Chapter 12 – Transgenic - PowerPoint Presentation

Chapter 12 – Transgenic Animals Transgenic mice: methodology Transgenic mice: applications Transgenic livestock Transgenic poultry Transgenic fish

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 12.1 Establishing transgenic mice by DNA microinjection. Note that only 5% or less of the microinjected fertilized eggs become transgenic progeny.

Creating a transgenic mouse using theDNA microinjection methodSee https:// www.youtube.com/watch?v=ysq-lqp1-Ho

Figure 12.3 Establishing transgenic mice with retroviral vectors.

Figure 12.6 Establishing transgenic mice with genetically engineered embryonic stem 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

Transgenic Animals: Using the Cre-loxP recombination system for tissue or time-specific gene knockouts

Figure 12.14 Editing animal genomes using the CRISPR-Cas9 system.Adapted from Williams et al., 2016, Cold Spring Harb Protoc ; doi:10.1101/pdb.top087536.

Figure 12.16 RNA interference (RNAi) to knock down expression of a target gene in transgenic mice. This DNA construct can be introduced into mice embryos by DNA microinjection or by using a retrovirus (e.g., lentivirus).

Figure 12.17 Conditional knockdown of target gene expression using RNA interference. This strategy takes advantage of the Cre-loxP recombination system.

Transgenic Mice: Applications Transgenic mouse 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., using RNAi to reduce levels of proteins [amyloid-b peptide] that contribute to Alzheimer disease, correcting disease-causing mutations in the dystrophin gene using CRISPR-Cas9, protection against infectious, such as pseudorabies virus, by expressing soluble virus receptor proteins) 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 aApplication: 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 Livestock Cloning livestock by somatic cell nuclear transfer Pharmaceuticals Donor organsDisease resistant livestock Milk quality Animal production traits Transgenic poultry Transgenic fish

Cloning livestock bysomatic cell nuclear transfer (e.g., sheep)- “ Hello Dolly”https://www.youtube.com/watch?v=AGxdWG6GO3k Figure 12.29

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.

Figure 12.29 Cloning livestock by somatic cell nuclear transfer as depicted here with sheep. The first cloned sheep was produced in 1996 and was named “Dolly”.

Transgenic cattle, sheep, goats, and pigsUsing 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 diseasesReduce phosphorous excretion

Table 12.3

Exogenous proteins expressed in the mammary glands of transgenic animalsErythropoietin Factor IX Factor VIII FibrinogenGrowth hormoneHemoglobinInsulinMonoclonal antibodiesTissue plasminogen activator (TPA) a 1 - antitrypsin Antithrombin ( ATyrn )-prevents clotting; 1 st approved recombinant drug produced in an animal (goat) ; approved by the FDA in 2009 See https:// www.youtube.com/watch?v=eJCReJbGZKs

Table 12.2

Figure 12.30 Construct for the expression of human antithrombin in goats’ milk leading to the production of the first recombinant drug produced in an animal and approved by the FDA in 2009.

“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 Enviropig TM an environmentally friendly breed of pigs that utilizes plant phosphorus efficiently.

And then there is “transgenic art” with GFP…

Figure 12.41 Transgenic chickens engineered with a small RNA molecule the inhibits RNA-dependent RNA polymerase required for production of new avian influenza virus.From Lyall et al., Science. 331: 223–226, 2011.

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 resistanceGenetically 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 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 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

The genetically engineered AquaBounty salmon shown here is about twice the size of its wild kin, although both are roughly the same age. This salmon was approved by the US FDA in November 2015 and by Canadian authorities in May 2016. The salmon is now sold in Canada, but not in the US due to political battles, including a provision in the US government’s budget law for 2017 for labeling this genetically engineered salmon. Transgenic Salmon Update: August 2017

Figure 12.42 Construct for the overexpression of growth hormone in Atlantic salmon used the Chinook salmon growth hormone cDNA under the control of the promoter and transcription terminator-polyadenylation signals from the the antifreeze protein (AFP) gene from another fish, the ocean pout.

Figure 12.43 Transgenic medaka fish as biosensors of environmental pollutants. The vitellogenin gene promoter was used to drive expression of the GFP reporter gene in medaka fish. Note that the vitellogenin promoter is activated only in the presence of natural and synthetic estrogenic compounds.

Some Other Genetically Modified Organisms See https://www.youtube.com/watch?v=bmi45JLJOgU I would encourage you to try to find the primary research papers on these GMOs if you have an interest in them in order to get more and better information on them.