transgenic animals: methodology and...

Transgenic Animals:Methodology and Applications

•Transgenic mice: methodology (Retrovirus vector, DNAmicroinjection, Engineered embryonic stem cell, Cre-loxPrecombination 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

•Transgenic mice: methodology (Retrovirus vector, DNAmicroinjection, Engineered embryonic stem cell, Cre-loxPrecombination 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 21Transgenic Animals

Figure 21.1

Retroviral vectors can be used to createtransgenic animals

Copyright © 2010 ASM PressAmerican Society for Microbiology

1752 N St. NW, Washington, DC 20036-2904

Molecular Biotechnology: Principles and Applications of Recombinant DNA, Fourth EditionBernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten


Figure 21.3

DNA microinjection is the mainmethod used to create transgenicanimals




Fig. 21.3 Establishing transgenicmice by DNA microinjection

• Most commonly used method

• Only 5% or less of the treated eggsbecome transgenic progeny

• Need to check mouse pups for DNA(by PCR or Southerns), RNA (bynortherns or RT-PCR), and protein (bywestern or by some specific assaymethod)

• Expression will vary in transgenicoffspring: due to position effect andcopy number

• Most commonly used method

• Only 5% or less of the treated eggsbecome transgenic progeny

• Need to check mouse pups for DNA(by PCR or Southerns), RNA (bynortherns or RT-PCR), and protein (bywestern or by some specific assaymethod)

• Expression will vary in transgenicoffspring: due to position effect andcopy number


Figure 21.4

Less than 5% of themicroinjectedfertilized eggsbecome transgenicprogeny




Creating a transgenic mouse using theDNA microinjection method

• Seehttp://bcs.whfreeman.com/lodish7e/#800911__812052__


Figure 21.5

Genetically engineered embryonicstem (ES) cells can be used to createtransgenic animals, but this method islabor intensive and used to allow forgene targeting via homologousrecombination.




Establishingtransgenic animalsusing engineeredembryonic stem

(ES) cellsBut what are ES

cells?

Establishingtransgenic animalsusing engineeredembryonic stem

(ES) cellsBut what are ES

cells?

Transgenic animals-Engineered embryonic stem cellmethod (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: Placeengineered ES cells

into an early embryo(Fig. 21.5)

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

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Step 3: Placeengineered ES cells

into an early embryo(Fig. 21.5)

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

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1000|22000|23000|99000|&ns=486

Transgenicanimals-Using Cre-loxP for tissue or

time-specific geneknockouts

Transgenic mice can be produced with highcapacity vectors

• Generally done by microinjection of numerous genescontained in a YAC

• Production of mice that can produce humanantibodies is one notable example

• Generally done by microinjection of numerous genescontained in a YAC

• Production of mice that can produce humanantibodies is one notable example

Transgenic mice/animal: applications

• Transgenic models for Alzheimer disease, amyotrophic lateralsclerosis, Huntington disease, arthritis, muscular dystrophy,tumorigenesis, hypertension, neurodegenerative disorders,endocrinological dysfunction, coronary disease, etc.

• Using transgenic mice as test systems (e.g., protein [CFTR] secretioninto milk, protection against mastitis caused by Staphylococcusaureus using a modified lysostaphin gene)

• Conditional regulation of gene expression (tetracycline-induciblesystem in Fig. 21.19)

• Conditional control of cell death (used to model and study organfailure; involves the organ-specific engineering of a toxin receptorinto the mice and then addition of the toxin to kill that organ)

• Transgenic models for Alzheimer disease, amyotrophic lateralsclerosis, Huntington disease, arthritis, muscular dystrophy,tumorigenesis, hypertension, neurodegenerative disorders,endocrinological dysfunction, coronary disease, etc.

• Using transgenic mice as test systems (e.g., protein [CFTR] secretioninto milk, protection against mastitis caused by Staphylococcusaureus using a modified lysostaphin gene)

• Conditional regulation of gene expression (tetracycline-induciblesystem in Fig. 21.19)

• Conditional control of cell death (used to model and study organfailure; involves the organ-specific engineering of a toxin receptorinto 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, thegenetically enhanced “marathon” mice (above, on thetreadmill in San Diego) ran twice as far and nearly twiceas long as ordinary rodents. The peroxisomeproliferator-activated receptor (PPAR-delta) gene wasoverexpressed in these transgenic mice. For details, seehttp://www.salk.edu/otm/Articles/PLoSBiology_October2004.pdf

Dr. Ron Evans and one of his genetically engineered“marathon” mice. The enhanced PPAR-delta activitynot only increased fat burning, but transformedskeletal muscle fibers, boosting so-called "slow-twitch" muscle fibers, which are fatigue resistant,and reducing 'fast-twitch' fibers, which generaterapid, powerful contractions but fatigue easily.

And then there is “transgenic art” with GFP…

Fig. 21.22 Cloninglivestock by nucleartransfer (e.g., sheep)

“Hello Dolly”

And now there is pet cloning for a “small” fee…

Nine-week-old "Little Nicky" peers out fromher carrying case in Texas. Little Nicky,a cloned cat, was sold to its new ownerby Genetic Savings and Clone for $50,000in December 2004.

August 07, 2008 | Bernann McKinney with one ofthe 5 puppies cloned from Booger, her late petpit bull. It cost her $50,000. When Booger wasdiagnosed with cancer, a grief-stricken McKinneysought to have him cloned -- first by the now-defunct Genetic Savings and Clone, and then bySouth Korean company RNL Bio.

Transgenic cattle, sheep,goats, and pigs

• Using the mammary gland as abioreactor (see adjacent figure)

• Increase casein content in milk

• Express lactase in milk (to removelactose)

• Resistance to bacterial, viral, andparasitic diseases

• Reduce phosphorous excretion

• Using the mammary gland as abioreactor (see adjacent figure)

• Increase casein content in milk

• Express lactase in milk (to removelactose)

• Resistance to bacterial, viral, andparasitic diseases

• Reduce phosphorous excretion

Table 21.2 Some human proteins expressed inthe mammary glands of transgenic animals

• Erythropoietin• Factor IX• Factor VIII• Fibrinogen• Growth hormone• Hemoglobin• Insulin• Monoclonal antibodies• Tissue plasminogen activator (TPA)• α1-antitrypsin• Antithrombin III (the first transgenic animal drug, an

anticlotting protein, approved by the FDA in 2009)

• Erythropoietin• Factor IX• Factor VIII• Fibrinogen• Growth hormone• Hemoglobin• Insulin• Monoclonal antibodies• Tissue plasminogen activator (TPA)• α1-antitrypsin• Antithrombin III (the first transgenic animal drug, an

anticlotting protein, approved by the FDA in 2009)

“Enviropigs”

• Transgenic pigs expressing thephytase gene in their salivary glands

• The phytase gene was introduced viaDNA microinjection and used theparotid secretory protein promoterto specifically drive expression in thesalivary glands

• Phytate is the predominant storageform of phosphorus in plant-basedanimal feeds (e.g., soybean meal)

• Pigs and poultry cannot digestphytate and consequently excretelarge amounts of phosphorus

• “Enviro-pigs” excrete 75% lessphosphorus

• Microinjected an E. coli phytasegene under the control of a mouseparotid secretory protein promoter

• Transgenic pigs expressing thephytase gene in their salivary glands

• The phytase gene was introduced viaDNA microinjection and used theparotid secretory protein promoterto specifically drive expression in thesalivary glands

• Phytate is the predominant storageform of phosphorus in plant-basedanimal feeds (e.g., soybean meal)

• Pigs and poultry cannot digestphytate and consequently excretelarge amounts of phosphorus

• “Enviro-pigs” excrete 75% lessphosphorus

• Microinjected an E. coli phytasegene under the control of a mouseparotid secretory protein promoter

EnviropigTM an environmentally friendlybreed of pigs that utilizes plantphosphorus efficiently.

Fig. 21.32 Establishingtransgenic chickens bytransfection of isolated

blastoderm cells• Resistance to viral, bacterial,

and coccidial diseases

• Better feed efficiency

• Lower fat and cholesterollevels in eggs

• Better meat quality

• Eggs with pharmaceuticalproteins in them

• Resistance to viral, bacterial,and coccidial diseases

• Better feed efficiency

• Lower fat and cholesterollevels in eggs

• Better meat quality

• Eggs with pharmaceuticalproteins 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/)

• Genes are introduced into fertilized eggs by DNA microinjection orelectroporation

• 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 genepromoter and 3’ untranslated region (currently under FDA consideration)

• Madaka fish were genetically engineered to serve as biosensors forenvironmental pollutants (e.g., estrogens) by using an estrogen-induciblepromoter (the vitellogenin promoter) to control expression of the GFP gene

Fig. 21.33 Fig. 21.34

transgenic animals: methodology and...

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