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

TT2011 program and abstracts published in Transgenic Research

TT2011 Programs and Abstracts published in Transgenic Research
TT2011 Programs and Abstracts published in Transgenic Research

The full scientific program and abstracts that will be presented at the forthcoming 10th Transgenic Technology (TT2011) meeting have been published in the latest issue of the scientific journal Transgenic Research (2011) 20(5):1139-89, associated to the International Society for Transgenic Technologies (ISTT).

ISTT Members are entitled to access the full contents of the scientific journal Transgenic Research, associated to ISTT, through the members-only of the ISTT web site.

The TT2011 meeting will be held at the TradeWinds Island Grand Resort, in St Pete Beach, Florida, USA, on October 24-26, with the welcome get-together pre-conference dinner taking place on Sunday October 23. Over 400 delegates coming from 34 countries, all over the world, from US to Europe, from Asia to Oceania and South America too, will gather in Florida to present, share and discuss the latest developments in Transgenic Technologies. This will be a very special TT meeting, our 10th TT meeting of this series, and we will celebrate in Florida the 10th anniversary with the publication of a booklet with contribiutions by several members of our transgenic field commemorating this event.

EFSA launches public consultation on draft guidance for the risk assessment of food and feed derived from GM animals and related animal health and welfare aspects

European Food Safety Authority
European Food Safety Authority

In its guidance document, the European Food Safety Authority (EFSA) outlines specific data requirements and the methodology to be followed for risk assessment should applications for food and feed derived from GM animals be submitted for market authorisation in the European Union (EU). The risk assessment approach compares GM animals and derived food and feed with their respective conventional counterparts integrating both food and feed safety as well as animal health and welfare aspects. All stakeholders and interested parties are invited to provide their comments through an online public consultation that runs until 30 September 2011.

8th UC Davis Transgenic Animal Research Conference

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

Once again, and correspondig this year to its 8th edition, the biennial UC Davis Transgenic Animal Research Conference, organized by Prof. James Murray, UC Davis and ISTT Member, will be held at the beautiful location of Lake Tahoe, in Northern California, starting next Sunday, August 7, until August 10. Once again, the International Society for Transgenic Technologies (ISTT) is pleased to co-sponsor and promote this event.

This will be the eight 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. The upcoming conference will again focus on state-of-the-art science in the field of transgenic research. Presentations will address cutting-edge methodology, technical improvements, and current progress towards producing transgenic animals for biomedical and agricultural applications. The intent of these conferences is to bring together scientists to discuss progress, problems, and potential application of transgenic technology for animal applications.

The list of accepted speakers and topics include:

  • Goetz Laible, New Zealand, Opening talk
  • Daniel Salamone, Argentina, methods to increase efficiency of transgenesis
  • Scott Fahrenkrug, Minnesota, USA, transposon for making GE pigs
  • Lluis Montoliu, Spain, Boundary elements
  • Mark Tizard, Australia, miRNA tgs to mature immunity in chick embryos
  • Michael Roberts (Bhanu Telugu) Missouri, USA, reprogramming msenchymal stem cells
  • Elizabeth Maga, Davis, USA, In vivo bacteriacidal effects of lysozyme milk
  • Mike McGrew, Roslin, UK, germline transmission of TG chickens from PGCs
  • Rob Etches, Crystal Biosciences, interspecies use of PGC cells in birds
  • Jacob Bentzon, Denmark, hypercholesterolemic minipigs
  • Björn Petersen, Germany, ZFN pigs
  • Zsuzsanna Bosze, Hungary, transgenic rabbit systems
  • Cesare Galli, Italy, European xenotransplantation pig project
  • Charlotte Sorenson, Denmark, HR in nuclear donor cells for SCNT cloning in pigs
  • Darek Moreau, Canada, GXE interaction of GH Atlantic Salmon
  • Kevin Wells, Missouri, USA, targeting-dependent selection
  • Mingjun Lui, China, Lentivirus vectors in sheep
  • Xavier Lauth, AquaBounty, USA, GE sterile fish
  • Roland Buelow, OMT, USA, antibodies from TG rats
  • David Stolz, Iowa, USA, cystic fibrosis pigs
  • Eddie Sullivan, Hematech, USA, Increasing Transgenic Protein production when using transgenic animals as bioreactors
  • Helen Sang, Roslin, UK, Flu resistant chickens
  • Ron Stotish, AquaBounty, USA, Science for regulatory approval
  • Li Ning, China, shRNA resistance to foot and mouth in pigs
  • Heiner Niemann, Germany, Closing Talk

Early Bird Registration for TT2011 ends on July 31st !!!

Early Bird Registration for the TT2011 meeting ends on July 31st !!!
Early Bird Registration for the TT2011 meeting ends on July 31st !!!

Have you not yet registered for attending the 10th Transgenic Technology (TT2011) meeting? You can’t miss the best forum where to discuss the latest advances in animal transgenic technology, for the generation and the analyses of genetically-modified animals!. If you haven’t registered please don’t forget to do so by this next Sunday, July 31st, the announced deadline for early bird registration at reduced fees. Thereafter, and just before the meeting, to be held in Florida, on 24-26 October 2011, you still be able to register, though at slightly increased fees (+70 USD). Please, hurry up and don’t miss this opportunity to meet again all your colleagues from around the world, to openly discuss with them your problems and solutions in your daily work with transgenic animals. Hope to seeing you all in Florida!

Publication of the Academy of Medical Sciences Report on Animals containing human material

Publication of the Academy of Medical Sciences Report on Animals containing human material
Publication of the Academy of Medical Sciences Report on Animals containing human material
The Academy of Medical Sciences has published the report of a working group study on ‘animals containing human material (ACHM)’ today. The report was prepared by a working group, chaired by Professor Martin Bobrow CBE FRS FMedSci. The working group of experts included Prof. Robin Lovell-Badge (NIMR-MRC, UK), member of the International Society for Transgenic Technologies (ISTT). The Academy of Medical Sciences promotes advances in medical science and campaigns to ensure these are translated into healthcare benefits for society. The report examines the use of ACHM from scientific, social, ethical, safety and regulatory perspectives, and highlights how ACHM are used both in investigational research, and in the development and production of therapeutics. The study was informed by open call for evidence, expert evidence, and a commissioned public dialogue.
The report concludes that the majority of ACHM research does not pose ethical or regulatory difficulties, but identifies 3 areas that will need careful oversight in future:
  • Modification of an animal’s brain which might lead to human-like cognition;
  • Changing an animal so it has human appearance or characteristics (e.g. skin, facial or limb features, speech); and the
  • Development of human-derived sperm or eggs in an animal (especially if fertilisation may occur).
It recommendations include:
  • That the Home Office puts in place an expert oversight body, within the current system of animal research regulation, to oversee the most sensitive types of ACHM research.
  • Close alignment of several regulatory bodies that oversee aspects of ACHM research (particularly the Home Office and HFEA).
  • Raising international awareness of ACHM, promoting international consistency in research practice, and the development of international standards and guidance.
These recommendations should ensure that valuable and justifiable ACHM research can proceed within a robust, proportionate regulatory system, which is capable of responding to developing scientific knowledge and social attitudes, and which avoids undue bureaucracy and duplication of regulation.
The study was supported by the Department for Business, Innovation and Skills’ Sciencewise-ERC programme, the Department of Health, Medical Research Council, and Wellcome Trust. A report synopsis has been prepared by Dr Geoff Watts FMedSci.
Further information is available on The Academy of Medical Sciences website

TT2011 meeting: abstracts and awards deadlines coming soon

TT2011 Meeting: 24-26 October 2011, Trade Winds Island Grand Resort, St Pete Beach, Florida, USA
TT2011 Meeting: 24-26 October 2011, Trade Winds Island Grand Resort, St Pete Beach, Florida, USA

The 10th Transgenic Technology meeting (TT2011) will be held at the Trade Winds Island Grand Resort, in St Pete Beach, Florida, USA, on 24-26 October 2011, organized by the International Society for Transgenic Technologies (ISTT). The first deadline for submitting abstracts and applying for the various awards (Registration and Young Investigator Awards) will be due in about one month, on 30th June 2011. Accepted abstracts will be published in the scientific journal Transgenic Research, associated with the ISTT. Results from the various awards will be communicated by 16th July 2011.

Please, mark the TT2011 meeting in your agendas and don’t miss this opportunity to discuss the latest developments on Transgenic Technologies, at the International Level, with the world experts in the field. Looking froward to receive your latest work, experiments, designs, developments, your hottest research achievements using genetically modified animals.

ISTT Members entitled to purchase Springer books with a 33% discount

ISTT Members entitled to purchase Springer books with a 33% discount
ISTT Members entitled to purchase Springer books with a 33% discount

The International Society for Transgenic Technologies (ISTT) is most pleased to publicly announce the renewal of the agreement with the publisher Springer, for mutual cooperation, for an additional period of five years. As part of this agreement, ISTT members will continue benefitting from a privileged access to full online contents of the scientific journal Transgenic Research, to which ISTT is associated, through the members-only area of the ISTT web site. As a new benefit for ISTT members, this current agreement includes a list of books on animal transgenesis and animal genetics, published by Springer, which ISTT members are entitled to purchase with a 33% discount, also through the members-only area of the ISTT web site. Of course, these books by Springer include the forthcoming title on “Advanced Protocols for Animal Transgenesis. An ISTT Manual” edited by Shirley Pease and Thom Saunders, promoted by the ISTT and due to be published in August 2011.

Transgenic Rat Nantes Meeting, 6 June 2011

Transgenic Rat Nantes meeting, 6 June 2011
Transgenic Rat Nantes meeting, 6 June 2011

The Transgenic Rats common facility of IFR 26, Biogenouest and IBiSA organizes its third meeting in Nantes, France, on June 6, 2011, about Transgenesis and genome analysis. This one-day meeting, co-sponsored by the International Society for Transgenic Technologies (ISTT), aims to provide an update on transgenesis developments in the generation of transgenic animals and in genome analysis. The meeting is chaired by Ignacio Anegon (ISTT member) and organized by Séverine Menoret (ISTT member), Séverine Rémy, Laurent Tesson, Claire Usal and Tuan. H. Nguyen. Deadline for registration is 25 May 2011. ISTT members are entitled to reduced registation fees.

The meeting is intended for Master, PhD and medical students with a background in molecular biology and genetics as an introduction to future work in these rapidly developing areas of research. It is also intended for post-docs and scientists already working in certain of these fields and who are interested in expanding their knowledge on the potential applications of these new techniques to their models or in neighbouring pathophysiological models of analysis of genes or diseases using genetically modified animals.

Advanced Protocols for Animal Transgenesis. An ISTT Manual (Due: August 2011)

Advanced Protocols for Animal Transgenesis. An ISTT Manual. Shirley Pease & Thomas L. Saunders (eds.), Springer 2011, 1st edition (Due: August 2011)
Advanced Protocols for Animal Transgenesis. An ISTT Manual. Shirley Pease & Thomas L. Saunders (eds.), Springer 2011, 1st edition (Due: August 2011)

The International Society for Transgenic Technologies (ISTT), in collaboration with Springer, is pleased to announce the next publication of the book entitled “Advanced Protocols for Animal Transgenesis. An ISTT Manual“, edited by Shirley Pease and Thomas L. Saunders, whose 1st edition is expected to be published by August 2011.

This laboratory manual, published by Springer in cooperation with the International Society for Transgenic Technology (ISTT), provides almost all current methods that can be applied to the creation and analysis of genetically modified animals. The chapters have been contributed by leading scientists who are actively using the technology in their laboratories, most of them members of the ISTT. Based on their first-hand experience the authors also provide helpful notes and troubleshooting sections.

Topics range from standard techniques, such as pronuclear microinjection of DNA, to more sophisticated and modern methods, such as the derivation and establishment of embryonic stem (ES) cell lines, with defined inhibitors in cell culture medium. In addition, related topics with relevance to the field are addressed, including global web-based resources, legal issues, colony management, shipment of mice and embryos, and the three R’s: refinement, reduction and replacement.

Table of contents:

  • Karen S. Canady: Patent and licensing issues in transgenic technology.
  • Lluís Montoliu: Global Resources: Including Gene Trapped ES Cell Clones: Is Your Gene Already Knocked Out?.
  • Eduardo Moltó, Cristina Vicente-García and Lluís Montoliu: Designing Transgenes for Optimal Expression.
  • Thomas L. Saunders: Gene Targeting Vector Design for Embryonic Stem Cell Modifications.
  • Thomas J. Fielder and Lluis Montoliu: Transgenic Production Benchmarks.
  • Katja Becker and Boris Jerchow: Generation of Transgenic Mice by Pronuclear Microinjection.
  • Séverine Ménoret, Séverine Remy, Laurent Tesson, Claire Usal , Anne-Laure Iscache and Ignacio Anegon: Generation of Transgenic Rats using Microinjection of Plasmid DNA or Lentiviral vectors.
  • Almudena Fernández, Diego Muñoz and Lluís Montoliu: Generation of Transgenic Animals by Use of YACs.
  • Michael G. Zeidler, Margaret L. Van Keuren and Thomas L. Saunders: BAC Transgenes, DNA Purification, and Transgenic Mouse Production.
  • Carlos Lois: Generation of Transgenic Animals with Lentiviral Vectors.
  • Aron Geurts, Lajos Mates and Darius Balciunas: Vertebrate Transgenesis by Transposition.
  • Karen M. Chapman, Dalia Saidley-Alsaadi, Andrew E. Syvyk, James R. Shirley, Lindsay M. Thompson and F. Kent Hamra: Rat Spermatogonial Stem Cell Mediated Gene Transfer.
  • Sayaka Wakayama, Nguyen Van Thuan and Teruhiko Wakayama: Mouse Cloning by Nuclear Transfer.
  • Elizabeth D. Hughes and Thomas L. Saunders: Gene Targeting in Embryonic Stem Cells.
  • Wojtek Auerbach and Anna B. Auerbach: The Importance of Mouse ES Cell Line Selection.
  • Marina Gertsenstein: Tetraploid Complementation Assay.
  • Elizabeth Williams, Wojtek Auerbach, Thomas M. DeChiara and Marina Gertsenstein: Combining ES cells with Embryos.
  • Kristina Nagy and Jennifer Nichols: Derivation of Murine ES Cell Lines.
  • Ping Li, Eric N Schulze, Chang Tong and Qi-Long Ying: Rat Embryonic Stem Cell Derivation and Propagation.
  • Han Li, Katerina Strati, Verónica Domínguez, Javier Martín, María Blasco, Manuel Serrano and Sagrario Ortega: Induced pluripotency: generation of iPS cells from mouse embryonic fibroblasts.
  • Anna B. Auerbach, Peter J. Romanienko and Willie H. Mark: The Preparation and Analysis of DNA for Use in Transgenic Technology.
  • Karen Brennan: Colony Management.
  • Belen Pintado and Juan Hourcade: Cryopreservation.
  • Shirley Pease: Shipment of Mice and Embryos.
  • Jorge M. Sztein, R.J. Kastenmayer and K.A. Perdue: Pathogen Free Mouse Rederivation by IVF, Natural Mating and Hysterectomy.
  • Jan Parker-Thornburg: Refinement, Reduction and Replacement

ISTT Members are entitled to a 33% discount on the book price.