Meeting report: IX Transgenic Animal Research Conference. Granlibakken Conference Center, Tahoe City, California, USA, 11-15 August 2013

Lake Tahoe, CA, USA
Lake Tahoe, CA, USA

The IX Transgenic Animal Research Conference, organized by ISTT member Jim Murray (UC Davis), was held last week at the Granlibakken Conference Center, Tahoe City, California, USA. The unique and beautiful location of this meeting series, by Lake Tahoe, in Northern California, surrounded by woods and mountains (and sporting chipmunks and bears), triggered its magic again and, hence, this ninth TARC was a rewarding success. The conference was attended by about 100 delegates from academia and industry, representing groups primarily interested in the generation, analysis or marketing of non-rodent transgenic animal models, as well as regulators and representatives from governmental agencies. This conference was co-sponsored by the ISTT.

The meeting started with a most passionate keynote address by Matt Wheeler (University of Illinois, USA) who reminded us about our responsibility and the mission we all have as biotechnologists to improve the efficiency of food production in cattle, pigs, and also poultry (as adequately reminded by Helen Sang [Roslin Institute, UK]) , using our unique genetic tools and techniques. Dr. Wheeler provided a number of striking figures to highlight the extraordinary need for food in the near future: “estimates have suggested that we will need to increase our current food production by 70% by 2050. This means that we will have to produce the total amount of food each year that has been consumed by mankind over the past 500 years”. He also expressed regret at how transgenic large animal programs were declining in the US, in part due to the lack of trust in a regulatory process that has been witholding the approval of some early transgenic animals. One major example of this is the ongoing saga of the fast growing AquAdvantage transgenic salmon, produced by AquaBounty, not yet approved, more than 20 years after being first generated. Finally, he openly referred to the unacceptable cost for the world, of not using the most advanced genetic engineering techniques to improve food production. He concluded that “hunger is a curable disease”.

Scott Fahrenkrug (Recombinetics Inc., USA) continued with a most interesting talk describing how the new gene editing tools (i.e. TALENs) can be applied for direct livestock genetics. Using illustrative examples in pigs and cattle he demonstrated the efficient introduction of single and multiple subtle genetic changes, often found as rare alleles in some breeds and difficult to introduce in the animal of choice by standard genetic breeding program, where the segregation of traits would require tens of thousands of animals and many generations. This first of several talks on genome editing tools was followed by that of Emmanuelle Charpentier (Helmholtz Center for Infection Research, Germany), one of the pioneers and discoverer of the CRISPR-Cas9 system in bacteria, and its application for the efficient gene edition in mammals. She suggested that new applications will come from the use of new variants of the RNA-guided Cas9 endonuclease.

Emerald Bay, Lake Tahoe, CA, US
Emerald Bay, Lake Tahoe, CA, USA

The second session started with a talk by Daniel Carlson (Recombinetics Inc., USA), who gave technical details of the experiments described briefly by Scott Fahrenkrug, highlighting the factors that can influence success when attempting to precisely edit the genome of livestock species (pig and cattle) with TALENs. Next, Charlotte Brandt Sorensen (Aarhus University, Denmark) reported on the efficient genome engineering in pigs using both recombinant adeno-associated virus (rAAV) and TALENs in order to generate swine animal models of breast cancer and Type II diabetes. The session concluded with a technical lecture delivered by Colin Fox (Genentech, USA), on their approaches to systematically and efficiently genotype complex genetic alterations in transgenic animals affecting multiple alleles.

The third session was focused on the use of pigs for a variety of purposes. First, Kevin Wells (University of Missouri, USA), reported on their advances in a gene stacking project, where the use of phiC31 integrase and its corresponding target sites was evaluated, in parallel to standard homologous recombination approaches, for the efficient cointegration of multiple alleles at discrete genomic locations. The session was completed with talks from two German groups, where Nikolai Klymiuk (Ludwig-Maximilian University, Germany) and Angelika Schnieke (Technische Univ. Muenchen, Germany) shared their progress in xenotransplation and the modeling of cancer disease in pigs, respectively.

The fourth session, on the conference’s second day, started with a talk by Liangxue Lai (Guangzhou Institutes of Biomedicine and Health, China) reporting on their progress with a series of pig models of human degenerative diseases including Parkinson, Ataxia (ALS), Huntington and Alzheimer. Liangxue Lai had also participated as invited speaker at the TT2013 meeting in Guangzhou, held previously this year. Chuck Long (Texas A&M University, USA) presented work from his lab using lentiviral transgenes in cattle to knock-down the myostatin locus by RNA-interference. He also reported on a new model for muscle steatosis (marbling) in pigs. The session ended with a totally different animal system: chickens and avian primordial germ cells (PGCs), delivered by Mike McGrew (Roslin Institute, UK). Mike reported progress made in his lab to establish efficient conditions to culture chicken PGCs and his attempts to generate inducible knock-down of target genes using transposons and the TET-system.

Lake Tahoe, CA, USA
Lake Tahoe, CA, USA

The fifth session, with two talks, was entirely devoted to further evaluate risk assessment on the transgenic goat model producing lysozyme in milk, generated by Jim Murray and collaborators at UC Davis. First, Elizabeth Maga (UC Davis, USA) systematically analyzed whether there were any unintended effects associated with the mammary-specific expression of the lysozyme transgene in the host (lactating goats) and in a non-targeted organism (kid goats consuming the milk from transgenic goats). Even though they found some statistically significant differences among the many tests conducted, these were considered of no biological relevance, more due to time of expression and not due to the presence of the transgene. She concluded that there were no unintended effects as revealed in these analyses. Second, Caitlin Cooper (UC Davis, USA) shared her analysis on the effects of consumption of milk containing lactoferrin (from transgenic cows) and/or lysozyme (from transgenic goats) on the intestinal health in young pigs. Her studies concluded that lactoferrin and lysozyme exhibit both shared and unique mechanisms and highlighted the relevance of dosage in the positive effects observed in the intestinal villi architecture and the overall balance of several cells of the immunity system in the gut.

The sixth session presented two different but equally-interesting advances obtained by two agrobiotech companies. First, AgResearch’s researcher Goetz Laible (New Zealand) described their success in reducing the contents of beta-lactoglobulin (BLG) in ovine and cow milk, hence aiming to produce a less allergenic milk for eventual human consumption. They tested their strategy using RNA-interference in mice, with the help of some transgenic mice producing BLG in their milk. Finally, they generated a cow producing milk with reduced allergens. Next, Benjamin Schusser (Crystal Bioscience, Inc., USA) shared their advances towards producing therapeutic monoclonal antibodies against human proteins in chickens. In this regard, he documented the creation of the first chicken knockouts, for the IgL and IgH loci, by inserting the corresponding variable regions of human Ig loci.

The seventh session was also devoted to advances in chicken genetic engineering. Tim Doran (CSIRO, Australia) began with a description of an alternative way of genetically modifying chicken PGCs with transposon-type transgenes by direct in vivo transfection, thus avoiding the need to isolate, culture and reinsert these cells in host chicken embryos. This talk was followed by that of Mark Tizard (CSIRO, Australia), illustrating how the use of innovative RNA-interference approaches could be used for efficient trait control and disease resistance in poultry.

Lake Tahoe, CA, USA
Lake Tahoe, CA, USA

The conference’s last day started with three new large animal models for human diseases. First, Irina Polejaeva (Utah State University, USA) described her transgenic goat models that overexpress the profibrotic factor TGF-ß1 in cardiomyocytes, designed to study the relationship between cardiac fibrosis and atrial fibrillation. Next, Chris Rogers (Exemplar Genetics, USA) presented pig models for human hypercholesterolemia and atherosclerosis generated by disrupting the LDL receptor gene in the pig genome. The LDLR deficient pigs are currently being used to test new cholesterol-lowering drugs and to develop detection and treatment strategies for atherosclerosis. The session finished with a talk by Zhong Wang (University of Michigan, USA) and their new approaches to study heart development and regeneration in pigs.

The eighth session was devoted to the progress of animal products generated using biotechnology with regard to regulation and the expected path to market, once the product is investigated, validated and eventually approved by the relevant regulatory bodies. This process was described by Ronald Stotish (AquaBounty Technologies, USA), who shared the extremely long and as-yet unsuccessful attempt to obtain required FDA approval for marketing the AquAdvantage salmon. This fast-growing transgenic fish can grow to expected market size in half of the time required for non-transgenic salmon using standard aquaculture procedures. The apparent science-based regulatory process has been repeatedly interrupted by not only anti-technology groups but other groups with obvious political and economic interests conflicting with the marketing of these salmon. More than 20 years have passed since this transgenic salmon was first generated, and yet, after numerous scientific studies demonstrating that this product is as safe as non-transgenic salmon and after concluding that it does not pose a significant threat for the environment, the final approval by the FDA has not been issued. The seminar on the transgenic salmon issue was followed by a nice summary talk by Alison van Eennennaam (UC Davis, USA) where she presented how the regulation of genetically-modified animals is interpreted in different countries/continents, such as US, Europe or Australia, and the consequences these definitions have on the overall regulatory process aiming to obtain a permission to market a given transgenic animal or a product derived from them. Furthermore, she challenged the current regulatory scenario with the new gene editing tools (i.e. ZFNs, TALENs, or CRISPRs-Cas) where, in most cases, the genetic alterations leave no specific footprints and are undistinguishable from other similar genetic alleles that can be found in the nature, among the different breeds of a given species. Knowing in advance whether these precise genetic engineering processes will or will not be regulated through the current laws or whether they would require an adaption of current norms is of paramount importance for the progress of the animal biotechnology field.

The final session held two great but totally different talks. First, Derric Nimmo (Oxitec Inc., UK) described their elegant and innovative solution to efficiently down-regulate wild populations of mosquitoes (Aedes aegypti) This mosquito species survives by constantly feeding on human blood, and also serve as a vector to transmit serious diseases such as dengue or yellow fewer. He reported their approach using their RIDL strategy (Release of Insects with Dominant Lethality). The mechanism is based on a modified TET-off system where the tTA-VP16 activator is strongly expressed under several tet-op sequences unless the effector, Doxycycline (Dox), is provided in the diet. Hence, male transgenic mosquitoes can be raised in the laboratory, where the expression of the transgene is prevented with Dox, but, upon release in the wild, the lack of Dox triggers the expression of the transgene and the accumulation of the powerful transcriptional activators which cause irreversible damage to transgenic male mosquitoes, rending them sterile. Release of these sterile males and their subsequent mating with female populations is an efficient way to downsize wild mosquito populations. Approved open field tests have been already conducted in Cayman Islands, Malaysia and Brazil with success. The company is currently awaiting approval by the FDA and other equivalent agencies in order to apply their strategies in the US and other countries. This talk also illustrated the positive and rewarding effect accomplished by investing in informing people, affected populations, hospitals, governments, schools, etc… about this biotechnological approach to reduce disease-transmitting mosquitoes, which resulted in increased acceptance by the local populations. This community engagement approach appears to be the most promising and effective manner of gaining society’s acceptance for genetically-engineered animals and/or products.

Emerald Bay, Lake Tahoe, CA, USA
Emerald Bay, Lake Tahoe, CA, USA

The honor of the traditional concluding talk was given this time to Bruce Whitelaw (Roslin Institute, UK) with the challenge to envisage what the fourth decade would bring, after three decades of genetically engineered animals. After referring to the predicted needs for safe and more efficient food that this planet will need in the immediate future, Bruce divided the four decades as follows, identifying in each of them some major technological milestones: 1984-1993 (decade of the first transgenic animals produced by standard DNA pronuclear injection); 1994-2003 (decade of nuclear transfer, when Dolly was created and laid the foundation to generate many cloned and genetically-engineered mammals, using a technique currently referred as SCNT. At this point, Bruce kindly offered a tribute to the work done by Keith Campbell, instrumental in the creation of Dolly, who recently passed away); 2004-2013 (decade of a revolution in technologies including the use of lentivirus, transposons, SMGT, bird PGCs, ZFNs, TALENs and CRISPRs, and also, the decade of the first large animal models of human disease being effectively produced and tested). For the fourth decade, 2014-2023 Bruce speculated that the balance will re-equilibrate efforts and investments in both agricultural and biomedical sciences, after two decades where the genetic-engineering of animals was mostly dominated by projects and applications in biomedicine. He left us with the following thought: “The 4th decade of GE livestock is going to be good for those who work with this technology and for those – both man and animal – who benefit from it”.

Emerald Bay, Lake Tahoe, CA, USA
Emerald Bay, Lake Tahoe, CA, USA

All participants left home on August 15, after having enjoyed yet another fantastic conference put together by Jim Murray, who must be praised for his unrelenting commitment to this great meeting series, where the generation and application of non-rodent transgenic animals are discussed in depth, before, during and after the talks.
The next TARC meeting, the 10th Transgenic Animal Research Conference, will be held, at the same place, on August 9-13, 2015. We would encourage you to experience these meetings first hand, (and not through these meeting reports). Please make sure to book these dates on your agenda and not miss the next meeting by beautiful Lake Tahoe.

Lluis Montoliu & Jan Parker-Thornburg

First transgenic rabbits born in Turkey

First transgenic rabbits born in Turkey
First transgenic rabbits born in Turkey

ISTT member Gul Bakirer, from the University of Istambul, informed us about the first transgenic rabbits born in Turkey, as the result of an international research project where she has collaborated. These genetically-modified rabbits were produced by pronuclear microinjection of a DNA hyperactive transposon carrying the green-fluorescent protein reporter gene. This is a collaborative research project led by Prof. Sema Birler (Istanbul University, Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination) involving up to 13 researchers, research assistants and PhD students from two universities in Turkey (Istambul and Marmara), as well as two researchers from the Hawai’i University Manoa John A. Burns School of Medicine (Stephan Moisyadi, former ISTT member and Associate Professor at the UH Institute for Biogenesis Research [IBR] and Joel Marh, IBR Transgenic Facility Director). Emeritus Professor Ryuzo Yanagimachi, the founder of IBR, and Stefan Moisyadi travelled to Istanbul in November 2011 to set up the collaboration with the University of Istanbul and Marmara University. Stefan Moisyadi and Joel Marh returned to Istanbul this past June to further assist their collaborators in Turkey for the successful generation of these transgenic rabbits. The aim of the experiment was to show that the transposon-transgenes devised in Hawai’i could also be used in rabbits, as a first step towards the generation of additional transgenic rabbits producing proteins of interest in their milk. Two of the eight rabbits obtained in the litter (25%) were found to shine fluorescent green, and hence to be transgenic. Additional ongoing experiments, by the same Hawai’i-Turkish collaborative effort, are expected to produce similar transgenic sheep soon.

Some of the researchers from the Istambul University involved in this achievement. From left to right: Ramazan Arici (PhD student), Ayse Can (PhD student), Prof. Sema Birler (coordinator of this study) and Prof. Serhat Alkan.
Some of the researchers from the Istambul University involved in this achievement. From left to right: Ramazan Arici (PhD student), Ayse Can (PhD student), Prof. Sema Birler (coordinator of this study) and Prof. Serhat Alkan.