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

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)

Transgenic Technologists form a network across Oceania

Transgenic Technologists form a network across Oceania
Transgenic Technologists form a network across Oceania

The 1st Oceania Transgenic/Cryopreservation Symposium took place at the University of Queensland, Brisbane Australia on the 31st October -1st November 2013. This meeting was initiated by Elizabeth Williams and has grown out of workshops on cryopreservation of reproductive materials and in-vitro fertilization (IVF) that were run in conjunction with ANZLAA annual conferences (Hobart 2011 & Brisbane 2012). Through these activities it was recognized that there was a need for more local networking, collaboration and communication amongst transgenic technologists across Australia and New Zealand.

The meeting drew 53 participants representing twenty-six different organisations. 14 of the participants were locals from the Brisbane area, but there were also 33 interstate and 6 international delegates; mostly from New Zealand, but with one colleague visiting from Japan. The Australian Institute for Biotechnology and Nanotechnology generously provided the meeting venue and facilities.

The organizers Elizabeth Williams (University of Queensland Biological Resources, Brisbane), Karen Brennan (Victor Chang Cardiac Research Institute, Sydney), Andrew Brown (University of Auckland, Auckland), Paul Scowen (University of Tasmania, Hobart) and Kevin Taylor (Australian Bioresources, Moss Vale), all ISTT members, had canvassed topics from potential participants ahead of the meeting and ran informal discussions under the session headings Cryopreservation, Transgenic and Knock-Out Production, Administrative Issues, Husbandry, New Technologies/Techniques, and Rederivation.

Read the full meeting report prepared by ISTT council member Karen Brennan.

Transgenic Technologists form a network across Oceania
Transgenic Technologists form a network across Oceania

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.

Mouse sperm cryopreservation: satellite workshop at the 2013 FELASA-SECAL Congress in Barcelona, Spain, 10 June 2013

Mouse sperm cryopreservation: satellite workshop at the 2013 FELASA-SECAL Congress in Barcelona, Spain, 10 June 2013
Mouse sperm cryopreservation: satellite workshop at the 2013 FELASA-SECAL Congress in Barcelona, Spain, 10 June 2013

The International Society for Transgenic Technologies (ISTT) is pleased to announce the approved co-sponsorship of the Mouse Sperm Cryopreservation satellite workshop, organized by Jorge Sztein (NIH, Rockville, MD, USA) and Jesús Martínez Palacio (CIEMAT, Madrid, Spain), both ISTT members, in Barcelona (Spain) on 10 June 2013, within the activities associated to the 2013 FELASA-SECAL Congress in Barcelona, Spain, 10-13 June 2013. The main objective of this workshop is to acquaint students on reliable methods of mouse sperm cryopreservation, Jax and Nakagata, without requiring appliances or large investments to establishing a program at their centers. This course involves manipulation of animals and LN2.

This half-day satellite workshop will be held on 10 June 2013 from 09.00 to 12.00 h. at the Faculty of Medicine, University of Barcelona, Experimental Animal Unit. Satellite workshop registration fee: €120. Seats are limited to 24 participants. ISTT members, including those already registered to attend this workshop, are entitled to 25% discount. Interested participants should contact: felasa2013-registrations@mondial-congress.com

 

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)

The 2nd ISTT Young Investigator Award goes to Toru Takeo

Dr. Toru Takeo (Center for Animal Resources and Development, Kumamoto University, Japan). 2nd ISTT Young Investigator Award.
Dr. Toru Takeo (Center for Animal Resources and Development, Kumamoto University, Japan). 2nd ISTT Young Investigator Award.

The International Society for Trangenic Technologies (ISTT) is pleased to announce the second (2nd) ISTT Young Investigator Award, generously sponsored by inGenious Targeting Laboratory. The Award Committee carefully evaluated the excellent merits presented by all nominated candidates and decided to select Dr. Toru Takeo (Center for Animal Resources and Development, Kumamoto University, Japan) for the second ISTT Young Investigator Award. Dr. Toru Takeo was nominated by Dr. Jan Parker-Thornburg (ISTT Member, ISTT Council). The Award committee considered that Dr. Toru Takeo’s work represented a major improvement of mouse sperm cyropreservation and IVF techniques, thereby greatly facilitating the archiving and sharing of many mouse models produced by the transgenic community.

Dr. Toru Takeo, working in Dr. Naomi Nakagata’s laboratory, at CARD-KU, in Kumamoto (Japan), described three major technical improvements for boosting the efficiency of mouse sperm cryopreservation and the subsequently required IVF technique. The three fundamental modifications he reported in his recent publications tackled all three important steps in sperm cryopreservation and IVF, namely: (1) improving the cryopreservation of spermatozoa by adding L-Glutamine to the cryoprotective agent (CPA); (2) improving the number of capacitated sperm, upon thawing and pre-incubating them with Methyl-Beta-CycloDextrin (MBCD) by reducing the cholesterol levels in plasma membrane and increasing fertilization rate; and, (3) improving the in vitro fertilization (IVF) step itself, by adding reduced glutathione (GSH) to the IVF medium thereby increasing periviteline space in oocytes and the number of free thiols in the zona pellucida (ZP), greatly facilitating ZP penetration by sperm cells and, hence, fertilization. The three modifications, when jointly applied to some common mouse inbred strains, traditionally difficult to archive due to known poor sperm cryopreservation performance and limited IVF success, like C57BL/6J mice, boosted IVF efficiencies to >80%, and resulted in a true revolution in the transgenic mouse community and public archives. For example, at the European Mouse Mutant Archive (EMMA), a number of nodes have already updated their routine sperm cryopreservation and IVF methods according to Dr. Toru Takeo’s reports and succeeded in reproducing CARD innovations.

Dr. Toru Takeo’s three main publications on this subject are:

Takeo T, Nakagata N.
Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin.
Biol Reprod. 2011 Nov;85(5):1066-72.

Takeo T, Nakagata N.
Combination medium of cryoprotective agents containing L-glutamine and methyl-{beta}-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6J mouse sperm.
Lab Anim. 2010 Apr;44(2):132-7.

Takeo T, Hoshii T, Kondo Y, Toyodome H, Arima H, Yamamura K, Irie T, Nakagata N.
Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells.
Biol Reprod. 2008 Mar;78(3):546-51.

Dr. Toru Takeo was born in 1979. In 2003, he obtained a Bachelor degree in Pharmaceutical Sciences at the University of Kumamoto and, subsequently, he continued his studies at the Graduate School of Pharmaceutical Sciences obtaining a Master degree in Pharmaceutical Sciences in 2005 and his PhD degree in Pharmaceutical Sciences in 2008. Thereafter, he has worked as postdoctoral researcher at the Division of Reproductive Engineeting, of the Center for Animal Resources and Development (CARD)-University of Kumamoto and, in 2010, he was promoted to Assistant Professor. He currently holds several professional memberships at various Societies, including the Japanese Association for Laboratory Animal Science, the Japan Pharmaceutical Association, The Pharmaceutical Society of Japan and the Society for Reproduction and Development, Japan, and his excellent work has already been awarded several times, including his most recent 2012 Young Investigator Award, by the Japanese Association for Laboratory Animal Science.

The 2012 ISTT Young Investigator Award Committee was formed by Dr. Lluis Montoliu (President of ISTT)-Chair, Dr. Thom Saunders (Vice-President of ISTT), Dr. Boris Jerchow (ISTT Council member), Dr. Paul Sheiffele (iTL CEO) and Dr. Lisa Aronov (iTL).

Dr. Toru Takeo will attend the next 11th Transgenic Technology (TT2013) meeting where he will receive the corresponding diploma and will deliver a short talk summarizing his scientific achievements that led him to receive this 2nd ISTT Young Investigator Award, sponsored by inGenious Targeting Laboratory (iTL).

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the ISTT Young Investigator Award, sponsored by iTL
the ISTT Young Investigator Award, sponsored by iTL

	

EMMA Cryopreservation Workshop in Madrid: a meeting report

EMMA Cryopreservation Workshop: a meeting report
EMMA Cryopreservation Workshop: a meeting report

The EMMA (European Mouse Mutant Archive) Cryopreservation Workshop took place earlier this week in Madrid, May 7-8, at the main campus of CSIC, with an excellent success, organized by EMMA, supported by the EC-7th Framework Programme and co-sponsored by the International Society for Transgenic Technologies (ISTT). Sixty participants from many countries around the world gathered to present and discuss, in depth, the latest approaches, methodologies and techniques available for the efficient cryopreservation of mouse strains, through embryo, sperm and ovary cryopreservation. In addition to invited speakers and invited participants, the workshop was attended by delegates from EMMA nodes and 12 ISTT members. As many as 21 talks were delivered, by selected invited speakers, representing the different major archiving iniatives currently existing (EMMA, MMRRC, The Jackson Laboratory, RIKEN, APN, CMMR, AMMRA) and the research and development initiatives, as well as state-of-art protocols in the field. Presentations, abstracts and pictures from this EMMA Cryopreservation Workshop are freely available to anyone interested, from the meeting web site, and can be also accessed from ISTT and EMMA web sites. The remaining presentations will be progressively added upon receiving the approval from the corresponding guest speakers.

At EMMA, we envisaged this cryopreservation workshop as a forum to brainstorm and discuss in depth the latest technological advances in cryopreservation, including sperm and embryo cryopreservation, updated IVF methods and related techniques as ovary cryopreservation, laser-assisted and piezo-driven ICSI, transportation of frozen material and other technical and logistic challenges relevant to the operation of current mouse embryo/sperm archives. We believed we entirely fulfilled the expectations and all participants went back home, to their research institutions, loaded with new ideas, updated solutions and suggested improvements that can be explored and applied for a most efficient management of a mouse embryo/sperm cryopreservation bank. All participants agreed to continue organizing this type of focused workshops in the near future. The ISTT will be always there, ready to support these very interesting initiatives.

Lluis Montoliu, EMMA Spanish node

Slides Available for Webinar on Optimizing Quality Assurance for Mouse Sperm Freezing and Rederivation using Computer Assisted Sperm Analysis

Hamilton-Thorne Webimar, 29 March 2012
Hamilton-Thorne Webimar, 29 March 2012

On Thursday, March 29th, 2012, our colleagues at Hamilton Thorne hosted a webinar titled: “Optimizing Quality Assurance for Mouse Sperm Freezing and Rederivation using Computer Assisted Sperm Analysis” This webinar was delivered by ISTT members, Lily Reed and Ian Rosewell as well as Hamilton Thorne’s Kate Craig.

Hamilton Thorne has been generous enough to provide ISTT members with the slides from this webinar presentation which you can find in the Methods section of the Members-Only portion of the ISTT website:

Thank you to Hamilton Thorne for your continued support for the ISTT and it’s members!