

wt tomato
-insecticide

wt tomato
+insecticide

Bt-tomato
-insecticide

Bt-tomato
+insecticide

Tobacco
hornworm

48

Tomato
fruitworm

20

nd

nd

Tomato
pinworm

100

95

94

80

nd, not determined

Strategies to avoid Bt resistant insects


Use of inducible promoters (that can be turned on
only when there is an insect problem)
Construction of hybrid Bt toxins
Introducing more than one Bt gene (stacking)
Introduction of the Bt gene in combination with
another insecticidal gene
Spraying low levels of insecticide on Bt plants
Use of spatial refuge strategies

Genetically engineered Bt-plants in the field


Product

Institution(s)

Engineered Trait(s)

Sources of New
Genes

Name

Corn

Bayer

Resist glufosinate herbicide to control weeds/Bt toxin to control insect pests (European corn borer)

Bacteria, virus

StarLink-1998 (animals
only)

Corn

Dow/Mycogen

Bt toxin to control insect pests (European corn borer)

Corn, bacteria, virus

NatureGard-1995

Corn

Dow/Mycogen

Resist glufosinate herbicide to control weeds/Bt toxin to control insect pests (Lepidopteran)

Corn, bacteria, virus

Herculex I-2001

DuPont/Pioneer
Corn

Monsanto/DeKalb

Bt toxin to control insect pests (European corn borer)

Bacteria

Bt-Xtra-1997

Corn

Monsanto

Bt toxin to control insect pests (European corn borer)

Bacteria

YieldGard-1996

Corn

Monsanto

Resist glyphosate herbicide to control weeds/Bt toxin to control insect pests (European corn borer)

Arabidopsis, bacteria,
virus

?-1998

Corn

Syngenta

Bt toxin to control insect pests (European corn borer)

Bacteria

Bt11-1996

Corn

Syngenta

Bt toxin to control insect pests (European corn borer)

Corn, bacteria, virus

Knock Out-1995

Corn (pop)

Syngenta

Bt toxin to control insect pests (European corn borer)

Corn, bacteria, virus

Knock Out-1998

Corn
(sweet)

Syngenta

Bt toxin to control insect pests (European corn borer)

Bacteria

Bt11-1998

Cotton

Monsanto/Bayer

Resist bromoxynil herbicide to control weeds/Bt toxin to control insect pests (cotton bollworms

Bacteria

?-1998

and tobacco budworm)


Cotton

Monsanto

Bt toxin to control insect pests (cotton bollworms and tobacco budworm)

Bacteria

Bollgard-1995

Potato

Monsanto

Bt toxin to control insect pests (Colorado potato beetle)

Bacteria

NewLeaf-1995

Potato

Monsanto

Bt toxin to control insect pests (Colorado potato beetle)/resist potato virus Y

Bacteria, virus

NewLeaf Y-1999

Potato

Monsanto

Bt toxin to control insect pests (Colorado potato beetle)/resist potato leafroll virus

Bacteria, virus

NewLeaf Plus-1998

Fig. 18.7 Procedure for putting CuMV


coat protein into plants

Virus-resistant plants
Overexpression of the virus
coat protein (e.g. cucumber
mosaic virus in cucumber
and tobacco, papaya ringspot
virus in papaya and tobacco,
tobacco mosiac virus in
tobacco and tomato, etc.)
Expression of a dsRNase
(RNaseIII)
Expression of antiviral
proteins (pokeweed)

Genetically engineered Papaya to resist the Papaya


Ringspot-Virus by overexpression of the virus coat protein

Herbicides and herbicide-resistant plants

Herbicides are generally non-selective (killing both weeds and


crop plants) and must be applied before the crop plants
germinate
Four potential ways to engineer herbicide resistant plants
1. Inhibit uptake of the herbicide
2. Overproduce the herbicide-sensitive target protein
3. Reduce the ability of the herbicide-sensitive target to
bind to the herbicide
4. Give plants the ability to inactivate the herbicide

Chapter 19
Engineering Plants To Overcome Biotic and Abiotic Stress

Table 19.3

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Herbicide-resistant plants:
Giving plants the ability to inactivate the herbicide

Herbicide: Bromoxynil
Resistance to bromoxynil (a photosytem II inhibitor) was
obtained by expressing a bacterial (Klebsiella ozaenae)
nitrilase gene that encodes an enzyme that degrades this
herbicide

Herbicide-resistant plants:
Reducing the ability of the herbicide-sensitive target to bind to the
herbicide

Herbicide: Glyphosate (better known as Roundup)


Resistance to Roundup (an inhibitor of the enzyme EPSP
involved in aromatic amino acid biosynthesis) was obtained by
finding a mutant version of EPSP from E. coli that does not bind
Roundup and expressing it in plants (soybean, tobacco,
petunia, tomato, potato, and cotton)
5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a
chloroplast enzyme in the shikimate pathway and plays a key
role in the synthesis of aromatic amino acids such as tyrosine
and phenylalanine

How to make a Roundup Ready Plant

Fungus- and bacterium-resistant plants

Genetic engineering here is more challenging; however, some


strategies are possible:
Individually or in combination express pathogenesis-related (PR)
proteins, which include b1,3-glucanases, chitinases, thaumatinlike proteins, and protease inhibitors
Overexpression of the NPR1 gene which encodes the master
regulatory protein for turning on the PR protein genes
Overproducing salicylic acid in plants by the addition of two
bacterial genes; SA activates the NPR1 gene and thus results in
production of PR proteins

Development of stress- and senescence-tolerant plants:


genetic engineering of salt-resistant plants

Overexpression of the
gene encoding a Na+/H+
antiport protein which
transports Na+ into the
plant cell vacuole
This has been done in
Arabidopsis and tomato
plants allowing them to
survive on 200 mM salt
(NaCl)

Development of stress- and senescence-tolerant plants:


genetic engineering of flavorful tomatoes

Fruit ripening is a natural aging or senescence process that involves two independent
pathways, flavor development and fruit softening.
Typically, tomatoes are picked when they are not very ripe (i.e., hard and green) to allow
for safe shipping of the fruit.
Polygalacturonase is a plant enzyme that degrades pectins in plant cell walls and
contribute to fruit softening.
In order to allow tomatoes to ripen on the vine and still be hard enough for safe shipping
of the fruit, polygalacturonase gene expression was inhibited by introduction of an
antisense polygalacturonase gene and created the first commercial genetically engineered
plant called the FLAVR SAVR tomato.

Flavor development pathway


Red

Green

Fruit softening pathway


Hard

polygalacturonase

Soft
antisense polygalacturonase

Fig. 20.18 Genetic manipulation of flower pigmentation

Manipulation of the
anthocyanin
biosynthesis pathway
Introduction of maize
dihydroflavonol 4reductase (DFR) into
petunia produces a brick
red-orange transgenic
petunia
Novel flower colors in
the horticultural
industrial are big money
makers!
Note a blue rose would
make millions!

New pathway in
petunia created by
the maize DFR gene

Modification of plant nutritional content

Amino acids (corn is deficient in lysine, while legumes are


deficient in methionine and cysteine)
Lipids (altering the chain length and degree of unsaturation is
now possible since the genes for such enzymes are known)
Increasing the vitamin E (a-tocopherol) content of plants
(Arabidopsis)
Increasing the vitamin A content of plants (rice)

Modification of plant nutritional content: increasing the


vitamin E (a-tocopherol) content of plants
Plants make very little a-tocopherol
but do make g-tocopherol; they do
not produce enough of the
methyltransferase (MT)
The MT gene was identified and
cloned in Synechocystis and then in
Arabidopsis
The Arabidopsis MT gene was
expressed under the control of a
seed-specific carrot promoter and
found to produce 80 times more
vitamin E in the seeds

Dean DellaPenna, Michigan State Univ. Professor


B.S. 1984, Ohio University

Modification of plant nutritional content: increasing the


vitamin A content of plants (Fig. 20.7)
124 million children worldwide are
deficient in vitamin A, which leads
to death and blindness
Mammals make vitamin A from bcarotene, a common carotenoid
pigment normally found in plant
photosynthetic membranes
Here, the idea was to engineer the
b-carotene pathway into rice
The transgenic rice is yellow or
golden in color and is called
golden rice

GGPP
Daffodil phytoene synthase gene

Phytoene
Bacterial phytoene desaturase gene

Lycopene
Daffodil lycopene b-cyclase gene

b-carotene
Endogenous human gene

Vitamin A

Plants as bioreactors
Production of therapeutic agents (proteins)
Production of recombinant vaccines or edible
vaccines
Production of antibodies

Chapter 20
Engineering Plant Quality and Proteins

Table 20.6

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 20
Engineering Plant Quality and Proteins

Table 20.7

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 20
Engineering Plant Quality and Proteins

Table 20.8

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 20
Engineering Plant Quality and Proteins

Table 20.9

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Plants are also being genetically engineered for:


Biofuel production (e.g., lower lignin, lower
recalcitrance)
Phytoremediation (i.e., bioremediation using
plants)
Biopolymers (i.e., biodegradable plastics)

Transgenic Animals: Methodology and


Applications
Transgenic mice: methodology (Retrovirus vector, DNA
microinjection, Engineered embryonic stem cell, Cre-loxP
recombination system, High capacity vectors)
Transgenic mice: applications (Alzheimer disease, test systems,
conditional regulation, control of cell death)
Cloning livestock by nuclear transfer
Transgenic cattle, sheep, goats and pigs
Transgenic birds
Transgenic fish

Chapter 21
Transgenic Animals

Figure 21.1

Retroviral vectors can be used to create


transgenic animals

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Chapter 21
Transgenic Animals

Figure 21.3
DNA microinjection is the main
method used to create transgenic
animals

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Fig. 21.3 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

Chapter 21
Transgenic Animals

Figure 21.4
Less than 5% of the
microinjected
fertilized eggs
become transgenic
progeny

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Creating a transgenic mouse using the


DNA microinjection method
See
http://bcs.whfreeman.com/lodish7e/#800911__812052__

Chapter 21
Transgenic Animals

Figure 21.5
Genetically engineered embryonic
stem (ES) cells can be used to create
transgenic animals, but this method is
labor intensive and used to allow for
gene targeting via homologous
recombination.

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth Edition


Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten

Copyright 2010 ASM Press


American Society for Microbiology
1752 N St. NW, Washington, DC 20036-2904

Establishing
transgenic animals
using engineered
embryonic stem
(ES) cells
But what are ES
cells?

Transgenic animals-Engineered embryonic stem cell


method (used for gene knockouts)
Step 1: Get the ES cells (Fig. 21.5)

Step 2: Genetically engineer the ES cells


(Figs. 21.5 and 21.6)

Step 3: Place
engineered ES cells
into an early embryo
(Fig. 21.5)
see

http://bcs.whfreeman.com/lodish5e/pages/bcs-

main.asp?v=category&s=00020&n=09000&i=09020.01&o=|00510
|00610|00520|00530|00540|00560|00570|00590|00600|0070
0|00710|00010|00020|00030|00040|00050|01000|02000|030
00|04000|05000|06000|07000|08000|09000|10000|11000|12
000|13000|14000|15000|16000|17000|18000|19000|20000|2
1000|22000|23000|99000|&ns=486

Transgenic
animals-Using CreloxP for tissue or
time-specific gene
knockouts

Transgenic mice can be produced with high


capacity vectors
Generally done by microinjection of numerous genes
contained in a YAC
Production of mice that can produce human
antibodies is one notable example

Transgenic mice/animal: 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 in Fig. 21.19)
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)

And then there is transgenic art with GFP

Fig. 21.22 Cloning


livestock by nuclear
transfer (e.g., sheep)
Hello Dolly

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

Table 21.2 Some human 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 III (the first transgenic animal drug, an
anticlotting protein, approved by the FDA in 2009)

Enviropigs
Transgenic pigs expressing the
phytase gene in their salivary glands
The phytase gene was introduced via
DNA microinjection and used 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 consequently excrete
large amounts of phosphorus
Enviro-pigs excrete 75% less
phosphorus
Microinjected an E. coli phytase
gene under the control of a mouse
parotid secretory protein promoter

EnviropigTM an environmentally friendly


breed of pigs that utilizes plant
phosphorus efficiently.

Fig. 21.32 Establishing


transgenic chickens by
transfection of isolated
blastoderm cells
Resistance to viral, bacterial,
and coccidial diseases
Better feed efficiency
Lower fat and cholesterol
levels in eggs
Better meat quality
Eggs with pharmaceutical
proteins in them

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
Genetically engineered for a novel pet (Glofish-see http://glofish.com/)

Transgenic fish (more detail)


Salmon were genetically engineered for more rapid growth using the growth
hormone gene under the control of the ocean pout antifreeze protein gene
promoter and 3 untranslated region (currently under FDA consideration)
Madaka fish were genetically engineered to serve as biosensors for
environmental pollutants (e.g., estrogens) by using an estrogen-inducible
promoter (the vitellogenin promoter) to control expression of the GFP gene

Fig. 21.33

Fig. 21.34