Preface Publication of a Guide to Techniques in Mouse Development is timely in view of the already enormous and rapidly growing interest in the mouse as an experimental organism. Perhaps nowhere is the impact of technol- ogy on developmental biology seen so clearly as in the case of research on mouse development. The recently acquired ability to add specifically en- gineered genes to the mouse genome by the production of transgenic animals, as well as to remove ("knockout") specific genes from the mouse genome by homologous recombination in embryonic stem (ES) cells, has virtually revolutionized the field. Genetic manipulations that not so long ago were feasible only with nonmammals, such as fruit flies and worms, are now performed routinely with mice. A plethora of new meth- odology is available that can be applied to classical questions involving cellular behavior during mouse development; such questions can now be addressed at the level of individual genes, messenger RNAs, and pro- teins. Our purpose in assembling this volume is to create a source of state-of- the-art experimental approaches in mouse development useful at the labo- ratory bench to a diverse group of investigators. The aim is to provide investigators with reliable experimental protocols and recipes that are described in sufficient detail by leaders in the field. Although technology in this area is changing rapidly, it is likely that much of the Guide will remain relevant for many years to come. We extend our thanks to the authors for their contributions as well as for their cooperation and patience during the preparation of the volume. Also, we are grateful to our colleagues Tom Gridley, Andy McMahon, and Colin Stewart who provided good advice throughout this long ven- ture. PAUL M. WASSARMAN MELVIN L. DEPAMPHILIS xvii Contributors to Volume 225 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. SUSAN J. ABBONDANZO (49), Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Re- search Center, Nutley, New Jersey 07110 MAY! Y. ARCELLANA-PANLILIO (18), De- partment of Medical Biochemistry, Uni- versity of Calgary Health Sciences Cen- ter, Calgary, Alberta, Canada T2N 4N1 CHRISTOPHER P. AUSTIN (56), Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 SHEILA C. BARTON (44), Wellcome/CRC In- stitute of Cancer and Developmental Biol- ogy, University of Cambridge, Cam- bridge CB2 1QR, England ANTHONY R. BELLVI~ (6, 7), Departments of Anatomy and Cell Biology and Urology, and Center for Reproductive Sciences, College of Physicians and Surgeons, Co- lumbia University, New York, New York 10032 JOHN D. BIGGERS (9), Department of Cellu- lar and Molecular Physiology, Labora- tory of Haman Reproduction and Repro- ductive Biology, Harvard Medical School, Boston, Massachusetts 02115 JEFFREY D. BELIE (14), Department of Mo- lecular Biology, The Scripps Research In- stitute, La Jolla, California 92037 CLAIRE BONNEROT (27, 28), Unitd de Biolo- gie Moldculaire du Ddveloppement, Unitd Associde 1148 da Centre National de la Recherche Scientifique, 75724 Paris Ce- dex 15, France ALLAN BRADLEY (51), Institute for Molecu- lar Genetics, Baylor College of Medicine, Houston, Texas 77030 GERARD BRADY (36), Molecular Pharma- cology, School of Biological Sciences, University of Manchester, Manchester M13 9PT, England PASCALE BRIAND (27), Laboratoire de Gdndtique et Pathologie Expdrimentales, INSERM, lnstitut Cochin de Gdndtique Mol(culaire, 75014 Paris, France MIA BUEHR (4), Institut for Molekylaer Biologi, ,~rhus Universitet, DK-8000 Arhus C, Denmark QIu PING CAO (19), Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545 RICHARD A. CARDUELO (8), Department of Biology, University of California, River- side, Riverside, California 92521 CONSTANCE L. CEFKO (56), Department of Genetics, Harvard Medical School, Bos- ton, Massachusetts 02115 KERRY B. CLEGG (15), Developmental Biol- ogy Laboratory, Veterans Administration Medical Center, Sepulveda, California 91343 RONALD A. CONLON (23), Samuel Lunen- reid Research Institute, Mount Sinai Hos- pital, Toronto, Ontario, Canada M5G 1X5 JULIE E. COOKE (3), Wellcome/CRC Insti- tute, Cambridge University, Cambridge CB2 1QR, England ROGER D. Cox (38), Genome Analysis Lab- oratory, Imperial Cancer Research Fund, London WC2A 3PX, England WILLIAM R. CRAIN (19), McLaughlin Re- search Institute for Biomedical Sciences, Great Falls, Montana, 59401 ANN C. DAVIS (51), Institute of Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030 CLAYTUS A. DAVIS (31), Division of Biol- ogy, California Institute of Technology, Pasadena, California 91125 xi xii CONTRIBUTORS TO VOLUME 225 JULIE A. DELOIA (35), Department of Phys- iology, University of Pittsburgh, Magee- Womens Hospital, Pittsburgh, Pennsyl- vania 15213 MELVIN L. DEPAMPHIEIS (25, 26, 30), De- partment of Cell and Developmental Biol- ogy, Roche Institute of Molecular Biol- ogy, Roche Research Center, Nutley, New Jersey 07110 JOHN J. EPPIG (5), The Jackson Laboratory, Bar Harbor, Maine 04609 DONNA M. FEKETE (56), Department of Bi- ology, Boston College, Chestnut Hill, Massachusetts 02167 CHARLES FFRENCH-CONSTANT (3), Wellcome/CRC Institute, Cambridge Uni- versity, Cambridge CB2 1QR, England P. A. FLECKNELL (2), Comparative Biology Centre, University of Newcastle upon Tyne, Medical School, Newcastle upon Tyne NE2 4HH, England HARVEY M. FLORMAN (8), Worcester Foun- dation for Experimental Biology, Shrews- bury, Massachusetts 01545 LYNN R. FRASER (13), Anatomy andHuman Biology Group, Biomedical Sciences Divi- sion, King's College London, University of London, Strand, London WC2R 2LS, England GLENN FRIEDRICH (41), Fred Hutchinson Cancer Research Center, Program in Mo- lecular Medicine, Seattle, Washington 91809. INDER GADI (49), Department of Genetics, Roche Biomedical Laboratories, Inc., Raritan, New Jersey 08869 JAMES I. GARRELS (29), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 MICHELLE F. GAUDETTE (19), Cell Biology Group, Worcester Foundation for Experi- mental Biology, Shrewsbury, Massachu- setts 01545 BRIAN J. GAVIN (39), Department of Recep- tor Mechanisms, Sandoz Research Insti- tute, Sandoz Pharmaceuticals Corpora- tion, East Hanover, New Jersey 07936 MAUREEN GENDRON-MAGUIRE (48), De- partment of Cell and Developmental Biol- ogy, Roche Institute of Molecular Biol- ogy, Roche Research Center, Nutley, New Jersey 07110 ISABELLE GODIN (3), Institut d'Embryolo- gie, Centre National de Recherche Scien- tifique et Colldge de France, Nogent-sur- Marne 94736, France JON W. GORDON (12, 45), Department of Obstetrics, Gynecology, and Reproduc- tive Science, Mount Sinai School of Medi- cine, New York, New York 10029 MARK GRANT (33), St. Edmund's College, Cambridge CB3, England THOMAS GRIDLEY (48), Department of Cell and Developmental Biology, Roche Insti- tute of Molecular Biology, Roche Re- search Center, Nutley, New Jersey 07110 JANET HEASMAN (3), Wellcome/CRC Insti- tute, Cambridge University, Cambridge CB2 1QR, England BERNHARD G. HERRMANN (23), Max- Planck Institut fiir Entwicklungsbiologie, Abteilung Biochemie, D-7400 Tiibingen, Germany DAVID P. HILL (40), Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Si- nai Hospital, Toronto, Ontario, Canada MSG 13(5 JOAQUIN HUARTE (21), Institute of Histol- ogy and Embryology, University of Ge- neva Medical School, CH-1211 Geneva 4, Switzerland NORMAN N. ISCOVE (36), Ontario Cancer Institute, Toronto, Ontario, Canada M4X 1K9 JOEL JESSEE (35), Life Technologies, Inc., Gaithersburg, Maryland 20877 DABNEY JOHNSON (35), Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Ross A. KINEOCH (17), Department of Cell and Developmental Biology, Roche Insti- tute of Molecular Biology, Roche Re- search Center, Nutley, New Jersey 07110 CONTRIBUTORS TO VOLUME 225 xiii BARBARA B. KNOWLES (35), The Jackson Laboratory, Bar Harbor, Maine 04609 FRANK KONTGEN (52), Cellular Immunol- ogy Unit, The Walter and Eliza Hall Insti- tute of Medical Research, The Royal Mel- bourne Hospital, Victoria 3050, Australia ZOIA LARIN (37, 38), Department of Bio- chemistry, University of Oxford, Oxford OX1 3QU, England KEITH E. LATHAM (29, 43), Fels Institute for Cancer Research and Molecular Biol- ogy, Temple University School of Medi- cine, Philadelphia, Pennsylvania 19140 JOEL A. LAWITTS (9), Transgenic Facility, Beth Israel Hospital, Boston, Massachu- setts 02115 HANS LEHRACH (37, 38), Genome Analysis Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, England SADHAN MAJUMDER (26), Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Re- search Center, Nutley, New Jersey 07110 JEFFREY R. MANN (46, 47), Division of Biol- ogy, Beckman Research Institute of the City of Hope, Duarte, California 91010 YORDANKA S. MARTINOVA (7), Institute of Cell Biology and Morphology, Bulgarian Academy of Sciences, 1113 Sofia, Bul- garia ANNE McLAREN (4), MRC Mammalian De- velopment Unit, Wolfson House, Univer- sity College London, London NW1 2HE, England K. J. MCLAUGHLIN (55), Division of Biol- ogy, Beckman Research Institute of the City of Hope, Duarte, California 91010 ANDRE P. MCMAHON (39, 46), Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 SEBASTIAN MEIER-EWERT (37, 38), Genome Analysis Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, England MIRIAM MIRANDA (25, 26), Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Re- search Center, Nutley, New Jersey 07110 ANTHONY P. MONACO (37, 38), Human Ge- netics Laboratory, Imperial Cancer Re- search Fund, Institute of Molecular Medi- cine, John Radcliffe Hospital, Oxford OX3 9DU, England MAmLYN MONK (33), Institute of Child Health, London W1C1N 1EH, England JEAN-FRANCOIS NICOLAS (27, 28), Unit( de Biologie Mol(culaire du D(veloppement, Unit( Associ~e 1148 du Centre National de la Recherche Scientifique, 75724 Paris Cedex 15, France M. ANGELA NIETO (22), MRC Laboratory of Eukaryotic Molecular Genetics, Na- tional Institute for Medical Research, London NW7 1AA, England G. P. PFEIFER (34), Department of Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010 LAJOS PIKe5 (15, 16), DevelopmentalBiology Laboratory, Veterans Admnistration Medical Center, Sepulveda, California 91343 RAMIRO RAMfREz-SoLIS (51), Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030 WILLIAM G. RICHARDS (21), Department of Pharmacology, State University of New York at Stony Brook, Stony Brook, New York 11794 A. D. RIGGS (20, 34), Department of Biol- ogy, Beckman Research Institute of the City of Hope, Duarte, California 91010 RICHARD J. ROLLER (17), Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Re- search Center, Nutley, New Jersey 07110 NADIA ROSENTHAL (24), Cardiovascular Research Center, Massachusetts General Hospital East, Charlestown, Massachu- setts 02129 xiv CONTRIBUTORS TO VOLUME 225 MARK ROSS (38), Genome Analysis Labora- tory, Imperial Cancer Research Fund, London WC2A 3PX, England JAY L. ROTHSTEIN (35), Departments of Mi- crobiology~Immunology and Otolaryngol- ogy, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Penn- sylvania 19107 ELIZABETH F. RYDER (56), Department of Genetics, Harvard Medical School, Bos- ton, Massachusetts 02115 EERNANDO J. SALLI~S (21), Department of Pharmacology, State University of New York at Stony Brook, Stony Brook, New York 11794 DAVID SASSOON (24), Department of Bio- chemistry, Boston University School of Medicine, Boston, Massachusetts 02118 BRIAN SAUER (53), Biotechnology, Du Pont Merck Pharmaceutical Company, Wil- mington, Delaware 19880 GERALD SCHATTEN (32), Department of Zo- ology, University of Wisconsin, Madison, Wisconsin 53706 GILBERT A. SCHULTZ (18), Department of Medical Biochemistry, University of Cal- gary Health Sciences Center, Calgary, Alberta, Canada T2N 4N1 RACHEL I). SHEPPARD (42), Genetic Ther- apy, Inc., Immunology Group, Gaithers- burg, Maryland 20878 LEE M. SILVER (1, 42), Department of Mo- lecular Biology, Princeton University, Princeton, New Jersey 08544 CALVIN SIMERLY (32), Department of Zool- ogy, University of Wisconsin, Madison, Wisconsin 53706 J. SINGER-SAM (20, 34), Department of Biol- ogy, Beckman Research Institute of the City of Hope, Duarte, California 91010 JACEK SKOWRONSKI (35), Cold Spring Har- bor Laboratory, Cold Spring Harbor, New York 11724 DAVOR SOLTER (29, 35, 43), Max-Planck In- stitut fiir Immunbiologie, D-7800 Frei- burg-Ziihringen, Germany PHILIPPE SORIANO (41), Fred Hutchinson Cancer Research Center, Program in Mo- lecular Medicine, Seattle, Washington 91809 COLIN L. STEWART (49, 50, 52), Depart- ment of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 SIDNEY STRICKEAND (21), Department of Pharmacology, State University of New York at Stony Brook, Stony Brook, New York 11794 KARIN STURM (10), Embryology Unit, Chil- dren's Medical Research Institute, Uni- versity of Sydney, Wentworthville NSW 2145, Australia M. AZIM SURANI (44), Wellcome/CRCInsti- tute of Cancer and Developmental Biol- ogy, University of Cambridge, Cam- bridge CB2 1QR, England PATRICK P. L. TAM (10, ll), Embryology Unit, Children's Medical Research Insti- tute, University of Sydney, Wentworth- ville NSW 2145, Australia KENT D. TAYLOR (16), Developmental Biol- ogy Laboratory, Veterans Administration Medical Center, Sepulveda, California 91343 EVELYN E. TELFER (5), Institute of Ecology and Resource Management, University of Edinburgh, School of Agriculture, Edin- burgh EH9 3JG, Scotland JEAN-DOMINIQUE VASSALLI (21), Institute of Histology and Embryology, University of Geneva Medical School, CH-1211 Ge- neva 4, Switzerland MURIAL VERNET (27), Laboratoire de Gdn~tique et Pathologie Expdrimentales, INSERM, Institut Cochin de Gdndtique Mol(culaire, 75014 Paris, France CHRISTOPHER WALSH (56), Department of Neurology, Beth Israel Hospital, Boston, Massachusetts 02115 CONTRIBUTORS TO VOLUME 275 xv PAUL M. WASSARMAN (17), Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Roche Re- search Center, Natley, New Jersey 07110 MARIA WIEKOWSKI (26, 30), Department of Molecular Biology, Schering-PIough Cor- poration, Kenilworth, New Jersey 07033 MICHAEL V. WILES (54), Basel Institute for Immunology, CH-4005, Basel, Switzer- land DAVID G. WILKINSON (22), MRC Labora- tory of Eukaryotic Molecular Genetics, National Institute for Medical Research, London NW7 1AA, England WOLFGANG WURST (40), Division of Molec- ular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Si- nai Hospital, Toronto, Ontario, Canada M5G 1X5 CHRISTOPHER C. WYLIE (3), Wellcome/CRC Institute, Cambridge University, Cam- bridge, CB2 1QR, England WENXIN ZHENG (7), Department of Pathol- ogy, New York Hospital Cornell Medical Center, New York, New York 10021 MAUR1ZIO ZUCCOTTI (33), Dipartimento Biologia Animale and Centro Studio per L'istochimica del CNR, University of Pa- via, Pavia 27100, Italy [1] MOUSE COLONY DATABASE MANAGEMENT 3 [1] Recordkeeping and Database Analysis of Breeding Colonies By LEE M. SILVER General Strategies for Recordkeeping Requirements A breeding mouse colony differs significantly from a static one in the type and complexity of information that is generated. In a nonbreeding colony, there are only the animals and the results obtained from experi- ments on each one. In a breeding colony, there are animals, matings, and litters, with specific connections among various members of each of these data classes. Classical genetic analysis is based on the transmission of information between generations, and, as a consequence, the network of associations among individual components of the colony is often as important as the components in and of themselves. An ideal recordkeeping system would allow one to keep track of (1) individual animals, their ancestors, siblings, and descendants; (2) experi- mental material (tissues and DNA samples) obtained from such animals; (3) matings between animals; (4) litters born to such matings, and the animals derived from such litters used in experiments or to set up the next generation of matings. Ideally, one would like to maintain records in a format that readily allows one to determine the relationship, if any, that exists between any two or more components of the colony, past or present. Based on these general requirements, two different systems for record- keeping have been developed by mouse geneticists over the last 60 years. The "mating unit" system centers on the mating pair as the primary unit for recordkeeping. The "animal/litter" system treats each animal and litter as a separate entity. As discussed below, there are advantages and disadvantages to each. In a later section, I describe a computer software version of the animal/litter system that can be implemented on a per- sonal computer. Mating Unit System With the mating unit system, each mating unit is assigned a unique number and is given an individual record. When recordkeeping is carried out with a notebook and pencil, each mating pair is assigned a page in Copyright © 1993 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 225 All rights of reproduction in any form reserved. 4 GENERAL METHODOLOGY [1] the book. The cage that holds the mating pair can be identified with a simple card on which the record number is indicated; this provides immedi- ate access to the corresponding page in the record book. When litters are born, they are recorded within the mating record. Each litter is normally given one line on which the following information is recorded: (1) a number indicating whether it is the first, second, third, or a subsequent litter born to the particular mating pair; (2) the date of birth; (3) the number of pups; and (4) other information of importance to the investigator. At a later time, individual mice within a litter can be identified (and recorded, if necessary, on further lines within the record page) by the mating number along with a secondary simple number or letter combination to distinguish siblings from each other. For example, the fourth pup in the third litter born to mating unit 7371 could be numbered 7371-3d, where "3" indicates the litter and "d" indicates the pup within it. This system provides for the individual numbering of animals in a manner that immediately allows one to identify siblings. At the outset, parental numbers are incorporated into each mating record, and since these are linked implicitly to the litters from which they come, it becomes possible to trace a complete pedigree back from any starting individual. It also becomes possible to trace pedigrees forward if, as a matter of course, one cross-references all new matings within the litter records from which the parents derive. For example, if one sets up a new mating assigned the number 8765 with female 5678-2e and male 5543-1c, the number 8765 would be inscribed on appropriate lines in re- cords 5678 and 5543. There are several important advantages to a recordkeeping system based on the mating unit: (1) only a single set of primary record numbers is required; (2) one can easily keep track of the reproductive history of each mating pair; and (3) information on siblings is readily viewed within a single location. The major disadvantage to this recordkeeping system is that, for most investigators, it is impossible to determine ahead of time how much space will be required ultimately for any one record. One mating may yield no litters, while another may be highly prolific and require more record space than was originally set aside. A second disad- vantage comes into play in those colonies where mating units are not infrequently taken apart and re-formed with new combinations of animals. In this situation, where the mating unit is not sacrosanct, the animal/litter system described below is more amenable for recordkeeping. Animal~Litter System In a second system developed originally by the geneticist L. C. Dunn, there are two primary units for recordkeeping the individual animal and [1] MOUSE COLONY DATABASE MANAGEMENT 5 the individual litter. Each breeding animal is assigned a unique number (at the time of weaning) that is associated with an individual record occupy- ing one line across one or two facing pages within an "animal record book." Each animal record contains the numbers of both parents, and through these numbers it is possible to trace back pedigrees. Each litter that is born is also assigned a unique number with an individual one-line record in a separate "litter record book." Both animal numbers and litter numbers are normally assigned in sequence. A third independent set of numbers are those assigned to individual cages. Cage numbers can be assigned in a systematic manner so that related matings are in cages with related numbers. For example, different matings that derive from the same founder of a particular transgenic line may be placed in cages numbered from 2311 to 2319. A second set of matings that carry the same transgene from a different founder could be placed into cages numbered 2321 to 2329, and so on. Thus, the cages between 2300 and 2399 would all have animals that carried the same transgene; however, different sets of ten would be used for different founder lines. For matings of animals with a second transgene, one might choose to use the cages numbered 2400 to 2499. This type of numbering allows one to classify cages, which represent matings, in a hierarchical manner. Although at any point in time, every cage in the colony will have a different number, once a particular cage is eliminated, its number can be reassigned to a new mating. Cage cards from dismantled matings are saved in numerical order. When a litter is born, the litter record is initiated with an identifying number, the birth date, the numbers of the parents, the number of the cage in which the litter was born, and any other important information. In addition, the litter number is inscribed on the cage card (which may or may not have additional information about the mating pair). When an animal is weaned from a litter for participation in the breeding program, an animal record is initiated. The most important information in the animal record is the number of the litter from which it came, the cage that it goes into, and the date of that move. The cage number is particularly important in allowing one to trace pedigrees forward from any individual at a future date. If an animal is moved from one cage to another at some later date, this can easily be added to the record. With three unrelated systems of number assignment and the need for extensive cross-referencing, the animal/litter system is complex, and implementation on paper is labor intensive. However, it does provide the investigator with additional power for analysis. For example, by choosing cage numbers wisely and saving cage cards in numerical order, it becomes possible to go back at any point in the future and look at all of the litters born to a particular category of matings over any period of time. With 6 GENERAL METHODOLOGY [1] the mating unit system, this can only be accomplished by using different record books, or different sections of a book, for different categories of matings. However, with a complex breeding program, it is often difficult to predict how much space a particular category of matings is likely to subsume over an extended period of time. This problem could be solved with the use of a loose-leaf book in which pages (representing mating units) can be added without limits to any particular section. Another difference between the mating unit system and the animal/ litter system is the ease with which it is possible to keep track of animals that are moved from one mating unit to another. The mating unit system is most effective for colonies where "animals are mated for life." The animal/litter system is effective for colonies of this type as well, but it is also amenable to those where animals are frequently switched from one mate to another. Electronic Mouse Colony Recordkeeping System Overview of Program The animal/litter system for recordkeeping has been incorporated into a more extensive computer software package that greatly simplifies data entry, with automatic cross-referencing and built-in error checking. This software package is called the "Animal House Manager" or AMAN and can be licensed for use by Princeton University as described at the end of this chapter. AMAN is a specialized database program that allows users to record and retrieve information on animals, litters, tissues, DNA samples, and restriction digests generated from one or more breeding mouse colonies. Data are entered through a series of queries and answers. With automatic cross-referencing, the same information never has to be entered more than one time. Hard copy printouts can be obtained for cage cards, individual records, or sets of records uncovered through searches for positive or negative matches to particular parameters. Search protocols are highly versatile; for example, it is possible to print out a cage-ordered list of litters that are old enough for weaning or a list of live mice ordered according to birth cage. AMAN provides investigators with the ability to maintain control over a complex breeding program with instant access to each record, current and past. AMAN can store 100,000 records in each of four files for (1) animals, (2) litters, (3) DNA/tissue samples, and (4) restriction digests. In the sections that follow, a detailed description is provided for the utilization of various components of this software package. Principles of data entry are described first, followed by protocols for data retrieval. [...]... for intramuscular injection should not exceed 0.05 ml, to minimize muscle trauma and to ensure rapid absorption Intravenous injection can be made into the lateral tail vein, using a 25- or 26-gauge needle Injection is made easier if the mouse has first been warmed by placing it in an incubator maintained at around 30° This results in vasodilation of the tail veins Volumes for injection should be in. .. according to the same general principles just described for animal searches In addition, you can limit the search to litters having a certain minimum age and/or a certain maximum age Lists of litters can be printed either according to litter number or according to cage number Hardware and Licensing Information The Animal House Manager (AMAN) can be licensed for use through Princeton University (Princeton,... cross-referenced, in both directions, within the database By maintaining litter records, it is possible to keep track of the complete breeding history associated with each mating group In some cases, it may not be necessary to maintain such detailed records of breeding when all the investigator wants to do is keep track of the pedigrees along a particular line In these cases, it is possible to skip the litter... animals directly into the animal file AMAN allows the investigator to choose both approaches within the same database The two approaches are discussed separately in the following two sections Entering New Animals Directly The quickest way to maintain breeding data is to bypass the litter entry protocol and enter information directly only on those colony offspring that are of particular interest and/or... first and females next The cage card option in the same submenu allows the printing of cage cards in a 3 by 5 inch format (again with animals ordered according to sex) If you would just like to print up cage cards for a new set of matings put together on a single date, you can set the data parameter accordingly Another useful option is printing according to cage of birth This option is very useful... (10-60 min) can be provided either by use of a volatile anesthetic, by intraperitoneal or intramuscular administration of injectable anesthetics, or by continuous intravenous infusion of a short-acting anesthetic The most useful combinations for achieving medium duration surgical anesthesia are either fentanyl/fluanisone (Hypnorm) combined with midazolam or ketamine in combination with xylazine or medetomidine... wish in the information, but be sure to include a *k if the litter was killed or a *d if the litter died This will cause AMAN to change the status of the litter to K I L L E D or DEAD, respectively It is important to carry out this protocol in order to keep the database up to date General Error-Checking Routines Through the entry of breeding information, you will notice the various error-checking routines... Surgical anesthesia, but postoperative mortality possible Sedative, some analgesia Duration of anesthesia Sleep time b 20-30 min 5 min 2-4 hr 10 min 60 min 15-60 min 120 min 2-4 hr 5 min 10 min 10 min 15 min 15-25 min 1-2 hr 15-60 min 1-2 hr a Considerable variation in response between different strains of mice can be anticipated Always undertake a pilot study when changing to a new anesthetic regime... each record To see individual animal and litter records in their entirety, use the view/edit option To print any screen full of information, be sure to start up with this option when you first begin, and then use the shift-PrtSc combination All printing occurs through the COM1 port A printer should be hooked up directly to this port If you wish to print through a networked printer, be sure to purchase... not maintained for more than 1 year The remaining fields are all optional The request for further information will place any entered data (up to 78 characters) into the information 1 field (INF1) This field is useful for sentence-like descriptions of unique characteristics or any other data Certain investigators, especially those involved in transgenic work, may use the same inbred or F~ strains over . names including "Princeton Mouse Recordkeeping Pro- gram"). To receive further information, contact Dr. Lee M. Silver, Depart- ment of Molecular Biology, Princeton University, Princeton,. Preface Publication of a Guide to Techniques in Mouse Development is timely in view of the already enormous and rapidly growing interest in the mouse as an experimental organism are incorporated into each mating record, and since these are linked implicitly to the litters from which they come, it becomes possible to trace a complete pedigree back from any starting individual.