Transgenic Animal Technology. A Laboratory Handbook (3rd edition, 2014)

Transgenic Animal Technology. A Laboratory Handbook (3rd edition, 2014)
Transgenic Animal Technology. A Laboratory Handbook (3rd edition, 2014)

Twenty years after the publication of the first edition and twelve years after the release of the second edition of this book, Carl A. Pinkert (Auburn University, College of Veterinary Medicine, Auburn, AL, USA) in association with Elsevier, releases now the third edition of his famous transgenic manual: “Transgenic Animal Technology. A Laboratory Handbook. 3rd edition, 2014“. As it will be familiar to readers of the two previous editions of this useful and unique handbook, this is not only a manual to understand how to make a transgenic mouse. This handbook looks beyond mice and it contains protocols to prepare a wide variety of genetically-modified animals, including: rats, rabbits, poultry, fish, pigs, ruminants and non-human primates. In addition, this compilation of helpful methods includes a number of chapters devoted to understand and improve all steps of transgenesis, from vector design, analysis of transgene integration and the evaluation of transgene expression. Finally, the book also includes cryopreservation methods, an embryo culture section, a review of standard nomenclature and a selection of databases and internet resources currently available in the field.

This handbook is a worth addition to any library, laboratory or transgenic facility, complementary to other available manuals on the subject, but unique in the sense that it exquisitely illustrates current transgenic methods that can be applied to a wide variety of animal species, beyond mice.

Carl A. Pinkert has been helped in his outstanding Editorial task by a large group of co-authors, experts in their subjects, including some ISTT members: Satoshi Akagi, Anna V. Anagnostopoulos, Benjamin P. Beaton, Cory F. Brayton, Steve Brown, Anthony W.S. Chan, Tom Doetschman, Rex A. Dunham, David A. Dunn, Janan T. Eppig, Almudena Fernandez, Tatiana Flisikowska, Vasiliy Galat, Robert A. Godke, Philip Iannaccone, Michael H. Irwin, Larry W. Johnson, Yoko Kato, Teoan Kim, Alexander Kind, Bon Chul Koo, Mo Sun Kwon, Daniel J. Ledbetter, Michael J. Martin, Kazutsugu Matsukawa, Colin McKerlie, Lluis Montoliu, Paul E. Mozdziak, Akira Onishi, Paul A. Overbeek, James N. Petitte, L. Philip Sanford, Jorge A. Piedrahita, Wendy K. Pogozelski, H. Greg Polites, Edmund B. Rucker III, Marina Sansinena, Angelika Schnieke, Kumiko Takeda, James A. Thomson, Ian A. Trounce, Yukio Tsunoda, Cristina Vicente-Garcia, Kevin D. Wells, Richard N. Winn and Curtis R. Youngs.

SALAAM: Sharing Advances on Large Animal Models

SALAAM: Sharing Advances on Large Animal Models
SALAAM: Sharing Advances on Large Animal Models

The EU-COST action SALAAM (Sharing Advances on Large Animal Models) was launched yesterday in Brussels, at a kick-off meeting attended by most of its members. This 4-year EU-COST action is currently formed by 17 countries and more than 44 participants, including many experts in the fields of animal genetics, physiology, transgenesis, bioethics, welfare and animal science, with a focus on large (i.e. non-rodent) animal models. This EU-COST action is chaired by Prof Eckhard Wolf (Germany) and vice-chaired by Dr. Pascale Chavatte-Palmer (France) and it includes various ISTT members such as Bruce Whitelaw (UK), Zsuzsanna Bosze (Hungary), András Dinnyes (Hungary), Cesare Galli (Italy) and Lluis Montoliu (Spain). In addition, another participant in this EU-COST action, Angelika Schnieke (Germany) is one of the invited speakers at the forthcoming 12th Transgenic Technology (TT2014) meeting to be held in Edinburgh (Scotland, UK).

EU-COST (European Cooperation in Science and Technology) is one of the oldest European initiatives in Science, an intergovernmental framework for European Cooperation in Science and Technology, allowing the coordination of nationally-funded research on a European level. SALAAM EU-COST action, as its acronym indicates, aims to sharing advances in genetic engineering and phenotyping of non-rodent mammals to develop predictive animal models for translational medicine. While recognizing the value of small and most popular animal models (mouse, rat, zebrafish, Drosophila, C. elegans, …) and its powerful genetics for increasing our knowledge on complex biological systems and for proof-of-concept-type experiments, this EU-COST action SALAAM focuses on large (i.e. non-rodent) mammalian models, since these may bridge the gap between proof-of-concept studies and more effective clinical trials, leading to better translational animal models for the study of human diseases. The research projects undertaken using rodent and non-rodent animal models should not be perceived as competition or opposed initiatives, rather as complementary studies, where each animal species is selected according to its particular value and expected benefits for the ultimate goal, that is, our understanding on the function of the mammalian (i.e. human) genome and the eventual development of effective treatments for many human diseases. During the course of this EU-COST action several conferences and training workshops will be organized, open to anyone interested in the field, to discuss about (1) new technologies (including the application of genome editing nucleases, i.e. CRISPR-Cas, for the generation of improved genetically altered animal models); (2) defining best animal models for specific phenotyping studies; (3) creation of databases for sharing information on animal models creates, tissues available and protocols; and (4) animal welfare, bioethics and communication to the public. All these conferences and training courses will be adequately advertised through the ISTT web site.

At the International Society for Transgenic Technologies (ISTT) we care about the generation and the analysis of “all” genetically altered animals, not only focused in the classical rodent models, but also including the work done with other species, with large animal models, in livestock. In this regard, the ISTT has been traditionally supporting conferences on non-rodent transgenic animals, organized in Tahoe by ISTT Member Jim Murray (UC Davis, USA) and has promoted a web page within the ISTT web site where most of the advances on livestock and other non-rodent genetically modified animal resources are shared. At the next 12th Transgenic Technology (TT2014) meeting, which will be held in Edinburgh on 6-8 October 2014, the Conference Organizers (Douglas Strathdee-Chair, Peter Hohenstein and Bruce Whitelaw) have scheduled a session on animal biotechnology, where the recent work accomplished using large animal models will be discussed. In addition, immediately following the TT2014 meeting, a hands-on workshop on zebrafish transgenesis methods will be offered to interested participants.

Livestock and other non-rodent genetically modified animal resources available from the ISTT web page

Livestock and other non-rodent genetically modified animal resources available from the ISTT web page
Livestock and other non-rodent genetically modified animal resources available from the ISTT web page

There is life beyond mice and rats, there are many additional interesting and useful genetically-modified animal models beyond those made using rodents. Rodents are great animal models for genetic/genomic analyses and for the first preliminary experimental tests. However, larger mammals are more suitable to study most human diseases and to develop therapies and treatments. Likewise, other vertebrates, such as chicken and fish, are also very interesting and useful to develop applications in animal biotechnology. At the International Society for Transgenic Technologies (ISTT) we have an increasing population of members working with all these other non-rodent genetically modified animals. The reference meeting in this area is the Transgenic Animal Research Conference (TARC), organized by ISTT Member James Murray (UC Davis, CA, USA) every two years, in August. The ISTT has had the pleasure to co-sponsor the last three editions of this conference series, in 2009, 2011 and 2013. In addition, in response to an increased interest by new ISTT members, a session devoted to non-rodent transgenic animals has been regularly scheduled in the last TT meetings (i.e. TT2011 and TT2013).

Now, from the ISTT web site, we would like to contribute disseminating and informing about all these other non-rodent transgenic animal models by launching a new web page with a collection of available resources for livestock and other non-rodent genetically modified animals. This page contains links to several academic and private institutions working with non-rodent transgenic animals. The list is not exhaustive and will be progressively updated and expanded with your suggestions and recommendations. Hence, if you are working in this field and your web page is not yet included in this web page, please contact us at webmaster@transtechsociety.org and we will correct, modify or add your suggested information. Thanks in advance for your expected collaboration!.

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

ISTT co-sponsors the 2009 Transgenic Animal Research Conference, hosted by UC Davis

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

The International Society for Transgenic Technologies (ISTT) has approved to co-sponsor the 2009 Transgenic Animal Research Conference, hosted by UC Davis (Animal Science Department), and organized by James D. Murray at the Granlibakken Conference Center (Lake Tahoe, CA, USA) on August 17-22, 2009.

This is the seventh international meeting hosted by UC Davis to bring together representatives from the leading laboratories worldwide doing cutting edge work on transgenic research in non-murine animals, including livestock, fish and poultry species.

Granlibakken Conference Center, Tahoe City, California, USA
Granlibakken Conference Center, Tahoe City, California, USA

The Transgenic Animal Research Conference VII will take place at Granlibakken Conference Center, Tahoe City, on the north shore of Lake Tahoe, California. The conference is hosted by the UC Davis Department of Animal Science. Granlibakken is located two hours from Davis, and 55 minutes from the Reno Airport.

The list of topics to be covered and invited speakers to this conference includes sessions on Technology Development, Animal Health and Safety, Antibody Production, Medical Models and applications.

Registration and detailed information can be obtained directly from the Conference WEB site.