Animal Genetic Engineering: Methodology and Applications Transgenic animals created by two methods DNA microinjection 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 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_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.

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 https://www.youtube.com/watch?v=mAfCauLF-14 EnviropigTM an environmentally friendly breed of pigs that utilizes 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 Cloning livestock by nuclear transfer (e.g., sheep)-“Hello Dolly” https://www.youtube.com/watch?v=AGxdWG6GO3k

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.