Chapter 21-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

Figure 21.1 Retroviral vectors can be used to create transgenic animals

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

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

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

Creating a transgenic mouse using the DNA microinjection method See http://bcs.whfreeman.com/lodish7e/#800911__812052__

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.

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 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|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=486

Transgenic animals-Using Cre-loxP 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)

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_October2004.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.

And then there is “transgenic art” with GFP…

Fig. 21. 22 Cloning livestock by nuclear transfer (e. g Fig. 21.22 Cloning livestock by nuclear transfer (e.g., sheep) “Hello Dolly”

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.

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