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!

TT2016 Deadlines and Poster

TT2016, to be held in Prague in late March, should prove to be an exciting meeting. The scientific portion of the meeting will be from 20-23 March. In addition, there are three hands-on workshops that are available, including a Mouse Cryopreservation Workshop (16-18 March), a course on CRISPR/Cas9 programmable nucleases (16-18 March) and a Fish Transgenesis Course (23-25 March).

Early registration will close on 15 December, 2015, so register soon! Please note that ISTT members have discounted registrations, and can apply for a limited number of registration and travel awards (as detailed in e-mails sent to ISTT members). Applications and documentation for these awards must be sent in by 15 December, 2015 to istt@transtechsociety.org.

We also encourage you to submit an abstract for the poster session. A number of poster abstracts will be chosen for oral presentation, so this is an excellent opportunity to share your science. Poster submissions are also eligible for the TT2016 Poster awards. Abstracts are due by 15 December, 2015

Please note that a printable poster has been appended to this blog post. Feel free to print and post widely! Looking forward to seeing you at TT2016!

TT2016_poster A3w workshops

CARD-RPCI Cryopreservation Course Report

CARD-RPCI Cryopreservation Course Report
CARD-RPCI Cryopreservation Course Report

During the past week, 15-19 September 2014, the CARD-RPCI Mouse Sperm and Embryo Cryopreservation Course was held at Roswell Park Cancer Institute, in Buffalo, NY, USA, organized by Naomi Nakagata (CARD-Kumamoto University, Japan), Aimee Stablewski (Roswell Park Cancer Institute, Buffalo, NY, USA) and Jan Parker-Thornburg (MD Anderson Cancer Center, Houston, TX, USA). This was the second CARD course organized overseas, outside Asia, after the first course organized in Madrid in October 2013. This new practical course in North America was co-sponsored by the International Society for Transgenic Technologies (ISTT). What follows is a brief course report prepared by Aimee Stablewski and Jan Parker-Thornburg, who deserve to be praised, along with Naomi Nakagata and his CARD team, for another most successful cryopreservation course. This course report can also be downloaded from here.

CARD-­RPCI Spermand Embryo CryopreservationWorkshop
September 15-­19, 2014
Roswell Park Cancer Institute, Buffalo NY, USA

MEETING REPORT

The CARD-RPCI Sperm and Embryo Cryopreservation Workshop was recently held in Buffalo NY USA on the campus of Roswell Park Cancer Institute. Eighteen trainees, fifteen instructors and lecturers, and numerous vendors were hosted by Naomi Nakagata (Kumamoto University), Aimee Stablewski (Roswell Park Cancer Institute) and Jan Parker-Thornburg (MD Anderson Cancer Center) from September 15-19, 2014. Drs. Naomi Nakagata and Toru Takeo brought their team from CARD to assist the trainees in learning the latest techniques in embryo vitrification, sperm cryopreservation, in vitro fertilization, and the new method of vitrifying oocytes. In addition, Jorge Sztein instructed the trainees in methods of ovary freezing and subsequent implant after thaw; Lluis Montoliu provided lectures in current methods of embryo cryopreservation and CRISPR/Cas9 methods of generating genetically engineered mice, and Barbara Stone provided training in using the NSET method of embryo implant.

This was the first time that the CARD team provided training in North America, and the team (supported by Kumamoto University and Kyudo Co., LTD.) delivered a truly memorable experience for the trainees. Hands-on sessions were intense and intended to provide trainees with the knowledge and experience of performing the exacting CARD techniques. The eighteen trainees were composed of fourteen ISTT members and four non-members. Of these, three were from Europe, one from Australia, one from New Zealand, and the remainder from all parts of North America.

Hands on training included isolating unfertilized mouse oocytes, isolating and cold storage/shipping of mouse cauda epididymis, freezing/thawing mouse sperm and IVF using CARD frozen sperm and legacy sperm, fresh mouse sperm and IVF, cold stored sperm and IVF, freezing/thawing 2-cell IVF-derived mouse embryos, vitrification of mouse oocytes and embryos, IVF of vitrified mouse oocytes, ovary transplantation/ovary freezing, and embryo transfer techniques in mice (oviduct, uterus via NSET).

Didactic lectures were given on the topics of historic and scientific perspectives of embryo and sperm cryopreservation (by Jorge Sztein), comparing current embryo and sperm cryopreservation methods (by Lluis Montoliu), vitrification of oocytes and their use for IVF (by Naomi Nakagata), new US guidelines for the use of animals in research/IACUC (by Sandra Sexton), CRISPR/Cas9 and gene editing endonucleases (by Lluis Montoliu), development of database for managing mouse banking system (by Shuuji Tsuchiyama), shipping mice, frozen or refrigerated embryos/sperm across the world (by Toru Takeo) and freezing and transplantation of ovaries (by Jorge Sztein).

On Wednesday of the workshop, all of the participants were treated to a trip to Niagara Falls followed by an exceptional gala dinner (arranged by Aimee Stablewski) at a local winery. This short respite enabled the participants to replenish their energies for subsequent long days of IVF, vitrifications and surgery.

In all, it proved to be an exceptional workshop, with all participants achieving exceptional results in most, if not all, of the practicals. In fact, the depth of appreciation for being taught these methods became clear both during the closing session, where all instructors, vendors, and participants thanked Dr. Nakagata with a standing ovation, and immediately afterward, where many compliments were given. Perhaps, one of the participants said it best in an e-mail immediately following the course:

a big thank you to Prof. Nakagata, Dr. Toru Takeo and the entire CARD team. The way they have organised is unbelievable. As we all know in biological practical experiments, how much ever we take care, they never go according to plan and there is always a blooper. I must confess, I had my doubts that we would ever stick to the time table. I am glad that I was completely proven wrong. They have given attention to every bit of details and must have put lot of rehearsals behind this. They were fantastic. They answered all the questions and made sure everyone understood plus followed it up with the practicals. The Entire Card team was simply amazing and no other way to express my gratitude than simply saying “I bow to the entire CARD team”.”
Prasanna Kallingappa
University of Aukland
New Zealand

Aimee Stablewski and Jan Parker-Thornburg, as hosts, would like to acknowledge not only our lecturers, but also the CARD team who assisted the students, including Shuuji Tsuchiyama, Kiyoko Fukumoto, Yukie Haruguchi, Tomoko Kondo, Yumi Takeshita, Yuko Nakamuta, Tomoko Umeno, (all from Kumamoto University and Kyudo Co. LTD); and the CARD “adoptees”—Kristy (Kinchen) Williams (University of Florida) and Amar Dasari (Taconic). In addition, technical assistance was ably provided by Dawn Barnas (ISTT), Karstin Webber, Sandra Sexton and Leslie Curtin (all RPCI), who treated over 600 mice with hormones!

The workshop was extremely fortunate to have extremely generous vendor support, including our platinum sponsors: Charles River Laboratories (who supplied all of the mice used in the course), Leica Microsystems (who provided all of the microscopes used for the course as well as onsite support by Louise Bertrand), and Kyudo Co., LTD who provided support for the CARD instructors as well as onsite support by Nobuyuki Mikoda; our gold sponsors: the ISTT (who sponsored Lluis Montoliu’s participation and contributed to the gala dinner), Taconic (who provided instructional support from John Couse and Amar Dasari), and Regeneron (who sponsored a lecture by Lluis Montoliu); our silver sponsors: Hamilton Thorne (who provided instruction in sperm analysis and laser-assisted IVF by Nancy Mutch), IDEXX Bioresearch for course support, Paratechs for sponsoring Dr. Barbara Stone’s participation, Cell Preservation Solutions who provided course support and Lifor media for cold-storage, CosmoBio who provided course support, Transposagen who provided course support, and our bronze sponsors: Millipore EMD and mofa for equipment, Tokai-Hit for equipment, Eppendorf (Mike Bady) for course support and equipment, and VWR and Sarstedt for course support.

Finally, we thank our participants for their hard work and dedication to bring the newest transgenic technologies back to their institutions.

Aimee Stablewski, Roswell Park Cancer Institute, Buffalo, NY, USA
Jan Parker-Thornburg, MD Anderson Cancer Center, Houston, TX, USA

Laboratory Course on Cryopreservation of Mouse Germplasm, Monterotondo, Italy, 20-24 October 2014

Laboratory Course on Cryopreservation of Mouse Germplasm, Monterotondo, Italy, 20-24 October 2014
Laboratory Course on Cryopreservation of Mouse Germplasm, Monterotondo, Italy, 20-24 October 2014

The Consiglio Nazionale delle Ricerche (CNR), EMMA-Monterotondo Campus International Development and The Jackson Laboratory (JAX) offer a comprehensive course on cryopreservation of mouse embryos, sperm and ovaries. This Course is financially supported by the European Union FP7 Capacities Specific Program, through the Infrafrontier-I3. The 2014 Edition of this traditional annual cryopreservation EMMA-JAX course is planned for 20-24 October 2014, organized by ISTT Member Marcello Raspa and his collaborators at CNR-EMMA Campus and with the participation of various faculty members, including Robert Taft and Jane Farley, from JAX; Ferdinando Scavizzi and Raffaele Matteoni, from CNR; Susan Marschall, from Munich; Martin Fray, from Harwell; Lluis Montoliu, from Madrid; and Kent Lloyd, from UC Davis, USA.

The 2014 edition of this course will be organized in memory of Stanley P. Leibo – Course leader 1999-2013.

The course is offered to teach methods in cryopreservation for banking of various strains of mice used in research, including inbred, transgenic and knock-out strains. Several methods of cryopreservation are now available and because no single method is adequate for all the various strains of mice being developed, a variety of methods are taught. The course is designed primarily as a “hands-on” laboratory program in which participants learn techniques for the cryopreservation of cleavage-stage embryos, spermatozoa and ovaries. Techniques include: embryo “two-step” equilibrium freezing in plastic straws, embryo non-equilibrium “ultra-rapid” cooling or “vitrification” in straws and sperm freezing and recovery of frozen sperm by in vitro fertilization. In addition, general principles of cryobiology, development of inventory databases for individual programs, and adaptation of long-term storage systems and cryogenic equipment for different situations will be presented and discussed.

Enrollment is limited to 10 participants and early application is advised. This Course is financially supported by the EU FP7 Capacities Specific Program, through the Infrafrontier-I3.

Course Fees (Fees do not include participant’s travel and lodging):

European Union’s and Associated Countries’ nationals:

  • Academic or Non-Profit Institutions: Euro 400.00
  • Other Institutions: Euro 950.00

Other Countries’ nationals:

  • Academic or Non-Profit Institutions: Euro 900.00
  • Other Institutions: Euro 1,200.00

Deadline for applications: September 10, 2014
Download here the Course application form

Course information:
Marcello Raspa, Ferdinando Scavizzi, Raffaele Matteoni
Consiglio Nazionale delle Ricerche
EMMA-Monterotondo Campus International Development
INFRAFRONTIER I3
e-mail: mraspa@emma.cnr.it

Course secretariat:
Giuliana Boera, Teresa Cuccurullo
Consiglio Nazionale delle Ricerche
EMMA-Monterotondo Campus International Development
INFRAFRONTIER I3
e-mail: cuccurullo@ibc.cnr.it

CARD-RPCI Mouse Sperm and Embryo Cryopreservation Course, Roswell Park Cancer Institute, Buffalo, NY, USA, September 15th-19th, 2014

CARD-RPCI Mouse Sperm and Embryo Cryopreservation Course, Buffalo, NY, USA, September 15-19, 2014
CARD-RPCI Mouse Sperm and Embryo Cryopreservation Course, Buffalo, NY, USA, September 15-19, 2014

The International Society for Transgenic Technologies (ISTT) has agreed to co-sponsor another CARD Cryopreservation Course. This time, for the first time, the CARD methods will be taught in North America: the CARD-RPCI Mouse Sperm and Embryo Cryopreservation Practical Course will be held at Roswell Park Cancer Institute (RPCI) in Buffalo, NY USA. The course will be held September 15th-19th, 2014, and will be a hands-on intensive workshop.

The course is organized by Naomi Nakagata (CARD-Kumamoto University, Japan, Coordinator of CARD), Aimee Stablewski (Roswell Park Cancer Institute, Buffalo, NY, USA, Co-Director of the Gene Targeting and Transgenic Resource) and Jan Parker-Thornburg (MD Anderson, Houston, TX, USA, Director of the Genetically Engineered Mouse Facility).

This course is open to anyone interested. Pre-application will be required, including, at least, a recent CV, completion of this application questionnaire, 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… Pre-applications should be submitted by email to: buffalo_card_cryocourse@transtechsociety.org

The maximum number of participants attending this course will be 18, distributed among countries and institutions, and according the documentation provided and the interests expressed. Pre-applications will be accepted starting April 1st, 2014 and will close on May 31st, 2014. The review and selection of participants will be done by the Organizers from June 1st-15th, 2014. Registrations and payments for selected participants will be accepted from June 16th, 2014 to August 31st, 2014. If required, an ordered waiting list will be prepared and any cancellation or unpaid registration by August 31st, 2014 will be readily substituted by the first available person from this waiting list, starting on September 1st, 2014.

The course registration fee is $1300 USD (with a reduced fee of $1100 USD for ISTT members). This fee includes participation in the entire course, all materials and reagents, lunches over the 5 days and one course official dinner. Hotel costs are not included in the registration fee but booking assistance will be provided, if required, at a hotel on Roswell Park Cancer Institute’s campus, where all instructors and lecturers will be also lodged, hence further promoting interaction from breakfast to dinner. The official language of the course will be English.

COURSE INFORMATION: Recent developments from the laboratory of Prof. Naomi Nakagata (CARD-Kumamoto University, Japan) have boosted the mouse cryopreservation field with improved methods for fresh and frozen sperm techniques and associated optimized IVF methods that have resulted in unparalleled increased efficiencies for the cryopreservation and rescue of relevant mouse lines.

The aim of this course is to introduce the new CARD methods to researchers and technicians involved in managing mouse archiving and/or transgenic facilities and willing to implement these new methods, directly taught by the team which devised them. Each participant will have one stereomicroscope and the entire set of tools, reagents and animals required to learn and practice all the methods included in the program of this course. 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. The number of instructors and lecturers appointed is 17.

Hands-on topics that will be covered during this cryopreservation course:

  • Isolating unfertilized mouse oocytes
  • Isolating and cold storage/shipping of mouse cauda epididymis
  • Isolating and cold storage of embryos
  • Freezing/thawing mouse sperm and IVF using CARD frozen sperm and legacy sperm
  • Fresh mouse sperm and IVF
  • Cold Storage sperm and IVF
  • Freezing/thawing 2-cell IVF-derived mouse embryos
  • Vitrification of mouse oocytes and embryos
  • IVF of vitrified mouse oocytes
  • Ovary transplantation/ovary freezing
  • Embryo transfer techniques in mice (oviduct, uterus via NSET)

Additional lectures on the following topics:

  • Historic and Scientific perspectives of embryo and sperm cryopreservation
  • Comparing current embryo and sperm cryopreservation methods
  • Vitrification of oocytes and their use for IVF
  • New US Guidelines for the use of animals in research/IACUC
  • Cold storage and transport of germplasm
  • Large archiving and distribution centers- challenges and solutions
  • Shipping mice, refrigerated and frozen material
  • CRISPR/Cas9
  • Freezing and transplantation of ovaries
  • NSET: non-surgical embryo transfer
  • Breeding strategies for cohort generation 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)
  • Jan Parker-Thornburg (MD Anderson, Houston, TX, USA)
  • Aimee Stablewski (Roswell Park Cancer Institute, Buffalo, NY, USA)
  • Dawn Barnas (Roswell Park Cancer Institute, Buffalo, NY, USA)
  • Amar Dasani (Taconic, Germantown, NY, USA)
  • Andrea Dunn (Taconic, Germantown, NY, USA)
  • Kristy Williams (USA)
  • Barbara Stone (ParaTechs, Lexington, KY, USA)
  • Jorge Sztein (Barcelona, Spain)

Additional lectures by:

  • Naomi Nakagata (CARD-Kumamoto University, Japan)
  • Toru Takeo (CARD-Kumamoto University, Japan)
  • Sandra Sexton (Roswell Park Cancer Institute, Buffalo, NY, USA)
  • Jorge Sztein (Barcelona, Spain)
  • Barbara Stone (ParaTechs, Lexington, KY, USA)
  • Lluis Montoliu (CNB-CSIC, Madrid, Spain)
  • Carlisle Landel (Transposagen Inc., Lexington, KY, USA)
  • Amar Dasani (Taconic, Germantown, NY, USA)

In memoriam: Stanley Leibo

Stanley Leibo in Rome, October 2013, at the official dinner of the annual EMMA-JAX cryopreservation course in Monterotondo, Italy (Photograph kindly provided by Jane Farley)
Stanley Leibo in Rome, October 2013, at the official dinner of the annual EMMA-JAX cryopreservation course in Monterotondo, Italy (Photograph kindly provided by Jane Farley)

It is with great sadness that we note the passing of Stanley Leibo, who died this morning, March 25, in the USA. Stanley Leibo was Professor at the Department of Biological Sciences, University of New Orleans, LA, USA. On behalf of the ISTT, we wish to offer our most sincere condolences to his family and friends.

Stanley Leibo was a renowned authority in cryobiology and one of the pioneers in the cryopreservation of mammalian germplasm. His seminal publication in Science in 1972, in collaboration with Peter Mazur and David Whittingham, described the first, successful cryopreservation of mammalian embryos, demonstrating the recovery of live pups from mouse embryos that had been stored in liquid nitrogen and liquid helium. His scientific work laid the foundation for the current widespread use of cryopreservation by human fertility clinics, agriculture, scientific resource repositories, and conservation efforts preserving endangered species. In addition to his outstanding scientific contributions, Stanley Leibo will be remembered for the joy and passion he brought to teaching. He was a fixture from the beginning in the Jackson Laboratory’s Cryopreservation Course, offered both in Bar Harbor and later also in partnership with EMMA in Monterotondo, Italy, where over the years hundreds of students, including many ISTT members, had the pleasure of hearing his enthusiastic lectures on the history, theory and practice of cryopreserving mammalian cells and germplasm.

Stanley Leibo was a mentor for many of us, a colleague, and most of all, also a friend. We will not forget him. May he rest in peace.

ISTT council

P.S. Additional information from Stanley Leibo’s career and achievements can be obtained from this document, published in Reproduction, Fertiliy and Development, when he received the 2009 IETS Pioneer Award

Stanley Leibo with Marcello Raspa, in Monterotondo, November 2009, at the 10th anniversary of the EMMA-JAX annual cryopreservation course (Photograph by Lluis Montoliu).
Stanley Leibo with Marcello Raspa, in Monterotondo, November 2009, at the 10th anniversary of the EMMA-JAX annual cryopreservation course (Photograph by Lluis Montoliu).
Stanley Leibo with Martin Fray, at the 10th annual EMMA-JAX cryopreservation course in Monterotondo, Rome, Italy, in November 2009 (Photograph by Lluis Montoliu)
Stanley Leibo with Martin Fray, at the 10th annual EMMA-JAX cryopreservation course in Monterotondo, Rome, Italy, in November 2009 (Photograph by Lluis Montoliu)
Stanley Leibo's handwriting and drawings illustrating his passion for teaching. These were made on the paper tablecloth by himself, while explaining several concepts in cryobiology to other colleagues, during the official dinner of the last annual EMMA-JAX cryopreservation course, held in October 2013, in a restaurant in Rome (Photograph by Lluis Montoliu)
Stanley Leibo’s handwriting and drawings illustrating his passion for teaching. These were made on the paper tablecloth by himself, while explaining several concepts in cryobiology to other colleagues, during the official dinner of the last annual EMMA-JAX cryopreservation course, held in October 2013, in a restaurant in Rome (Photograph by Lluis Montoliu).
Stanley Leibo's lecture at the 50th anniversary of the Society for Cryobiology's Annual Meeting, in Washington, DC, USA, in July 2013. http://www.societyforcryobiology.org/cryo-2013
Stanley Leibo’s lecture at the 50th anniversary of the Society for Cryobiology’s Annual Meeting, in Washington, DC, USA, in July 2013. http://www.societyforcryobiology.org/cryo-2013

Vitrification of unfertilized mouse oocytes and their efficient use for in vitro fertilization

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization. New vitrification method by Naomi Nakagata's laboratory at CARD-University of Kumamoto (Japan).
Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization. New vitrification method by Naomi Nakagata’s laboratory at CARD-University of Kumamoto (Japan).

The laboratory of Prof. Naomi Nakagata at CARD-University of Kumamoto (Japan) has achieved a new milestone in mouse cryopreservation techniques. After boosting the efficiencies of mouse in-vitro-fertilization (IVF) from fresh, refrigerated and, particularly, frozen sperm from C57BL/6J mice, they have focused their attention in establishing a robust, reproducible but simple and efficient method for cryopreserving unfertilized mouse oocytes using vitrification, and their subsequent use for IVF. Their results have just been published in Cryobiology:

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization.
Nakagata N, Takeo T, Fukumoto K, Kondo T, Haruguchi Y, Takeshita Y, Nakamuta Y, Matsunaga H, Tsuchiyama S, Ishizuka Y, Araki K.
Cryobiology. 2013 Oct;67(2):188-92.

The vitrification and thawing of unfertilized oocytes was one of the methods taught by the Nakagata-team, for the first time abroad, at the most recent CARD-CNB Mouse Sperm and Embryo Cryopreservation Course, held in Madrid two weeks ago. The efficient cryopreservation of unfertilized metaphase-II oocytes had remained a challenging procedure for investigators. Unfertilized oocytes are fragile germ cells that do not have nuclear membrane. However, these are most relevant cells and the ones to be used for somatic cell nuclear-transfer (SCNT), intracytoplasmic sperm injection (ICSI) and, in particular, for in vitro fertilization (IVF) techniques. Standard available vitrification methods failed to work, reproducibly, with unfertilized oocytes, whereas similar methods were routinely used with fertilized oocytes, for the efficient production of transgenic mice by pronuclear microinjection.

In 2013, a first report by Kohaya et al., described the generation of live offspring from vitrified C57BL/6J mouse oocytes for the first time. Now, Nakagata et al., show us in their most recent publication the efficient use of vitrified mouse unfertilized oocytes for their routine application to IVF techniques, in combination with fresh, refrigerated and frozen sperm, with high success. The resulting 2-cell embryos obained were transferred to recipients and also gave rise to pups at high rates. The popularization of these vitrification methods for unfertilized oocytes will enable a more efficient planification for IVF sessions. Using the stock of vitrified unfertilized oocytes, IVFs can be programmed daily, and should not be limited to the strict superovulation schedules that usually result in IVFs done only once (on Thursday) or twice (Tuesday and Thursday) per week.

 

CARD-CNB Cryopreservation Course Report

Organizers, instructors, lecturers and participants at the CARD-CNB Cryopreservation Course, held at CNB-CSIC in Madrid, Spain, on 7-11 October 2013 and organized by Naomi Nakagata (CARD-University of Kumamoto, Japan) and Lluis Montoliu (CNB-CSIC, Madrid, Spain)
Organizers, instructors, lecturers and participants at the CARD-CNB Cryopreservation Course, held at CNB-CSIC in Madrid, Spain, on 7-11 October 2013 and organized by Naomi Nakagata (CARD-University of Kumamoto, Japan) and Lluis Montoliu (CNB-CSIC, Madrid, Spain)

This past week, 7-11 October 2013, the CARD-CNB Mouse Sperm and Embryo Cryopreservation Course was held at CNB-CSIC, in Madrid, Spain, with great success and accompanied with also great sunny weather. This was the first cryopreservation course of this kind, co-organized by Naomi Nakagata (CARD-University of Kumamoto, Japan) and Lluis Montoliu (CNB-CSIC, Madrid, Spain), were the newest methods developed by CARD, at the Nakagata lab, were demonstrated in Europe, directly by the CARD team. The instructors at this CARD-CNB course were commanded by the CARD-University of Kumamoto team, from Japan (Toru Takeo, Kiyoko Fukumoto, Tomoko Kondo, Yukie Haruguchi, Yumi Takeshita, Yuko Nakamuta and Shuuji Tsuchiyama), and additional help and collaboration was provided from the Mouse Biology Program at UC-Davis, CA, USA (Kristy Kinchen), from INIA, Madrid, Spain (Raúl Fernández), from CIEMAT, Madrid, Spain (Jesús Martínez), from USA (Jorge Sztein), from Paratechs, Lexington, KY, USA (Barbara Stone) and from CNB-CSIC (Julia Fernández, María Jesús del Hierro, Marta Castrillo and Isabel Martín-Dorado), for several of the methods demonstrated. All instructors must to be praised for their deep knowledge, patience and extraordinary dedication and commitment towards the success of this course. Complementary and most interesting lectures were provided on a wide variety of topics related to the course main focus, including: animal welfare and regulations by Belén Pintado and Jorge Guillén; the history, fundaments and comparison of methods by Jorge Sztein; the effects of the in vitro culture of mouse embryos by Alfonso Gutiérrez-Adán; safety and handling issues of liquid nitrogen by Jesús Martínez; shipping frozen and refrigerated materials by Toru Takeo, databases in a cryopreservation lab, by Shuuji Tsuchiyama, about EMMA by Lluis Montoliu and CARD by Naomi Nakagata, as examples of mouse embryo and sperm archives, and, also, on the new editing nucleases for genome modification, by Kai Schönig (Mannheim, Germany), a talk sponsored by Sigma.

As many as 24 participants, coming from research institutions or companies located in 16 countries around the world (UK, Spain, Australia, USA, Canada, Czech Republic, Brazil, Finland, France, Denmark, Israel, Netherlands, Portugal, Sweden, Italy and Taiwan) were presented with the latest advances in mouse sperm and embryo cryopreservation and all associated mouse reproductive biology ancillary techniques.

The topics covered by the course included the following major areas: obtaining sperm from mouse cauda epididymis, obtaining unfertilized mouse oocytes, three different types of in vitro fertilization techniques (using fresh, frozen or refrigerated sperm), vitrification of unfertilized oocytes and 2-cell embryos (freezing and thawing), slow-method for freezing and thawing 2-cell embryos, refrigerated sperm and 2-cell embryos, abdominal and scrotal vasectomies, three types of embryo transfer (oviduct, uterus and non-surgical, with NSET tools), freezing and thawing mouse sperm and ICSI, among many additional common methodologies used to handle mouse embryos and gametes adequately.

The course was very intensive, but the kind atmosphere created by participants and instructors was excellent and, hence, all the tight and carefully devised demonstrations and practices, packed within a very busy schedule, could be run smoothly and successfully without problems. The vast experience in running this type of cryopreservation courses and the remarkable professionality of our colleagues from CARD-University of Kumamoto were key for the accomplished success. All methods followed their three-step learning process. At first, the theory and fundaments were briefly provided and summarized. Then, the method was demonstrated by instructors and, finally, the participants executed the procedures by themselves, with the help of instructors.

The participants left this cryopreservation course to return to their countries and institutions with the most satisfactory results obtained and with plenty of new information to digest, process and reproduce. All participants were given the task to spread the word and disseminate the use of these highly efficient and robust cryopreservation techniques that have boosted the field.

This CARDCNB cryopreservation course was sponsored by the International Society for Transgenic Technologies (ISTT) and received the co-sponsorship and support from a number of companies whose contributions need to be greatly acknowledged as well: Leica, Charles River, Sigma, Labotect, Cosmo-Bio, Kyudo, Harlan and Paratechs.

Numerous ISTT events in June 2013

Numerous ISTT events in June 2013
Numerous ISTT events in June 2013

The International Society for Transgenic Technologies (ISTT) will be participating and/or co-sponsoring numerous events during the month of June 2013. At first, on June 7, 2013, our ISTT colleagues from Nantes (France), Ignacio Anegon and Séverine Ménoret, experts in the generation of transgenic rats, will be holding their 2013 Nantes Transgenic meeting on “Technical advances in the generation of transgenic animals and in their applications“. Next, on June 10-13, 2013, in Barcelona (Spain), the 12th FELASA-SECAL congress will take place, where the ISTT will be participating in two ways. First, the ISTT will co-sponsor the satellite workshop on Mouse Sperm Cryopreservation, to be held within the 2013 FELASA meeting, on 10 June 2013, Barcelona, Spain, and organized by ISTT members Jorge Sztein (NIH, USA) and Jesús Martínez-Palacio (CIEMAT, Madrid, Spain). Second, the ISTT will be participating as exhibitor and will attend the 2013 FELASA meeting. The ISTT will have a booth in Barcelona (#230), manned by the ISTT administrative assistant, Alison Cameron, and where all ISTT members (and non-ISTT members) are welcome to visiting us. Finally, immediately next, our ISTT colleagues from The Netherlands, Marian Van Roon (VU, Amsterdam) and Sjef Verbeek (LUMC, Leiden), have organized their 2013 Workshop on Innovative Mouse Models (IMM2013). This 7th Workshop on Innovative Mouse Models will be held on 13-14 June 2013, at the Leiden University Medical Center, LUMC, Leiden, The Netherlands, and the ISTT will be co-sponsoring also this event. ISTT members will be entitled to reduced registrations at all these events, proudly co-sponsored by the ISTT.

CARD-CNB Mouse Sperm and Embryo Cryopreservation Practical Course, Madrid, Spain, 7-11 October 2013

CARD-CNB Mouse Sperm and Embryo Cryopreservation Practical Course, Madrid, Spain, 7-11 October 2013
CARD-CNB Mouse Sperm and Embryo Cryopreservation Practical Course, Madrid, Spain, 7-11 October 2013

The International Society for Transgenic Technologies (ISTT) has agreed to co-sponsor the CARD-CNB Mouse Sperm and Embryo Cryopreservation Practical Course that will be held at the National Centre for Biotechnology (CNB-CSIC), in Madrid, CSIC, during one week, on 7-11 October 2013, organized by Naomi Nakagata (CARD-Kumamoto University, Japan, Coordinator of CARD) and Lluis Montoliu (CNB-CSIC, Madrid, Spain, Coordinator of the Spanish EMMA node).

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… Pre-applications should be submitted by email to cryocourse@cnb.csic.es by 31 May 2013.

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. Pre-applications will be accepted until 31 May 2013. The review and selection of participants will be done by the Organizers from 1 to 15 June 2013. Registrations and payments for selected participants will be accepted from June 16, 2013 to August 31, 2013. If required, an ordered waiting list will be prepared and any cancellation or unpaid registration by 31 August 2013 will be readily substituted by the first available person from this waiting list, starting on 1 September 2013.

The course registration fee is 800 Euros (with a reduced fee of 750 Euros for ISTT Members). This fee includes participation in the entire course, all materials and reagents, lunches over the 5 days and one course official dinner. Hotel costs are not included in the registration fee but booking assistance will be provided, if required, at a convenient nearby hotel, close to CNB Campus, where all instructors and lecturers will be also lodged, hence further promoting interaction from breakfast to dinner. The official language of the course will be English.

COURSE INFORMATION: Recent developments from the laboratory of Prof. Naomi Nakagata (CARD-Kumamoto University, Japan) have boosted the mouse cryopreservation field with improved methods for fresh and frozen sperm techniques and associated optimized IVF methods that have resulted in unparalleled increased efficiencies for the cryopreservation and rescue of relevant mouse lines. At the CNB-CSIC in Madrid, hosting the Spanish EMMA node, these new CARD cryopreservation methods have been successfully implemented and, moreover, a fruitful collaboration has been established with the laboratory of Prof. Naomi Nakagata, eventually resulting in the signature of an institutional cooperation agreement between the CSIC and the University of Kumamoto, under the framework of which this cryopreservation course is organized. The aim of this course is to introduce the new CARD methods to researchers and technicians involved in managing mouse archiving and/or transgenic facilities and willing to implement these new methods, directly taught by the team which devised them. Each participant will have one stereomicroscope and the entire set of tools, reagents and animals required to learn and practice all the methods included in the program of this course. 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. The number of instructors and lecturers appointed is 20.

Hands-on topics that will be covered during this cryopreservation course

  • making pipettes and practising embryo handling
  • 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
  • vasectomy of male mice
  • demonstration of ICSI

Additional lectures on the following topics

  • new EU Directive on the protection of animals for experimentation
  • new US Guidelines for the use of animals in research
  • epigenetic effects of in vitro culture on mouse embryos
  • databases for handling information in cryopreservation facilities
  • shipping mice, refrigerated and frozen material
  • safety issues handling liquid nitrogen in a cryopreservation facility
  • EMMA and Infrafrontier-I3
  • MMRRC

Instructors

  • Naomi Nakagata (CARD-Kumamoto University, Japan)
  • Toru Takeo (CARD-Kumamoto University, Japan)
  • Kiyoko Fukumoto (CARD-Kumamoto University, Japan)
  • Tomoko Kondo (CARD-Kumamoto University, Japan)
  • Yukie Haruguchi (CARD-Kumamoto University, Japan)
  • Yumi Takeshita (CARD-Kumamoto University, Japan)
  • Yuko Nakamuta (CARD-Kumamoto University, Japan)
  • Shuji Tsuchiyama (CARD-Kumamoto University, Japan)
  • Raul Fernández (INIA, Madrid, Spain)
  • Lluis Montoliu (CNB-CSIC, Madrid, Spain)
  • Julia Fernández (CNB-CSIC, Madrid, Spain)
  • María Jesús del Hierro (CNB-CSIC, Madrid, Spain)
  • Marta Castrillo (CNB-CSIC, Madrid, Spain)
  • Isabel Martín-Dorado (CNB-CSIC, Madrid, Spain)
  • Kristy Kinchen (Mouse Biology Program, UC Davis, CA, USA)

Additional lectures by

  • Naomi Nakagata (CARD-Kumamoto University, Japan)
  • Toru Takeo (CARD-Kumamoto University, Japan)
  • Shuji Tsuchiyama (CARD-Kumamoto University, Japan)
  • Alfonso Gutiérrez-Adán (INIA, Madrid, Spain)
  • Kent Lloyd (Mouse Biology Program, UC Davis, CA, USA)
  • Belén Pintado (CNB-CSIC, Madrid, Spain)
  • Jesús Martínez Palacio (CIEMAT, Madrid, Spain)
  • Javier Guillén (AAALAC, Pamplona, Spain)
  • Lluis Montoliu (CNB-CSIC, Madrid, Spain)