11th ISTT Prize Awardee – Dr. Mario Capecchi

Dr. Mario Capecchi
Dr. Mario Capecchi

 

Houston TX, USA. 26 October, 2016.

The International Society for Transgenic Technologies (ISTT, Inc.) is delighted to announce that the 11th ISTT Prize will be awarded to Dr. Mario Capecchi for his seminal work on homologous recombination in embryonic stem cells, for which he was awarded the Nobel Prize in 2007. The ISTT Prize is awarded to investigators who have made outstanding contributions to the field of transgenic technologies. The selection of the 11th ISTT Prize winner, Dr. Capecchi, was made by the ISTT Prize Committee [composed of the ISTT President (Jan Parker-Thornburg), ISTT Vice-president (Benoit Kanzler), and the CEO of genOway (Alexandre Fraichard) (the company that generously sponsors the award), and all previous ISTT prize awardees].

The committee unanimously agreed that Dr. Capecchi’s work was essential for the field of transgenesis, having opened up the field to the possibility of generating exact genetic mutations in the mouse genome. At the time when his discoveries were published, the ability to generate transgenic animals by pronuclear injection had recently been published, and was rapidly becoming an essential method to flesh out how genes would both be regulated, and would function. However, while standard transgenesis could answer many genetic questions, it was still limited, as the gene being interrogated was still intact. To truly make leaps forward, it was essential to specifically mutate that gene, either by removing it (to generate a gene knock-out) or to mutate it (to recapitulate a genetic mutation by knock-in). With the culture and injection of ES cells described by Dr. Martin Evans, it became apparent to Drs. Smithies and Capecchi (all three being co-recipients of the 2007 Nobel prize) that such cells could be used for the introduction of mutations into the genome. Homologous recombination of an exogenous DNA into the exact gene to be replaced can occur in very rare circumstances. In his 1986 Cell publication, Dr. Capecchi showed that 1 in 103 cells in culture would undergo the process using homologous recombination. The following year, Capecchi successfully knocked out the Hprt gene in mouse ES cells and Smithies independently demonstrated repair of the gene. Dr. Capecchi gave the process the name by which we know it today, “gene targeting”. A critical aspect of gene targeting (and one that is still essential today) was that both positive and negative selection processes could be used to identify those cells that had undergone homologous recombination, a process published in 1988 in Nature by Dr. Capecchi.

Dr. Capecchi was born in Verona, Italy in 1937, the only child of an Italian father (lost in WWII) and an American mother (detained in and later released from a Nazi concentration camp). He immigrated to the USA in 1946 with his mother to live in Pennsylvania. After graduation from the George School, he attended Antioch College in Yellow Springs Ohio, USA where he majored in chemistry and physics. He began his graduate work at MIT in physics and mathematics, but after becoming interested in molecular biology, moved to Harvard University to study with James Watson, and changed the course of his career. Dr. Capecchi was on the faculty at Harvard until 1973, when he moved his laboratory to the University of Utah. His seminal work describing gene targeting was performed at that institution. During his extremely productive career, some of the awards that Dr. Capecchi has been given include the Kyoto Prize in Basic Sciences (1996), The Premio Phoenix-Anni Verdi for Genetic Research Award, Italy (2000), the 33rd Jiménez-Diaz Prize for Contributions to Medical Genetics, Spain (2001), the Albert Lasker Award for Basic Medical Research (2001), and the American Association of Cancer Research Lifetime Achievement Award (2015). He has been elected to the European Academy of Sciences (2002), the American Academy of Arts and Sciences (2009), the National Academy of Medicine (2015) and has been given honorary doctorate degrees from institutions in Italy, the UK, and Israel. Dr. Capecchi remains an active researcher, with seven research publications in 2015, and six current 2016 publications either printed, submitted, or in press.

We are most pleased that Dr. Capecchi has agreed to receive the ISTT Prize to be given at TT2017, thus joining the list of previously honored scientists, including Janet Rossant (2014), Allan Bradley (2013), Ralph Brinster (2011), Francis Stewart (2010), Brigid Hogan (2008), Charles Babinet (2007), Andras Nagy (2005), Qi Zhou (2004), Kenneth McCreath (2002) and Teruhiko Wakayama (2001). All ISTT Prize winners are given an honorary ISTT membership and a unique silver sculpture representing a mouse blastocyst created by the Hungarian artist Béla Rozsnyay. Dr. Capecchi will receive his prize at the 14th Transgenic Technology Meeting (TT2017) that will be held at the Snowbird Resort outside of Salt Lake City, Utah USA on 1-4 October, 2017.

Selected References from Dr. Capecchi’s lifetime achievements:

Folger, K. R., K. R. Thomas and M. R. Capecchi (1984). Analysis of homologous recombination in cultured mammalian cells. Cold Spring Harbor Symp. Quant. Biol. 49:123-138. PMID: 6099232.

Thomas, K. R., K. R. Folger and M. R. Capecchi (1986). High frequency targeting of genes to specific sites in the mammalian genome. Cell 44:419-428. PMID: 3002636.

Wong, E. A. and M. R. Capecchi (1986). Analysis of homologous recombination in cultured mammalian cells in a transient expression and a stable transformation assay. Somat. Cell Mol. Genet. 12:63-72. PMID: 3003931.

Thomas, K. R., and M. R. Capecchi (1986). Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene. Nature 324:34-38. PMID: 3785372.

Thomas, K. R. and M. R. Capecchi (1986). Targeting of genes to specific sites in the mammalian genome. Cold Spring Harbor Symp. Quant. Biol. 51:1101-1113. PMID: 3472755.

Wong, E. A. and M. R. Capecchi (1987). Homologous recombination between coinjected DNA sequences peaks in early to mid-S phase. Mol. Cell. Biol. 7:2294-2295. PMID: 3600663.

Thomas, K. R. and M. R. Capecchi (1987). Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503-512. PMID: 2822260.

Mansour, S. L., K. R. Thomas and M. R. Capecchi (1988). Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: A general strategy for targeting mutations to nonselectable genes. Nature 336:348-352. PMID: 3194019.

Capecchi, M. R. (1989). Altering the genome by homologous recombination. Science 244:1288-1292. PMID: 2660260.

Thomas, K. R., C. Deng and M. R. Capecchi (1992). High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol. Cell. Biol. 12:2919-2923. PMID: 1620105.

Deng, C. and M. R. Capecchi (1992). Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell. Biol. 12:3365-3371. PMID: 1321331.

Capecchi, M. R. (1995). A personal view of gene targeting. In Accomplishments in Cancer Research 1994. (J. G. Fortner and J. E. Rhoads, Ed.) Philadelphia: J. B. Lippincott, pp. 67-78.

Capecchi, M. R. (2000). How close are we to implementing gene targeting in animals other than the mouse? Proc. Natl. Acad. Sci. USA 97:956-957. PMID: 10655465.

Capecchi, M.R. (2001). Generating mice with targeted mutations. Nature Med. 7:1086-1090. PMID: 11590420.

Austin, C.P., J.F. Battey, A. Bradley, M. Bucan, M.R. Capecchi, F.S. Collins, W.F. Dove, G. Duyk, S. Dymecki, J.T. Eppig, F.B. Grieder, N. Heintz, G. Hicks, T.R. Insel, A. Joyner, B.H. Koller, K.C. Lloyd, T. Magnuson, M.W. Moore, A. Nagy, J.D. Pollock, A.D. Roses, A.T. Sands, B. Seed, W.C. Skarnes, J. Snoddy, P. Soriano, D.J. Stewart, F. Stewart, B. Stillman, H. Varmus, L. Varticovski, I.M. Verma, T.F. Vogt, H. von Melchner, J. Witkowski, R.P. Woychik, W. Wurst, G.D. Yancopoulos, S.G. Young and B. Zambrowicz (2004). The knockout mouse project. Nat Genet.36(9): 921-4. PMID: 15340423.

Wu, S., G. Ying, Q. Wu, and M.R. Capecchi (2007). Towards simpler and faster genome-wide mutagenesis in mice. Nat Genet. Jul;39(7):922-30. PMID: 17572674.

Li, S., Lan, H., Men, H., Wu, Y., Li, N., Capecchi, M.R., Bryda, E.C., and S. Wu (2016). Derivation of Transgene-Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells. Stem Cells Transl Med. Sep 13. [Epub ahead of print]

Du, X., Feng, T.,Yu, D., Wu, Y., Zou, H., Ma, S., Feng, C., Huang, Y., Ouyang, H., Hu, X., Pan, D., Li, N., Capecchi, M., and S. Wu(2015). Barriers for the Derivation of Germline Competent Porcine Pluripotent Stem Cells. Stem Cell and Development (submitted).

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

Dolly @ 20

Although she didn’t hit the limelight until February 1997 with a publication in Nature, Dolly-the-most-famous-sheep-in-the-world was born 20 years ago, on 5 July 1996 in The Roslin Institute in Edinburgh. Being the first mammal cloned from adult cells by a team led by Ian Wilmut, Dolly changed the way we look at the up-to-then-supposed irreversibility of development and paved the way for many other forms of reprogramming. Despite suffering from several illnesses early in life, including the well-known arthritis, Dolly was eventually humanely put down on 14 February 2003 when she was found to suffer from a viral form of lung cancer. Dolly’s remains are still on view in the National Museum of Scotland in Edinburgh. Concerns about a potential effect of cloning on premature or accelerated ageing have recently been addressed in a study from the University of Nottingham which shows that 13 cloned sheep, four of which clones from Dolly herself, aged normally.
To celebrate Dolly’s 20th birthday The Roslin Institute is organizing a series of scientific and public events to examine and celebrate her legacy. Public events include a debate in the ‘Cabaret of Dangerous Ideas’ as part of the Edinburgh Festival Fringe, a public lecture and discussion featuring Professor Sir Ian Wilmut, Professor Angelika Schnieke and Noble Prize laureate Professor Shinya Yamanaka in the Surgeon’s Hall Museum in Edinburgh, and a public lecture by Sir Ian at the Virginia-Maryland College of Veterinary Medicine, Blacksburg, Virginia, USA. A number of Dolly-related exhibits will also feature at The Roslin Institute Open Day on the 8 October. There will also be a scientific symposium on 2 September in The Roslin Institute Auditorium that will cover the latest advances in research fields that are linked to Dolly: animal biotechnology, stem cell pluripotency and regenerative medicine. Although the main auditorium is now full, it is still possible to register for a seat in adjacent rooms where the talks will be live-streamed.

Submitted by Peter Hohenstein

Dolly as a lamb with her Scottish Blackface surrogate mother.
Dolly as a lamb with her Scottish Blackface surrogate mother.
Dolly with Professor Sir Ian Wilmut, who led the research which produced her.
Dolly with Professor Sir Ian Wilmut, who led the research which produced her.

 

ISTT at FELASA

13th FELASA Congress, 13-16 June, 2016 – Brussels, Belgium
Respectfully submitted by Boris Jerchow:

Together with Board members Benoît Kanzler and Branko Zevnik, I had the chance to take part in the 2016 edition of the triennial conference of the Federation of European Laboratory Science Associations (FELASA), which took place in Brussels, Belgium, between June 13 and 16. With the kind support of our members Sandra Buhl and Kristin Evans we represented ISTT with a booth. The meeting mainly covered topics important for those involved in laboratory animal science. The conference was divided into six streams:
• Governance, including reports on the transposition of 2010 EU Directive aimed at harmonizing animal welfare standards throughout Europe that has a lot of impact on our work but is still in a phase in which the new regulations are being implemented in daily routines. The stream also addressed active information of the public and ways to conduct an ethical review process. Aurora Brønstad, who also presented at TT2016 in Prague, spoke on the important topic of harm-benefit analysis of animal experiments [1, 2].
• Joint programs across Europe, including education and training, competence management, and 3Rs programs, to name just a few.
• Safety issues with a strong focus on health monitoring of aquatic, amphibian, and rodent species. Moreover, this stream included topics such as occupational health and safety and best working practices but also best practices for husbandry and care, quality of feed, water, and enrichment.

• Common diseases of humans and animals. This stream addressed disease and disease models for metabolic disease, cancer, and also infectious disease including zoonoses. It also included presentations on BLS3 facilities and research with non-human primates.
• Animal well-being emphasized the importance of using the broad understanding of what is going on inside an animal to assess and improve its well-being. The stream contained presentations and discussions about behavioral and neural science, as well as severity assessment, prospectively and also during the time when while animals are being used experimentally by employing clinical signs to recognize pain, suffering distress or lasting harm. I found the latter notably important for our community as we are the ones generating and using genetically altered (GA) animals that may well present with a condition affecting their well-being that should be taken into account before starting the experiment and closely followed during their lifetime. GA animals should also be monitored for unforeseen effects that may compromise their well-being and measures to alleviate pain, suffering, distress or lasting harm should be taken

whenever possible.
• Animal models and animal experiments. For this session I had been asked to convene a session with the title “Genetic modification – technologies and pitfalls”. Together with Ann van Soom, Tom Vanden Berghe and Lluis Montoliu, we informed the audience about the latest technologies in the field, the relevance of the non-coding genome, the threat of passenger mutations, and epigenetic effects. Other sessions in this stream discussed experimental design and reporting, how to process, share, and review acquired as well as existing data, imaging techniques, and various experimental paradigms.
In general there was a strong focus on animal welfare, especially on refinement of procedures, which was present over all streams and sessions. Much of the conference was under the influence of the still ongoing implementation of the regulations of the EU Directive on animal experimentation. Due to the importance of European scientific activities in the world, the pressure on scientific publishers to adopt higher animal welfare and reporting standards, and tight cooperation with countries outside the EU, namely the United States, these regulations will impact on animal experimentation worldwide. The same is true for health standards where FELASA guidelines have already become a gold standard. Although FELASA Conferences draw an audience quite distinct from our TT Meetings, there is a small but important overlap in interest. I am convinced that the ISTT should keep on striving to foster a vivid exchange with FELASA and the laboratory animal science community.

1. Bronstad, A., et al., Current concepts of Harm-Benefit Analysis of Animal Experiments – Report from the AALAS-FELASA Working Group on Harm-Benefit Analysis – Part 1. Lab Anim, 2016. 50(1 Suppl): p. 1-20.
2. Laber, K., et al., Recommendations for Addressing Harm-Benefit Analysis and Implementation in Ethical Evaluation – Report from the AALAS-FELASA Working Group on Harm-Benefit Analysis – Part 2. Lab Anim, 2016. 50(1 Suppl): p. 21-42.

ISTT Booth at FELASA
Benoit Kanzler, Sandra Buhl and Boris Jerchow
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Pens and futbol at the ISTT Booth

ISTT at the 55th annual CALAS meeting

On June 11-14, 2016, the ISTT hosted a booth at the 55th CALAS annual symposium that was held in Toronto, Ontario, Canada. CALAS, the Canadian Association for Laboratory Animal Science, is a national association dedicated to providing high quality training and educational resources to animal care professionals across Canada. CALAS has almost 1,000 members and supports a diverse group of animal care attendants, animal health technicians, and veterinarians. It provides training and certification programs recommended by the Canadian Council on Animal Care (CCAC). The theme of this year symposium attended by 400 participants was: “The dirt on germs: the good, the bad, the unknown”.

Marina Gertsenstein attended the meeting, both to represent the ISTT at the booth and to organize a workshop on Current Technologies in Mouse Genome Manipulations at The Centre for Phenogenomics (TCP). At the booth, ISTT information flyers describing the benefits of the membership and pens with ISTT logo were handed out. Several attendees expressed interest in becoming the members of ISTT.

The highlight of the scientific session was the talk of Dr. Kevin C. Kain, Canada Research Chair in Molecular Parasitology. In his keynote address he described his research on host-parasite interactions responsible for major global health threats such as malaria and HIV and first-hand experience with the clinical problems. This leading researcher is developing effective therapeutic interventions using animal models to determine the molecular basis for clinical outcomes of life-threatening infections and to translate this knowledge into novel therapeutic interventions.

Meeting Report respectfully submitted by Marina Gertsenstein

CALAS logoISTT @CALAS2016CALAS meeting info

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

Genetic Engineering of Human Embryos- for discussion

Commentary by Ernst-Martin Füchtbauer

The new and accelerating technical development of the CRISPR/Cas9 system opens up for the possibility of targeted genetic modifications in germline competent human embryos. This is an avenue, which until very recently has been regarded as absolutely off limits. To cross the border between genetic modifications of somatic cells and germline cells was simply not conceivable, at least in most Western countries. Indeed, the border has not yet been crossed, but we are getting closer.

In two recent papers Chinese scientists used triploid human embryos as a ‘model’ to either treat ß-thalassemia [1] or to recapitulate a spontaneous mutation in the CCR5 gene [2], which results in resistance against HIV infections. Both targets are clearly chosen due to their potential for future therapeutic application.

Shortly after the first of the two papers was published, the Board of Directors of the ISTT posted a statement [3], which among other arguments contains the following sentence:

Uses of genetic engineering in human embryos should be limited to disease mitigation for those diseases where no other option is available; we reject the idea of “designer babies”.

This raises the question whether there are at all diseases where there are no other options (now or in the future). Hereditary diseases are rarely transmitted by homozygous parents, which makes preimplantation diagnostics (PID) an obvious safer and ethically far less disputed alternative. The example, ß-thalassemia reaches in very limited populations, like the Maldives, a frequency that puts about 1% of couples at risk to be double homozygous. But still, is not a CRISPR/Cas9 based hematopoietic stem cell therapy the obvious and much easier developed therapy?

However, the case of targeting CCR5 is fundamentally different. As no one can claim that being wild type for CCR5 is a disease, this is a clear designer approach. Given that we know relatively little about the function of CCR5, one might wonder how we can be sure that it is beneficial to mutate it in a world of ever changing microbial threats. It seems that developing a CCR5 blocking drug or somatic mutations of CCR5 in HIV patients is the obvious way forward.

It is my feeling that many colleagues, some of whom I greatly admire, are beginning to accept experiments with the obvious goal to modify the human germline ‘if it is the only cure for severe diseases’. However, I have not heard one convincing example of such disease that is not in principle “treatable” or “avoidable”. Finally we should keep in mind that the Hardy-Weinberg equation ridicules all eugenic attempts to clean the population from ‘disease’ alleles.

I am increasingly concerned because the discussion in our community has, within a few months, taken an almost purely technical turn about off target risks and efficiency. We neglect many decades of thorough philosophical and ethical literature on the issue. There is more at stake than the possible treatment of a few rare diseases.

These questions are too important to just wait and see. We as ISTT members are so close to the topic that we need to have an honest and open discussion about our opinions. This blog could be a starting point and I invite/encourage you to add to this discussion.

[1] Liang, P. et al. Protein Cell (2015) http://dx.doi.org/10.1007/s13238-015-0153-5

[2] Kang, X. et al. J. Assist. Reprod. Genet. (2016) http://link.springer.com/article/10.1007%2Fs10815-016-0710-8

[3] http://www.montonerin.es/isttlegacy/isttblog/?p=1581

TT2016 – President’s Synopsis

The opening night of TT2016 was momentous promising subsequent days filled with good friends and good science. We welcomed more than 700 delegates who attended, and then proceeded to hear a wonderful talk from Andras Nagy (2005 ISTT Prize winner). Andras’ talk was followed by a delicious buffet with wine, friends, colleagues and music. The opening proved to be an excellent portent of what was to come. Over the next three days, we heard many excellent talks—talks that encompassed the use of transgenic technologies, and especially CRISPR/Cas9 technology.

Charles Gersbach presents CRISPR/Cas9 modification of the dystrophin gene
Charles Gersbach presents CRISPR/Cas9 modification of the dystrophin gene

We heard about methods to ameliorate muscular dystrophy, to humanize large animals for xenotransplantation, to make swine resistant to an endemic disease, and to examine infertility in humans. We discussed technologies that would use epigenome to target the “regulome”, that would examine non-coding areas of the genome, that would recapitulate immune syndromes in ES cells, and that would allow us to assess phenotypic changes in embryonic lethal mutant mice using imaging. We learned both the history behind the CRISPR/Cas9 system, and newer CRISPR systems that are in development. We discussed ethics, gene drive, non-injection technologies, new injection technologies, and methods of generating many more oocytes in mice. There were seventeen abstracts chosen for full presentation, examining technological developments, large CRISPR/Cas9 initiatives, and transposon-mediated transgenesis. The remainder of the more than 125 abstract submssions were displayed throughout the meeting in the spacious poster room. Three were chosen as Poster Award winners, including Vera Jansen (Optogenetic tools to study cAMP signaling in cilia and flagella), Charles-Etienne Dumeau (Efficient method for the isolation of functional single cell from the ICM of mouse blastocyst), and Hiromi Miura (Generation of knockdown mice by CRISPR/Cas9-based targeted insertion of artificial miRNA sequence). On the last day, the ISTT Young Investigator Award (sponsored by inGenious Targeting Laboratory) was given to Pablo Ross based on his work developing ES cells in farm animals.

Charles River Representative, Iva Morse, and Jan Parker-Thornburg and Elizabeth Williams (ISTT, Inc.) present the Best Poster Awards to Vera Jansen (absent), Charlie
Iva Morse (Charles River), and Jan Parker-Thornburg and Elizabeth Williams (ISTT, Inc.) present the Best Poster Awards to Vera Jansen (absent), Charles-Etienne Dumeau, and Hiromi Miura (represented by Masato Ohtsuka).
Thomas Zeyda (inGenious Targeting Laboratory) and Jan Parker-Thornburg (ISTT President) present the Young Investigator Award to Dr. Pablo Ross, UC Davis.
Thomas Zeyda (inGenious Targeting Laboratory) and Jan Parker-Thornburg (ISTT President) present the Young Investigator Award to Dr. Pablo Ross, UC Davis.
TT2016 - Cryopreservation Workshop presentation by Lluis Montoliu.
TT2016 – Cryopreservation Workshop presentation by Lluis Montoliu.

The meeting was preceded by two workshops—one on programmable nucleases (headed by Radislav Sedlacek) and one on cryopreservation (led by Martin Fray, INFRAFRONTIER). Those who attended the workshops were very pleased with the learning opportunities that were afforded them. In addition, immediately following the meeting was one additional workshop on zebrafish transgenesis (Leads: Petr Bartunek, Zbynek Kozmik, Christian Mosimann and Graham Lieschke). All of the workshops were well-attended and greatly appreciated!

First Orbis pictus lecture given by Richard Behringer.
First Orbis pictus lecture given by Richard Behringer.

There were a number of new initiatives at TT2016. We had Orbis pictus lectures—lectures designed to use pictures and clear descriptions to demonstrate answers to a problem. Richard Behringer gave an excellent, encyclopedic presentation of methods of producing genetically modified animals in a vast variety of species. Later, Thomas Boehm described how lymphoid organs developed throughout evolution to the point where vertebrates now have a thymus. Also, for the first time, we had concurrent sessions. Delegates needed to choose whether to hear about ethics in animal use, or new injection and superovulation technologies. Overall, the scientific program was exceptional!

Departing Board members
Presentation of thank-you gifts to departing ISTT Board members – Wojtek Auerbach (absent), Boris Jerchow, and Tom Fielder.

The ISTT, Inc. held its third General Assembly just prior to the Gala Dinner. During that meeting, we sadly said goodbye to three departing Board members: Tom Fielder, Boris Jerchow and Wojtek Auerbach. We also reviewed ISTT finances, membership, committee activities, and interactions with our affiliated organizations. One new ISTT initiative that was presented was an outreach committee to our members (and non-members) who perform transgenic technologies in non-rodent (generally large-animal) species. The ISTT large animal group will be headed by Martina Crispo and Bruce Whitelaw. The meeting ended with a presentation inviting membership to attend TT2017 in Salt Lake City, Utah, USA, hosted by Susan Tamowski.

A wonderful time at the Zofin Palace.
A wonderful time at the Zofin Palace.

The social program prepared by our Czech colleagues was also amazing. Delegates enjoyed the opening buffet with traditional Czech music. However, it was the Gala Dinner that proved to be the high point of the social program. The Zofin Palace was full with partygoers. The wine flowed freely, the food was wonderful, and the string quartet (plus clarinet) fantastic as well. Overall, TT2016 can be considered as one of the best TT meetings ever, and I am proud, as ISTT President, that we helped to host such a wonderful meeting. Thanks so very much to the organizers—Radislav Sedlacek, Inken Beck and Nicole Chambers. Due to their amazing work, the ISTT has again had a successful TT meeting!

CARD – IP Mouse Sperm and Embryo Cryopreservation Course, Institut Pasteur, Paris, France, 20-24 June 2016

 

The International Society for Transgenic Technologies (ISTT) will proudly co-sponsor the CARD – IP Mouse Sperm and Embryo Cryopreservation Course that will be held at the renowned Pasteur Institute, in Paris, on 20-24 June 2016, organized by Naomi Nakagata (CARD-Kumamoto University, Japan, Coordinator of CARD) and Jean Jaubert (Pasteur Institute, Paris).

Recent developments from the laboratory of Prof. Naomi Nakagata (CARD-Kumamoto University, Japan) have pushed the envelope of mouse cryopreservation: i) improved female superovulation method; ii) fresh, frozen and cold storage sperm techniques; iii) optimized IVF methods. These improvements (see main references in program) have resulted in an unparalleled increase in efficiency of cryopreservation and rescue of relevant mouse lines.

The aim of this course is to introduce the newest CARD methods to researchers and technicians involved in mouse archiving and/or managing transgenic facilities and who are willing to implement these new methods in their work. These techniques will be taught directly by the team that devised them.

This course is open to anyone interested. Pre-application will be required, including, at least, a recent CV and a letter prepared by the intended participant describing how the applicant will benefit by attending this course and how relevant is the course material to his/her work. Additional documents are welcome, at the discretion of participants, including supporting letters by supervisors (where appropriate), reference letters, etc… A copy of the passport is mandatory. Applications should be submitted online, and will close on March 25th 2016.

The maximum number of participants attending this course will be 20, distributed among countries and institutions, and according the documentation provided and the interests expressed. Review and selection of participants will be done by the Teaching Committee and results will be communicated by April 15, 2016. The official language of the course will be English.

In addition to practical sessions, the course will also include several lectures of related interesting topics for the participants delivered by experts in each field.

See more at: http://www.pasteur.fr/fr/enseignement/ateliers/mouse-sperm-and-embryo-cryopreservation-course#sthash.BKcGh9VV.dpuf

 

Screen Shot 2016-02-02 at 15.20.55

Hands-on topics:

 

Making pipettes and embryo handling

Superovulating/Ultra-superovulating female mice

Isolating unfertilized mouse oocytes

Isolating and cold storage/shipping of mouse cauda epididymis

Freezing/thawing mouse sperm and IVF

Fresh mouse sperm and IVF

Freezing/thawing 2-cell IVF-derived mouse embryos

Vitrification of mouse oocytes and embryos

Embryo transfer techniques in mice (oviduct, uterus, NSET)

Vasectomy of male mice (scrotal and abdominal)

 

Additional lectures:

 

The laboratory mouse origin

Historic and scientific perspectives of transgenesis methods and the future of transgenic platforms

Historic and Scientific perspectives of embryo and sperm cryopreservation

Comparing current embryo and sperm cryopreservation methods

Vitrification of oocytes and their use for IVF

Cold storage and transport of germplasm

Shipping mice, refrigerated and frozen material

Managing and handling information in cryopreservation centers

CRISPR/Cas9 and the challenges in freezing these new GEM’s

NSET: non-surgical embryo transfer

Breeding, genotyping and back-ups of GEM’s

CARD

 

Instructors:

 

Naomi Nakagata (CARD-Kumamoto University, Japan)

Toru Takeo (CARD-Kumamoto University, Japan)

Shuuji Tsuchiyama (CARD-Kumamoto University, Japan)

Kiyoko Fukumoto (CARD-Kumamoto University, Japan)

Yukie Haruguchi (CARD-Kumamoto University, Japan)

Tomoko Kondo (CARD-Kumamoto University, Japan)

Yumi Takeshita (CARD-Kumamoto University, Japan)

Yuko Nakamuta (CARD-Kumamoto University, Japan)

Tomoko Umeno (CARD-Kumamoto University, Japan)

Hidetaka Yoshimoto (CARD-Kumamoto University, Japan)

Ayumi Mukunoki (CARD-Kumamoto University, Japan)

Mari Iwamoto (CARD-Kumamoto University, Japan)

Fumi Takahashi (CARD-Kumamoto University, Japan)

Kristy Kinchen (Gainesville, FL, USA)

Jean Jaubert (Institut Pasteur, France)

Franck Bourgade (Institut Pasteur, France)

Angélique Vincent (Institut Pasteur, France)

Claire Lecestre (Institut Pasteur, France)

Jorge Sztein (Barcelona, Spain)

Lluís Montoliu (CNB-CSIC, Madrid, Spain)

Barbara Stone (ParaTechs, Lexington KY, USA)

 

Additional lectures:

 

Naomi Nakagata (CARD-Kumamoto University, Japan)

Toru Takeo (CARD-Kumamoto University, Japan)

Shuuji Tsuchiyama (CARD-Kumamoto University, Japan)

Jorge Sztein (Barcelona, Spain)

Lluís Montoliu (CNB-CSIC, Madrid, Spain)

Fernando Benavides (MD Anderson, Smithville, USA)

Francina Langa Vives (Institut Pasteur, Paris, France)

Michel Cohen-Tannoudji (Institut Pasteur, Paris, France)

Xavier Montagutelli (Institut Pasteur, Paris, France)

Jean Jaubert (Institut Pasteur, Paris, France)

Barbara Stone (ParaTechs, Lexington KY, USA)

 

For any further information contact: enseignement@pasteur.fr

 

Tags: Cryopreservation course, embryo cryopreservation, ISTT co-sponsorisation, IVF, practical course, sperm cryopreservation, superovulation, vasectomy, vitrification

The TT2016 ISTT Registration and ISTT & Genentech Travel Award Winners

The ISTT is proud to announce the winners of the TT2106 ISTT Registration Awards and the ISTT & Genentech Travel Awards.

This year, the ISTT Board of Directors has awarded 12 ISTT members an ISTT Registration Award, which will cover their registration fee for TT2016.

Registration Awardees

Congratulations to:

Samy Alghadban

Katherine Bovariak

Carmen Cucarella

Paul Devenney

Denise Grant Lanza

Melissa Larson

Peter Major

Anna Novoa

Sara Ortica

Karolina Piotrowska-Nitsche

Lucas Pitt

Selin Yagcioglu

 

Travel Awardees

The ISTT and Genentech have also awarded 2 of the successful applicants an additional travel award. The awardees are: Melissa Larson from the University of Kansas Medical Center, Kansas City, Missouri, USA and Sara Ortica Gatti from the Paris-Saclay Institute of Neuroscience, Paris, France. Their award includes airfare to and accommodation at TT2016. Both travel award winners will be presenting their work at the meeting.

Melissa Larson (left), Sara Ortica (right)
Melissa Larson (left), Sara Ortica (right)

 

Post prepared and submitted by Elizabeth Williams on behalf of the ISTT Board of Directors.