Remembering Laura Pozzi

Laura Pozzi, pioneer of mouse transgenesis in Italy
Laura Pozzi, pioneer of mouse transgenesis in Italy

On August 7th 2016, Laura Pozzi, a pioneer of transgenesis in Italy, passed away at the age of 80.

She was associate professor at the University “La Sapienza” in Rome and during the 80’s of the last century was among the first researchers in Italy to set up, with minimal equipments, often handmade, and with great commitment and personal sacrifice, a laboratory for the generation of transgenic mice. This facility was for a long time one of the few reference points for anyone who wanted to get a transgenic mouse in Italy, so that colleagues jokingly called her “the mother of all the Italian transgenic mice”. Over the years she trained a lot of people on transgenic technologies. On them she had a profound influence and to them she left an irreplaceable legacy of theoretical and practical knowledge that has had a strong impact on their future professional life. Many people who are now operating in the field of transgenesis in Italy, were formed at her school. She was a strict and demanding teacher, but it was so clear to us, her trainees , that her ultimate goal was to provide the best training and formation possible, that we all loved and respected her as a mother and master.

Open-minded, cultivated and great traveler, conversing with her was always pleasant and interesting. Even in private life her main purpose was to be helpful to others: she spent herself as a teacher of Italian language to foreign refugees, like a grandmother she took care of children of friends and neighbours. And all this without bragging and always with her smile and her subtle English humor that she absorbed during a long stay in Great Britain.

After retirement she always remained in touch with her former students and she followed with pleasure and interest, even though she was not a member, the ISTT site using the password of one of us. When, with advancing age, she realized that it was impossible for her to be independent as she wanted and not wanting to be a burden to anyone, with great clarity, serenity and courage she decided that her moment had arrived and left this world, recommending to friends to remember her with a smile.

Elisabetta Mattei, Laura Tatangelo and Isabella Manni

*************************

It is with gread sadness that I learnt from Elisabetta Mattei about the recent passing of Laura Pozzi. She was my first mentor in mouse transgenesis. I was privileged to attend, as the only non-Italian trainee in a small group of five students, a two-weeks intensive practical course on the generation of transgenic mice organized in Siena in 1990, at the headquarters of the pharmaceutical company Sclavo. At the time I was still finishing my PhD in Plant Molecular Genetics, using maize as my experimental model, and I was already preparing myself to begin a future postdoctoral stay in Heidelberg as a mouse geneticist. I landed in Tuscany knowing nothing about transgenic mice and, thanks to the wisdom and teaching abilities of Laura and her team of collaborators, I left Italy with the required solid and robust starting knowledge to work with mice that has travelled with me since then. I went from maize to mice and Laura played a fundamental role in my transitioning between these two experimental models. Throughout the years I remained in touch with Laura and we often commented technical breakthroughs in the field. She was also happy to see the developments and success of the ISTT. I always had her as my first reference in mouse transgenesis. She deserves to be remembered as a most influential person for the mouse transgenics community in Italy and Europe.

Lluis Montoliu

Will the novel CRISPR/Cas9 technology for the generation of genetically modified animals increase the number of animals used and lead to a shift in the species used? Statement of the ISTT’s 3Rs Committee

The CRISPR/Cas9 technology emerged only recently. Still it is already obvious, that it makes the generation of genetically modified organisms more efficient than with conventional techniques. Moreover, species that were recalcitrant to those manipulations in the past are now amenable to genome editing.

What we initially saw in the rodent, especially the mouse research community, was a rush into the technique, where many scientists wanted to employ the new technology (me too). While there were a number of reports on off-target effects, it is now clear that off-target genome alterations are rarely found in an in vivo situation (Seruggia et al., 2015; Shen et al., 2014). Moreover modified nucleases are meanwhile available with no detectable off-target effects (Kleinstiver et al. 2016).

In the most widely used model organism in biomedical research, the mouse, the number of animals needed to generate a genetically modified line remains approximately the same with CRISPR/Cas9 as compared to conventional techniques. However, the ease at which new mutations are generated with the new system might lead to increased numbers of novel lines being produced (Williams et al., 2016). Moreover, numbers are also increasing for other species, especially zebrafish and rats (Auer and Del Bene, 2014; Hwang et al., 2013; Wang et al., 2015). As with all novel genetically modified lines, after their generation CRISPR/Cas9 generated animals need to be bred for experiments and are kept on the shelf for considerable time. Thereby, the increase in the number of lines generated will translate into larger numbers of additional animals bred.

At this time, conventional techniques still have their justification in the lab since precise modification of DNA via homologous recombination, especially with large constructs, is not as robust as necessary via CRISPR/Cas9. However, one has to take into account that this is a very recent technology, whilst work to improve the generation of genetically modified mice via homologous recombination in embryonic stem cells has been ongoing for more than 25 years. We therefore may see extensive improvements over the coming years once we gain a better understanding of the underlying mechanisms. With improvements in targeting efficiencies there could be opportunities for further reductions in the number of mice used for the generation of the animal model to be studied: In many cases mutations are still introduced into ES cells that are used for the generation of live mice. Even though culture conditions have been improved over the years, cell clones show a fairly high degree of aneuploidy so that multiple clones have to be injected to generate chimeric offspring that transmit the mutation to their progeny. Due to the non-chimeric nature of CRISPR/Cas9, mutated individuals will in most cases transmit. Therefore an improved ratio of mutant to wildtype offspring could directly reduce the number of animals produced. However, the degree of mosaicism could counteract this reduction. CRISPR/Cas9 technology has the potential that several mutations can be introduced on different alleles at the same time, even homozygously, once the mosaicism issue is solved. Alternatively, complex mutations can be generated by adding additional mutations in pronuclear stage embryos of mutant backgrounds. With a sufficient increase in efficiency, this promise does appear realistic (Williams et al., 2016). This would mean that instead of producing a plethora of unwanted mice with unwanted genotypes in the process of breeding compound mutants, one could proceed right to the desired combination of mutated alleles.

We expect to see an increase in the number of species that will undergo complex targeted genetic manipulation. Still, a significant increase in laboratory animal numbers is not expected. The research infrastructure has been developed and is still being developed for large scale mouse breeding. The breeding of rats takes up much more space and only a few centers have the infrastructure for larger animals. The applicability of the technology to non- human primates means they are also more often nowadays used for transgenic animal research, especially in countries where such research is not strictly regulated. This is another issue of public concern and will require an ethical evaluation process beyond the scope of the 3R approach. CRISPR/Cas9 technology is widely used to genetically manipulate zebrafish. An increase in zebrafish numbers similar to what is foreseen in the mouse can be expected. On the other hand there will be projects where the zebrafish is now amenable to the introduction of complex targeted genetic manipulation and can therefore replace the mouse as a model – a clear refinement in accordance with the 3Rs.

In summary, the ISTT’s 3Rs Committee acknowledges that the advent of the CRISPR/Cas9 technology has the potential to significantly increase the number of animals used and range of species genetically modified. However the committee believes that existing limits on space and other associated resources will inhibit the realization of this at the current time. In the long run, after the technology has been developed further and improved, the Committee is hopeful that opportunities for reducing the number of animals that are bred but not used will be realized.

Boris Jerchow, Chair
On behalf of the ISTT’s 3Rs Committee Berlin in March 2016

Auer, T.O., and Del Bene, F. (2014). CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods 69, 142-150.
Hwang, W.Y., Fu, Y., Reyon, D., Maeder, M.L., Tsai, S.Q., Sander, J.D., Peterson, R.T., Yeh, J.R., and Joung, J.K. (2013). Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31, 227-229.

Kleinstiver, B.P., Pattanayak, V., Prew, M.S., Tsai, S.Q., Nguyen, N.T., Zheng, Z., and Joung, J.K. (2016). High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off- target effects. Nature, 529, 490-495.
Seruggia, D., Fernandez, A., Cantero, M., Pelczar, P., and Montoliu, L. (2015). Functional validation of mouse tyrosinase non-coding regulatory DNA elements by CRISPR-Cas9- mediated mutagenesis. Nucleic acids research 43, 4855-4867.

Shen, B., Zhang, W., Zhang, J., Zhou, J., Wang, J., Chen, L., Wang, L., Hodgkins, A., Iyer, V., Huang, X., et al. (2014). Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature methods 11, 399-402.

Wang, L., Shao, Y., Guan, Y., Li, L., Wu, L., Chen, F., Liu, M., Chen, H., Ma, Y., Ma, X., et al. (2015). Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos. Scientific reports 5, 17517. Williams, A., Henao-Mejia, J., and Flavell, R.A. (2016). Editing the Mouse Genome Using the CRISPR-Cas9 System. Cold Spring Harbor protocols 2016, pdb top087536

Ultra-superovulation in C57BL/6 mice: 100 oocytes/female

New achievement in Reproductive Biology by Toru Takeo & Naomi Nakagata (CARD, University of Kumamoto, Japan). Ultra-superovulation of C57BL/6 mice: 100 oocytes/female obtained priming the animals with equine chorionic gonadotropin (eCG) and inhibin antiserum (IAS)
New achievement in Reproductive Biology by Toru Takeo & Naomi Nakagata (CARD, University of Kumamoto, Japan). Ultra-superovulation of C57BL/6 mice: 100 oocytes/female obtained priming the animals with equine chorionic gonadotropin (eCG) and inhibin antiserum (IAS)

Last September, in Buffalo (USA), at the CARD-RPCI Mouse Sperm and Embryo Cryopreservation course organized by Naomi Nakagata, Aimee Stablewski and Jan Parker-Thornburg, Naomi Nakagata himself presented the preliminary results of an amazing achievement in Reproductive Biology they had accomplished at the University of Kumamoto (Japan), namely: the obtention of more than 100 oocytes per C57BL/6 female after devising a new protocol for superovulation, a method they introduced as ULTRA-superovulation. Now, these totally unexpected results see the light in the form of a scientific manuscript, published yesterday in the PLOS ONE journal:

Toru Takeo & Naomi Nakagata (2015) Superovulation Using the Combined Administration of Inhibin Antiserum and Equine Chorionic Gonadotropin Increases the Number of Ovulated Oocytes in C57BL/6 Female Mice. PLOS ONE, Published: May 29, 2015DOI: 10.1371/journal.pone.0128330

In brief, in this publication, Toru Takeo and Naomi Nakagata describe their superovulation results using young (4-weeks old) C57BL/6 female after envisaging a new priming protocol. The combined used of equine chorionic gonadotropin (eCG) and inhibin antiserum (IAS), in a protocol they call IASe treatment, significantly increased the number of oocytes obtained per C57BL/6 females. On average, more than 100 oocytes/female were obtained, about 3-4 times the number of oocytes regularly obtained by classical superovulation protocols. Thereafter, the authors tested the quality of these oocytes and used them for IVF, obtaining high fertilization rates (~90%), comparable to the high values regularly obtained with the new CARD methods these authors also devised recently, which have boosted the field of cryopreservation of mutant mice. Furthermore, the authors verified that the number of pups obtained after transferring all these embryos, obtained from IASe-derived oocytes and IVF into recipients, was also 2-3 times higher.

The CARD YouTube channel has also released a movie showing ampullas of oviducts from IASe-treated C57BL/6 females literally full of oocytes.

The IAS reagent used by Takeo & Nakagata is not yet commercially available. In the paper, the authors produced the IAS by themselves and titrated the product until finding the optimal dose required for maximum output. On the contrary, eCG is commercially available and is commonly used in all mouse reproductive biology and transgenic labs to promote follicle growth. Subsequent experiments will be needed to explore the validity of these results in other mouse strains and species. In addition, a commercial reliable and validated source of IAS will greatly facilitate the dissemination of this new ultra-superovulation method among the scientific community. It is also remarkable to note that the application of the IASe treatment will logically reduce the number of superovulated donor females required to obtain oocytes for cryopreservation/IVF purposes, as nicely demonstrated in this first publication, and, likely, for other aims (i.e. microinjection of DNA or RNA/genome editors to produce genetically altered mice).

Congratulations once again to Toru Takeo and Naomi Nakagata for these impressing results and for their new spectacular achievement in mouse reproductive biology!.

Mouse Genetics. Methods and Protocols (2014)

Mouse Genetics. Methods and Protocols (2014)
Mouse Genetics. Methods and Protocols (2014)

This is yet another interesting book in our field that has been published this year, 2014. This manual, entitled “Mouse Genetics. Methods and Protocols“, edited by Shree Ram Singh and Vinzenzo Coppola, in association with the Publisher, Humana Press/Springer, contains a collection of useful protocols covering most of the methods that can be currently applied for the genetic modification of the mouse genome. According to its presentation at the Springer web page, this book “provides selected mouse genetic techniques and their application in modeling varieties of human diseases. The chapters are mainly focused on the generation of different transgenic mice to accomplish the manipulation of genes of interest, tracing cell lineages, and modeling human diseases. (…) Each chapter contains a brief introduction, a list of necessary materials, systematic, readily reproducible methods, and a notes section, which shares tips on troubleshooting in order to avoid known pitfalls.

The table of contents of this book illustrates the variety of highly sophysticated methods, beyond standard techniques, that are discussed here in detail, around mouse genetics, including: pronuclear injection-based targeted transgenesis through Cre-loxP specific recombination, the use of recombinase-mediated cassette exchange (RMCE) strategies, several approaches for preparing and analyzing conditional mutant alleles using tamoxifen-dependent Cre recombinases, the use of ICSI for the generation of transgenic mice, the use of BACs, mosaic analysis with double markers (MADM) in mice, transposon-mediated transgenesis, overexpression of microRNAs using Rosa26-mediated recombination, the isolation of various somatic and pluripotent cells, the generation of transgenic mice through spermatogonial stem cells in vivo, and, several illustrative examples of how different mouse engineered animal models are best suited to study a variety of human diseases. Hence, this book is also complementary to other recently published manuals, since it contains a careful detailed description of new methods that are not been covered in other similar titles in the field.

The editors of this book, Shree Ram Singh and Vinzenzo Coppola, have counted with the generous expertise shared and provided by a very large list of co-authors, including some ISTT members: Masato Ohtsuka, Kazuhito Sakamoto, Channabasavaiah B. Gurumurthy, Kay-Uwe Wagner, Petra Kraus, V. Sivakamasundari, Xing Xing, Thomas Lufkin, Anton J.M. Roebroek, Bart Van Gool, Kun-Hsiung Lee, Susanne Feil, Jana Krauss, Martin Thunemann, Robert Feil, Pedro N. Moreira, Lluis Montoliu, Jane Beil, Thorsten Buch, Sheng Ding, Tian Xu, Xiaohui Wu, Hui Zong, Claudia Piovan, Foued Amari, Francesca Lovat, Qun Chen, Olga Simmons, Esther M. Bolanis, Jian Wang, Simon J. Conway, Kanika Jain, Paul J. Verma, Jun Liu, Pollyanna Agnes Goh, Michael D. Williams, Wilson Wong, Amanda Rixon, Sarang N. Satoor, Anandwardhan A. Hardikar, Mugdha V. Joglekar, Andrei M. Vacaru, Joseph Vitale, Johnathan Nieves, Margaret H. Baron, Kristbjorn Orri Gudmundsson, Kevin Oakley, Yufen Han, Yang Du, Lalit Sehgal, Abul Usmani, Sorab N. Dalal, Subeer S. Majumdar, Spencer W. Luebben, Naoko Shima, Tsuyoshi Kawabata, Robert M. Hoffman, Viive M. Howell, Emily K. Colvin, Vishalakshi Chavali, Shyam Sundar Nandi, Paras Kumar Mishra, Julia Lorenz, Susanne Grässel, Ganesan Ramesh, Punithavathi Ranganathan, Santhakumar Manicassamy, Indumathi Manoharan, Deepak P. Patil, Holly D. Kristensen and Yogesh Shouche.

This new book will be added to the collection of Springer books published on animal transgenesis and animal genetics for which ISTT members are entitled to a 33% discount, as one of the many benefits associated with the ISTT membership.

 

 

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.

Manipulating the Mouse Embryo. A Laboratory Manual (4th edition, 2014)

Manipulating the Mouse Embryo. A Laboratory Manual (4th edition, 2014)
Manipulating the Mouse Embryo. A Laboratory Manual (4th edition, 2014)

A bit more than 10 years later, from the last edition (3rd, 2003) published, the classical manual, the “Bible” in our field has been renewed, updated, upgraded and recently published in its fourth edition. The new “Manipulating the Mouse Embryo. A Laboratory Manual. 4th edition, 2014” has just been released, published by Cold Spring Harbor Laboratory Press and edited by Richard Behringer, Marina Gertsenstein, Kristina Vintersten Nagy and Andras Nagy. The editors, the same team that produced the 3rd edition of this useful manual, should be praised once again for a brilliant work done, accommodating the latest techniques in mouse embryo manipulation, while not forgetting the traditional procedures that must be learnt properly by anyone joining this field of animal transgenesis for the first time. The editors counted with the help of many additional collaborators, who provided materials and protocols, including many ISTT members, as Marina and Andras are, such as: Wojtek Auerbach, Ralph Brinster, Jorge Cabezas, Tracy Caroll, Lluis Montoliu, Naomi Nakagata, Jan Parker-Thornburg, Shirley Pease and Thomas Saunders, just to name a few of the many colleagues that shared their expertise and contributed to this 4th edition of the CSHLP manual.

This fourth edition is dedicated to Anne McLaren (1927-2007), one of the brave and gifted pioneer in mouse genetics and embriology, and whom do we all owe a number of standard procedures that we routinely apply in a transgenic laboratory, such as mouse embryo culturing, pseudopregnancy and embryo transfer.

The structure of this 4th edition manual follows that of the 3rd edition, in the sense that chapters and methods are grouped logically and functionally, from a very good summary on mouse genetics and mouse embryo development, to mouse colony management, and followed by a variety of main sections dealing with in vitro and in vivo work, surgical procedures, the production of transgenic mice by pronuclear microinjection, derivation of ES cell lines, using ES cells to generate germline-transmitting chimeras, genotyping protocols, parthenogenesis and nuclear transfer, assisted reproduction techniques, cryopreservation methods, techniques for visualizing gene products and finishing by a chapter devoted to setting up a micromanipulation laboratory and the usual and convenient appendix with receipts for the common buffers and solutions.

Besides this excellent updated version of the CSHLP Mouse Manual, there are several books published on this subject, including the ISTT manual (Springer, 2011) edited by Shirley Pease and Thomas Saunders, and all are worth reading and having them around for consultation, since each one brings their own contribution, their view and their solutions for the adequate learning of how to best manipulate animal embryos, and not all methods and protocols are covered in a single book, hence they are largely complementary. I will be soon reporting in this ISTT blog about additional books recently published in this field.

 

Highlights of the TT2014 meeting in Edinburgh: a conference you can’t miss!

The TT2014 meeting in Edinburgh (6-8 October 2014)
The TT2014 meeting in Edinburgh (6-8 October 2014)

This year’s ISTT main activity is the 12th Transgenic Technology conference, the TT2014 meeting, which will be held in Edinburgh, Scotland, UK, on 6-8 October 2014, followed by a 2-day hands-on workshop on basic zebrafish transgenesis techniques. The ISTT promotes the TT meetings every 18 months, these being the most important activity of our Society. This year, the local Organizers and advisory committees, commanded by Douglas Strathdee, need to be praised for preparing a most appealing and interesting program, addressing hot topics, current and most up-to-date issues actively discussed nowadays by the transgenic animal community. Talks that will be presented by the most active and prestigious scientists in our field.

Why you shouldn’t miss the TT2014 meeting?

  • If you are interested in the new transgenic methods associated to nucleases (ZFNs, TALENs and CRISPRs-Cas9) there will be plenty of interesting talks where these new fantastic tools will be presented and discussed, directly by the key players in this rapidly-evolving field, including: Rudolf Jaenisch, William Skarnes, Angelika Schnieke, Kai Schönig, Ignacion Anegon, Pawel Pelczar, Francis Stewart, Keith Joung and Feng Zhang. And, most likely, these techniques will be referred and cited in many additional talks too, including the round table discussion about the future of transgenic core facilities, chaired by James Bussell.
  • If you are interested in ES cell biology and in innovative uses of ES cells and associated technologies there will be unique talks delivered by Jos Jonkers, Austin Smith, Ian Chambers, Janet Rossant and Alex Joyner
  • If you are interested in phenotyping your mouse animal models there will be fantastic talks delivered by Jacqueline White, Stephen Murray, David Adams, Daniel Murphy, Anna-Katerina Hadjatonakis and Vasilis Ntziachristos
  • If you are interested in non-rodent, large mammals and birds, animal models there will be great talks by James Murray, Angelika Schnieke, Mike McGrew and Adrian Shermann
  • If you are interested in rats there will be compelling talks by Kai Schönig and Ignacio Anegon
  • If you are interested in zebrafish animal models there will be fascinating talks by Stephen Ekker, Koichi Kawakami, Keith Joung and Elizabeth Patton
  • If you are interested in animal welfare and 3Rs, in the best use of our laboratory animals, there will be captivating talks by Peter Hohenstein, Sara Wells and Jan-Bas Prins.

Therefore, there will be really engaging talks interesting to everyone in our field. This is why you shouldn’t miss this great and unique opportunity!.

Register now for the TT2014 meeting. Submission of abstracts will be accepted up to June 30. Early-Bird registration at reduced fees will be promoted up to July 31. ISTT members are entitled to reduced fee registration.

See you all in Edinburgh in October!

 

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.

More than 27,000 messages on animal transgenesis available to ISTT members through ISTT_list and tg-l archives

More than 27,000 messages on animal transgenesis available through ISTT_list and tg-l archives
More than 27,000 messages on animal transgenesis available through ISTT_list and tg-l archives

One of the most important assets of the International Society for Transgenic Technologies (ISTT), is the amount of information on animal transgenesis accummulated through the archives of the ISTT_list and tg-l email lists. Currently, more than 27,000 messages are fully available to ISTT members, conveniently organized in searchable and dynamic archives. The traditional transgenic-list (tg-l), operative since 1996 and offered from the ISTT web server since the end of 2011, has distributed over 22,000 messages since then, whereas the ISTT_list, associated and born with our Society in 2006, has disseminated some 5,000 messages, discussing both lists on almost each and every topic, issue or situation related directly or indirectly with animal transgenesis. All this endless information resource is fully available to ISTT members, through powerful search engines. Non-ISTT members subscribing to tg-l have access only to the most recent messages distributed through the tg-l, using the simple search engine, which allows simple searches and outputs the 50 most recent messages discussed on the subject of interest. In contrast, ISTT members have access to more sophysticated searching engines and the output always contains all messages archived on the matter investigated.

Obtaining granted access to these rich sources of information is very easy and cheap. Simply apply for ISTT membership! Submit now your application to become a member of the ISTT and you will get immediate and full access to all these messages.

Workshop report: animals bred, but not used in experiments

Workshop:  “Animals bred, but not used in experiments”, October 18-20, 2013, Hotel Duin & Kruidberg, Santpoort, the Netherlands (Picture kindly provided by Fernando Benavides)
Workshop: “Animals bred, but not used in experiments”, October 18-20, 2013, Hotel Duin & Kruidberg, Santpoort, the Netherlands (Picture kindly provided by Fernando Benavides)

Workshop: “Animals bred, but not used in experiments”, October 18-20, 2013, Hotel Duin & Kruidberg, Santpoort, the Netherlands.

Experiments in biomedical science use large numbers of laboratory animals. It is a fact that to provide these animals, regularly more animals are bred than are finally used in the experiments planned. The Ministry of Economic Affairs as the competent body of the Netherlands had asked Prof. Coenraad Hendriksen and Dr. Jan-Bas Prins to organize a workshop to identify the reasons for the breeding of surplus animals and to devise recommendations as to how the number of animals that are bred but not used can be reduced to a minimum.

A number of experts from different fields of laboratory animal science were invited for a two day workshop to the Hotel Duin & Kruidberg in Santpoort, a town close to Amsterdam, to discuss these issues and to develop a paper for the Dutch authorities. Obviously, many of the laboratory animals bred are genetically altered (GA) animals. Moreover, techniques to cryopreserve GA animal lines could be a means to reduce the number of animals that are bred. The invitation was therefore extended to the ISTT to send a representative to take part in this workshop.

Here, I will give a short summary of the topics that have been discussed and of the outcomes. However, I refer you to the final report of the workshop, parts of which have been developed within individual small workgroups and will be put together into a final document by the kind efforts of Coenraad and Jan-Bas. I will inform you immediately upon the publication of this report.

A topic central to the discussion was the identification of reasons for the production of animals that are then not used in experiments. A major reason for this is the production of unwanted sexes and unwanted genotypes. The participants agreed that good planning can considerably reduce the number of surplus animals. At the same time, resources can be saved and either used for additional experiments or for cost reduction. However, breeding schemes with multiple alleles, as well as the organization of a facility, can be complex. A strong need for counseling as well as education of users of laboratory animals was identified, to make them competent to plan accordingly. The centralization of the breeding colonies under the responsibility of the facility management was discussed as a possibility to streamline breeding strategies. On the other hand, for the time being, this does not seem to be feasible for very many facilities. Local Animal Welfare Committees should evaluate local SOPs and develop a catalogue of best practices to help keep surplus animals to a minimum. GA animal lines should be cryopreserved immediately after their creation when there is no need to breed extra animals for this purpose and when animals from test rederivations can be used for experiments or for the breeding colony. Thereby, the lines are protected from disaster and from genetic drift at the same time, live mice can be terminated at any time, and the lines can be easily shipped to collaborators. Lines should be made available to collaborators as early as possibly to avoid generating the same line at different places. In case expertise for cryopreservation is lacking, lines can be donated to repositories like EMMA where they are cryopreserved free of charge. Investigators should always consider sharing lines with the scientific community through such repositories.

A second important topic discussed during the workshop was the use of new technologies for the generation of GA animals as well as for their experimental analysis. New lines should be directly generated on the desired background. In case backcrossing is needed, speed congenic strategies should be used to reduce the number of animals needed during that process. Technologies utilizing the targeting of nucleases to the locus of interest (ZFNs, TALENs, CRISPER/Cas9) promise to eventually allow the generation of GA lines with reduced numbers of animals directly on the desired background. Complex strategies for the generation of customized animals for specific experiments were presented. It was agreed that these should be freely available. However, individual scientists and institutes should evaluate whether it is worth adopting a new and complicated technique. Since the process of setting up complex protocols may well lead to the use of high numbers of animals, investigators should consider collaborating with colleagues who perform similar experiments at large scales.

Ethical considerations let us come to the understanding that there is an intrinsic value of life. We found that it is for this reason that it is morally wrong to kill more animals than absolutely necessary. Biomedical science is tasked with producing answers to pressing questions on the molecular functions of life and disease and finding new cures. It was pointed out that the principles of the 3R’s have to be respected at all times, but a number of animal experiments are indispensable. In this context, it is unavoidable to breed animals that are not used for these experiments, but it is important to ensure that their numbers are kept to a minimum.

Boris Jerchow
Member of ISTT’s Executive Council
October 23, 2013

List of participants and affiliations, excluding those who were unable to send permission for disclosure:

van der Broek, Frank, NVWA, The Netherlands; Aleström, Peter, The Norwegian Zebrafish Platform, Norway; Benavides, Fernando, University of Texas, USA*; Bussell, James, Wellcom Trust Sanger Institute, UK*; Chrobot, Nichola, MRC Harwell, UK; van Es, Johan, Hubrecht University, The Netherlands; Fentener van Vlissingen, Martje, Erasmus MC, The Netherlands; Hendriksen, Coenraad, InTraVacc, The Netherlands; Hohenstein, Peter, Roslin Intitute, UK*; Krimpenfort, Paul, NKI, The Netherlands; Morton, David, UK; Prins, Jan-Bas, LUMC, The Netherlands; Raspa, Marcello, EMMA, Italy*; Tramper, Ronno, Consultant, The Netherlands; van der Valk, Jan, NKCA; Wilbertz, Johannes, Karolinska Institutet, Sweden*; Ohl, Frauke, Utrecht University, The Netherlands; Pool, Chris, KNAW, The Netherlands; Witler, Lars, Max-Planck Institute Mol. Gen., Berlin, Germany*.

* ISTT members

Workshop: “Animals bred, but not used in experiments”, October 18-20, 2013, Hotel Duin & Kruidberg, Santpoort, the Netherlands (Picture kindly provided by Fernando Benavides)
Workshop: “Animals bred, but not used in experiments”, October 18-20, 2013, Hotel Duin & Kruidberg, Santpoort, the Netherlands (Picture kindly provided by Fernando Benavides)