Manual – LEGACY ISTT blog

New book published: Genetics of the Mouse (Springer, 2015)

New book published: Genetics of the Mouse. Editors: JL Guénet, F Benavides, JJ Panthier and X Montagutelli (Springer 2015)

A new book has been published in the field of mouse genetics, filling a gap created since the mid nineties, when the last similar books had been published (i.e. Lee Silver, 1995), devoted to this topic. This new book, entitled “Genetics of the Mouse” has been edited by four experiencied mouse geneticists: Jean-Louis Guénet, Fernando Benavides, Jean-Jacques Panthier and Xavier Montagutelli, and has been published by Springer (2015). The authors are all related with Institut Pasteur, in Paris, France, where Jean-Louis Guénet worked many years (currently retired), where Fernando Benavides (ISTT member and at MD Anderson Cancer Center, Smithville, TX, USA) had also worked, under the supervision of Jean-Louis Guénet, and where Jean-Jacques Panthier and Xavier Montagutelli have currently their laboratories. Jean-Louis Guénet and Fernando Benavides had also co-authored a previous text on a similar subject (Genetics of Rodents), in Spanish, published in 2003 and freely available from the ISTT web page since 2011.

This new book contains 10 chapters, as follows:

Origins of the Laboratory Mouse
Basic Concepts of Reproductive Biology and Genetics
Cytogenetics
Gene Mapping
The Mouse Genome
Epigenetic Control of Genome Expression
Mutations and Experimental Mutagenesis
Transgenesis and Genome Manipulations
The Different Categories of Genetically Standardized
Quantitative Traits and Quantitative Genetics

As indicated in the Springer’s book web page: ‘This book, written by experienced geneticists, covers topics ranging from the natural history of the mouse species, its handling and reproduction in the laboratory, and its classical genetics and cytogenetics, to modern issues including the analysis of the transcriptome, the parental imprinting and X-chromosome inactivation. The strategies for creating all sorts of mutations, either by genetic engineering or by using mutagens, are also reviewed and discussed in detail. Finally, a last chapter outlines the methodology used for the analysis of complex or quantitative traits. The authors also discuss the importance of accurate phenotyping, which is now performed in the mouse clinics established worldwide and identify the limits of the mouse model, which under certain circumstances can fail to present the phenotype expected from the cognate condition in the human model. For each chapter an up-to-date list of pertinent references is provided. In short, this book offers an essential resource for all scientists who use or plan to use mice in their research.‘

This book is available from Springer and from Amazon

Mouse Genetics. Methods and Protocols (2014)

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

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

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

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

Transgenic Animal Technology. A Laboratory Handbook (3rd edition, 2014)

Twenty years after the publication of the first edition and twelve years after the release of the second edition of this book, Carl A. Pinkert (Auburn University, College of Veterinary Medicine, Auburn, AL, USA) in association with Elsevier, releases now the third edition of his famous transgenic manual: “Transgenic Animal Technology. A Laboratory Handbook. 3rd edition, 2014“. As it will be familiar to readers of the two previous editions of this useful and unique handbook, this is not only a manual to understand how to make a transgenic mouse. This handbook looks beyond mice and it contains protocols to prepare a wide variety of genetically-modified animals, including: rats, rabbits, poultry, fish, pigs, ruminants and non-human primates. In addition, this compilation of helpful methods includes a number of chapters devoted to understand and improve all steps of transgenesis, from vector design, analysis of transgene integration and the evaluation of transgene expression. Finally, the book also includes cryopreservation methods, an embryo culture section, a review of standard nomenclature and a selection of databases and internet resources currently available in the field.

This handbook is a worth addition to any library, laboratory or transgenic facility, complementary to other available manuals on the subject, but unique in the sense that it exquisitely illustrates current transgenic methods that can be applied to a wide variety of animal species, beyond mice.

Carl A. Pinkert has been helped in his outstanding Editorial task by a large group of co-authors, experts in their subjects, including some ISTT members: Satoshi Akagi, Anna V. Anagnostopoulos, Benjamin P. Beaton, Cory F. Brayton, Steve Brown, Anthony W.S. Chan, Tom Doetschman, Rex A. Dunham, David A. Dunn, Janan T. Eppig, Almudena Fernandez, Tatiana Flisikowska, Vasiliy Galat, Robert A. Godke, Philip Iannaccone, Michael H. Irwin, Larry W. Johnson, Yoko Kato, Teoan Kim, Alexander Kind, Bon Chul Koo, Mo Sun Kwon, Daniel J. Ledbetter, Michael J. Martin, Kazutsugu Matsukawa, Colin McKerlie, Lluis Montoliu, Paul E. Mozdziak, Akira Onishi, Paul A. Overbeek, James N. Petitte, L. Philip Sanford, Jorge A. Piedrahita, Wendy K. Pogozelski, H. Greg Polites, Edmund B. Rucker III, Marina Sansinena, Angelika Schnieke, Kumiko Takeda, James A. Thomson, Ian A. Trounce, Yukio Tsunoda, Cristina Vicente-Garcia, Kevin D. Wells, Richard N. Winn and Curtis R. Youngs.

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

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

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

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

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

The new (2014) FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units have been released as open access document from Laboratory Animals journal web page

The new (2014) FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units have been released as open access document from Laboratory Animals journal web page. This updated reference document has been prepared by the FELASA working group on revision of guidelines for health monitoring of rodents and rabbits, formed by Michael Mähler (Convenor, GV-SOLAS), Marion Bérard (AFSTAL), Ricardo Feinstein (Scand-LAS), Alec Gallagher (LASA), Brunhilde Illgen-Wilcke (SGV), Kathleen Pritchett-Corning (AALAS) and Marcello Raspa (AISAL and ISTT Member). The previous and up-to-now current FELASA recommendations for the health monitoring of rodents and rabbits had been published in 2002 (Nicklas et al. 2002, Laboratory Animals). Therefore this new document, released in 2014, substitutes and updates the previous guidelines reported 12 years ago.

As it is stated in the abstract of this updated 2014 FELASA recommendations document: “These recommendations are aimed at all breeders and users of laboratory mice, rats, Syrian hamsters, guinea pigs and rabbits as well as diagnostic laboratories. They describe essential aspects of health monitoring, such as the choice of agents, selection of animals and tissues for testing, frequency of sampling, commonly used test methods, interpretation of results and health monitoring reporting“. FELASA and, in particular, the co-authors of this compelling updated recommendations for health monitoring of rodents and rabbits need to be praised for this work. This document will not only help and contribute to the desired harmonization of the health monitoring programmes across animal facilities but also it will provide important elements for the correct preparation and interpretation of Health Monitoring results.

Winner of ISTT Manual from bioterios.com

Ana Isabel Nieto (Granada, Spain), winner of a copy of the ISTT Manual (Springer, 2011) from bioterios.com

Ana Isabel Nieto (Granada, Spain) is the winner of a copy of the ISTT Manual:Advanced Protocols for Animal Transgenesis (edited by Shirley Pease and Thomas Saunders, published by Springer, 2011) from bioterios.com, the most popular South-American web portal for Laboratory Animal Science, in Spanish, organized and led by the ISTT member Juan Manuel Baamonde (CECs, Valdivia, Chile). The International Society for Trangenic Technologies (ISTT) and bioterios.com are mutually supporting and promoting their activities. The web portal bioterios.com has been referred previously from this ISTT blog, and also the launching of their new web layout, earlier this year. Likewise, the ISTT and the TT2014 meeting have also been promoted from bioterios.com.

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)

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.

BIOTERIOS.COM a web portal for Laboratory Animal Science in Spanish

The International Society for Transgenic Technologies (ISTT) recently co-sponsored a mouse meeting in Chile, at the CECs, in Valdivia, organized by Juan Manuel Baamonde (Manager of the CECs Animal Facility) and his colleagues. Juan M. Baamonde is also the creator and the soul behind a most interesting initiative worth knowing: the web portal BIOTERIOS.COM, an impressive web site containing plenty of information about many different aspects of laboratory animals, with texts, protocols, opinions, links to other sites and societies, meant to share information, to provide educational tools, resources and, in general, to trigger a discussion forum on the care and use of animals in research, and all the associated aspects that need to be taken into account, conforming what we usually know as Laboratory Animal Science.

One of the remarkable features of BIOTERIOS.COM is the language used, Spanish, uncommon for this type of initiatives in our field, mostly dominated by resources created in English, for obvious reasons. Therefore the BIOTERIOS.COM web portal is made in Spanish, a language that, according to most estimates, is the second most-spoken language in the world, after the Chinese (Mandarin), and close to English, the third most-used language. Some 400 million people use Spanish as their first language. This fact, along with all the rich and useful contents included, makes BIOTERIOS.COM a reference web site for Latin-American countries and Spain as a useful resource for Laboratory Animal Science in Spanish. Juan M. Baamonde, currently the General Editor of BIOTERIOS.COM, and a most recent member of the ISTT, must be praised for his pioneer idea and for his continuous efforts promoting contents and discussions in this web portal. More than 10% of ISTT Members commonly use or can understand Spanish (including our colleagues from Brazil and Portugal, whose mother language is Portughese). They all can benefit from this web portal, BIOTERIOS.COM, to follow the latest updates on Laboratory Animal Science.

The transgenic-list (tg-l) has been moved to ISTT

Today, December 21st, 2011, the historical transgenic-list (tg-l), created by Peter Sobieszczuk more than 15 years ago, in July, 1996, has been moved and successfully transferred to its new home at the ISTT web server, following the agreement signed on September 30th, 2011, between Peter and the ISTT. All current subscribers have been transferred automatically and they will be able to continue using and enjoying this useful resource within the field of transgenic animals. This is the new tg-l home web page, where additional information about this transfer and the new features of the tg-l at the ISTT can be browsed. At the ISTT, the tg-l will continue being public (anyone may subscribe, including ISTT members and non-members alike, but subject to email address verification), unmoderated (messages will not be altered by the list administrator) and closed (only subscribers may post messages). All questions, comments, requests, doubts should be addressed to the new tg-l administrators at: tglist_admin@transtechsociety.org.

Advanced Protocols for Animal Transgenesis. An ISTT Manual

The International Society for Transgenic Technologies (ISTT) is most pleased to announce the publication of its first book, entitled “Advanced Protocols for Animal Transgenesis. An ISTT Manual“, edited by Shirley Pease and Thom Saunders, and published by Springer (2011), in collaboration with the ISTT. This laboratory manual provides almost all current methods that can be applied to the creation and analysis of genetically modified animals. The chapters have been contributed by leading scientists, most of them members of the ISTT, who are actively using the technology in their laboratories. Based on their first-hand experience the authors also provide helpful notes and troubleshooting sections.

Topics range from standard techniques, such as pronuclear microinjection of DNA, to more sophisticated and modern methods, such as the derivation and establishment of embryonic stem (ES) cell lines, with defined inhibitors in cell culture medium. In addition, related topics with relevance to the field are addressed, including global web-based resources, legal issues, colony management, shipment of mice and embryos, and the three R’s: refinement, reduction and replacement. This book comprises 26 chapters, 109 illlustrations and 669 pages.

Table of contents, chapters of this ISTT Manual:

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

ISTT Members are entitled to purchase this ISTT Manual with a 33% discount, through the members-only area of the web site.