About the

Sundberg, D.V.M., Ph.D., is head of the pathology program and a senior staff scientist at The Jackson Laboratory in Bar Harbor, Maine. Dr. Sundberg graduated in 1973 from the University of Vermont with a B.S. degree in Animal Science (summa cum laude) and obtained his D.V.M. degree in 1977 from Purdue University School of Veterinary Medicine, West Lafayette, Indiana. Following a brief period in private practice, Dr. Sundberg earned a Ph.D. degree in comparative pathology and virology in...

B The Database

The whole purpose of this application is to store data in a fashion that leaves it readily accessible to the user in a desired fashion. In the case of this example, by the end of the modeling phase this project contained 31 tables, with a total of 353 columns requiring storage. Each of these tables also requires some form of relationship address to be stored along with it. To perform these tasks in the application as defined would require a tremendous amount of additional software effort. An...

Conclusions

Evaluation of the genetics and phenotype of spontaneous and induced mouse mutations is no longer a simple operation that can be done by a single individual. Groups of specialists working together are needed to carefully define the phenotype in the mouse, compare it with similar diseases in humans, integrate species differences, and correlate these findings with genetic and biochemical findings. In this way the spontaneous and induced mouse mutations will be carefully and rapidly evaluated and...

J Testing Candidate Genes

The markers that have been used for the mapping of the mutation were anonymous markers, most of which are not related to genes. When a small genetic interval has been defined where the mutant locus must lie (not necessarily with hundreds of mice), it is desirable to look for known genes already mapped to the same chromosomal region that could be candidates for the mutation on the basis of both map position and function. Testing a gene as a candidate can require a lot of effort, and it is...

BStandard Protocol for sem Specimen Preparation

Fixation 2 glutaraldehyde and 2 paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. Fix for 4 hours at 4 C. 2. Buffer Washes Wash three times for 15 minutes in phosphate buffer at 4 C. 3. Postfixation Postfix for 2 to 3 hours in 1 osmium tetroxide in phosphate buffer at 4 C. 4. Buffer Washes Wash three times for 15 minutes with phosphate buffer at 4 C. 5. Critical Point Dry Flush specimen gently for 5 minutes four times with CO2. Gradually increase temperature in the critical point drier to 41...

Cryopreservation resource

Cryopreservation of germplasm is widely recognized as the best means to ensure against loss of valuable strains and efficiently maintain strains when they are not in immediate demand. In the frozen state, embryos are unaffected by diseases, sudden reproductive failure, certain environmental accidents and genetic contamination that can threaten breeding colonies. Reconstitution of valuable strains that have been lost because of reproductive failure or genetic contamination has occurred nine...

CUser Configuration

There are always unforeseen items of data that are required but have not been incorporated into the data model. To accommodate such requirements, extensive user note storage can be added throughout the application, and user configurable drop-down lists can be added for faster input. All the previous modeling sets the guidelines for the development of the final application, but it does not create the design. From this point on, the requirements developed previously should be incorporated into a...

HRefining the Map Position

After the mutant locus has been assigned to a chromosomal region of 20 to 30 cM, it is necessary to type additional markers in this interval in order to refine the map position, in particular relative to known genes that could be candidates for the mutant locus. It is recommended that the chromosomal region be divided into smaller intervals of 5 cM by choosing more microsatellite markers. At this stage, the geno-typing will still be performed on the same limited set of animals (50 to 100) ....

Index

AEC, see also 3-Amino-9-ethylcarbazole Alkaline phosphatase, 135, 174 Allelism, 18 AMCA, see 7-Amino-4-methyl-coumarin- 3-acetic acid 3-Amino-9-ethylcarbazole (AEC), 136, see also Chromagens 7-Amino-4-methyl-coumarin-3-acetic acid (AMCA), 139 Analog technology, 92, see also Photography Ancestry information, 44 Animal models, comparative pathology, 102-103 fluorescent-labeled, 138 nonspecific binding, 139 sources for immunochemistry immuno-fluorescence methods, 132-133, 134 Asphyxiation, 68-69...

Information on laboratory mouse strains

TJL maintains a registry that lists all strains at the laboratory. A variety of reports are available to the scientific community, such as the Lane List of Named Mutations and Alleles of Polymorphic Loci of the Mouse, which lists spontaneous mutant genes and their genetic backgrounds. TJL publishes a Handbook on Genetically Standardized JAX Mice and a quarterly newsletter, JAX Notes, which contain information on mouse genetics and different types of strains. TJL's price list and product guide...

Cytogenetic models resource

The Cytogenetic Models Resource contains stocks carrying Robertsonian chromosomes, reciprocal translocations, and a segmental trisomy. The breeding colony in the Cytogenetic Models Resource focuses on chromosome aberrations that can be used to study aneuploidy for mouse Chromosome 16, in which many human Chromosome 21 genes are conserved. Included are selected reciprocal translocations, Robertsonian chromosome stocks that can be used to produce embryos with trisomy for the entire Chromosome 16,...

Phosphate buffered saline pH 74 pbs

For one liter, dissolve the following in DEPC-H2O. Adjust the pH to 7.4 with NaOH prepare NaOH in DEPC-H2O . Filter sterilize. Use at 1 x final concentration. Make fresh each day. For 100 ml, dissolve 4 g paraformaldehyde (Sigma No. P-6148) in 95 ml DEPC-H2O containing 50 jl of 1.25 N NaOH. Heat to 60 C while stirring do not overheat. When completely dissolved, add 5 ml 20 x PBS and chill on ice. Adjust the pH to 7.4, if necessary. For 50 ml, dissolve 12.3 g NaOAc (FW 82.03, anhydrous) in 40 ml...

The model

The basic requirement of the model is to describe an individual mouse in a colony of mice in a way that enables the user to store and extract as wide a range of pertinent data as possible. The model should be able to store all commonly required data concerning the mouse as well as sufficient user data to allow individual configurations to suit personal requirements. The model should allow filtering and sorting of the data and be able to present it in a fashion useful to either human or machine....

A Why Use Radiolabeled cRNA Probes

The overwhelming reasons to use radiolabeled cRNA for in situ hybridization are the excellent resolution and reproducibility of the method, and the high degree of specificity and sensitivity of these probes to detect relatively rare transcripts. Depending on the spatial distribution of transcripts within tissues, the sensitivity can approach that of reverse transcription-polymerase chain reaction, using total RNA as a template. Sequences having 90 identity are readily distinguished. For data...

Setting up a colony for gene mapping

Another important aspect of mutant mouse evaluation and colony management is the setting up of matings for genetic mapping purposes. If the mutant you are working with resembles a mutation that has already been characterized and mapped, you can save time by doing simple tests for allelism between your mutation and any other mutation that causes a similar phenotype. This type of testing is done by mating a homozygous or known heterozygous mouse, with a known, characterized, and mapped mutation,...

DConnecting to the Database

As the decisions to use a commercial database and a RAD language have been made, consideration must be given to how the two pieces of software are to communicate with each other. First, consider the database software. If the user already has site licenses for a commercial database, it would be a benefit not to purchase another database, but integrate the preexisting system. Also, if there were any reason to change the database sometime in the future, it would be preferable if it could be done...

Further information

This chapter is designed to give a basic overview of immunohistochemical applications and techniques. For more detailed information, there are several books that may be helpful. A Color Atlas of Dermatoimmunohistocytology17 has many fine color photographs and information on immunohistochemical and immunofluores-cent techniques as specifically related to the study of skin. The book Introduction to Immunocytochemistry18 contains everything you need to know to get started and become proficient at...

Interpreting results

A brown or red signal for immunohistochemistry or bright green, red, or other fluorochrome color for Immunofluorescence does not necessarily indicate a positive reaction. Often apparent immunoreactivity may not be indicative of antigen expression localization. The chromagen color may be indicative of nonspecific staining or even a cross reaction of the antibody to an epitope in another antigen. Molecular mimicry can be quite common. To determine if apparent specific staining is nonspecific, one...

BDatabase

The next step was to understand how individual researchers wanted to store their data. The common answer was in a database, but actually meaning, in some magical structure on my computer that can tell me everything I want to know about my colony without my entering any data. In reality, a database can take many forms, but it is usually some sort of file structure stored by a computer in some form of read-write memory. Some questions of concern were voiced regarding the location of these files...

C Phosphate Buffer pH

Solution A 0.2 M sodium phosphate, monobasic dissolve 27.6 g of NaH2PO4 H20 in 1000 ml of distilled water. Solution B 0.2 M sodium phosphate, dibasic dissolve 35.61 g of Na2HP04 2H20 in 1000 ml of distilled water. Prepare 2000 ml of 0.1 M buffer by mixing 280 ml of solution A with 720 ml of solution B and 1000 ml of distilled water. Check pH of buffer and adjust if necessary with 1 N NaOH or 1 M HCl.

Mouse Mutations

Dawnalyn Boggess, B.S. The Jackson Laboratory Bar Harbor, Maine Boca Raton London New York Washington, D.C. Library of Congress Cataloging-in-Publication Data Systematic approach to evaluation of mouse mutations editors, John P. Sundberg and Dawnalyn Boggess p. cm. Includes bibliographical references and index. ISBN 0-8493-1905-6 (alk. paper) 1. Mice - Genetics. 2. Genetics - Animal models. 3. Mice as laboratory animals. I. Sundberg, John P. II. Boggess,...

Introduction

By every potential user, so some sort of arbitration must be made in the design. Biological science has many common terms that are used to describe its data. For example, any mouse has a sex even if it is other and any mouse has a coat color even if it is transparent. Other data types that share commonality are items such as phenotypes, genotypes, and procedures. These common data types make up much of the data set required in a colony management system. Using these common data types but...

BImportation of Mutant Stocks

Mice arriving at TJL are isolated and quarantined in isolators in a dedicated facility. Sample mice are subjected to microbial and viral evaluations others are mated and their progeny rederived by hysterectomy. The derived mice are raised by foster mothers with defined flora, and, after pups are weaned, the foster mothers are tested for specific pathogens before the weaned litters are transferred to strict barrier mouse rooms. The importation of mice can be a costly and time-consuming...

Mouse mutant resource

The Jackson Laboratory holds the world's largest collection of spontaneous mouse mutants in its mouse mutant resource colonies. The majority of these mutants have been identified from the large breeding colonies at TJL where animal technicians are trained to recognize deviant phenotypic appearance and behavior. The identification and characterization of these mutants continues today as it has for the past 65 years. Until about 1980, the study of mammalian mutations relied almost entirely on the...

GView

Although inspecting the data does not affect the storage of the data model, the model must be easily accessible to inspection of any data stored in it. This means that the various tables that construct the database must be correctly referenced to each other to allow simple extraction of data from different tables at the same time. This is part of the database being considered normalized. The view aspect of the model is probably where the most advantage can be obtained from such a management...

Clinical pathology

Usually provide a readily renewable population to study, the latter approach is commonly used in most research laboratories. Blood is collected by retro-orbital bleeding, tail tip amputation, cardiac puncture, or decapitation. The reasons to use each method vary with age, purpose of the study, volume needed, etc. Methods are summarized below. This method is a valuable, nonterminal method of blood collection, normally used for small volumes of blood. The tip of a hematocrit tube is inserted into...

Contents

Copy Stands and Camera Supports VII. Photographing Necropsy Specimens VIII. Photographing Documents and Radiographs X. Conclusions Acknowledgments References Photodocumentation is an important aspect of defining a new mouse mutant pheno-type and comparing it with similar human diseases. It provides a permanent record of the findings, but more important, it serves as visual support for descriptions in the text. This is particularly important when molecular biologists, usually not fluent in...

References

Andersson, L., Archibald, A., Ashburner, M., Audun, S., Barendse, W., Bitgood, J., Bottema, C., Broad, T., Brown, S., Burt, D., Charlier, C., Copeland, N., Davis, S., Davisson, M., et al., Comparative genome organization of vertebrates The First International Workshop on Comparative Genome Organization, Mamm. Genome, 7, 717, 1996. 2. Davisson, M. T., Lalley, P. A., Peters, J., Doolittle, D. P., Hillyard, A. L., and Searle, A. G., Report of the comparative subcommittee for human, mouse, and...

Specimen collection and fixation

There are two basic methods of fixation immersion fixation and perfusion. During immersion fixation, the specimen is immersed in the fixative and the fixative penetrates the cells of the specimen. Because the rate of penetration of fixatives tends to be slow, the pieces should be as small as possible, 1 mm3 or less, and the fixation time can be fairly long. Alternatively, for tissues such as skin, very thin slices or strips may also be fixed in this manner as long as they are 1 mm thick or...

Films

Chemical-based films are available from a number of commercial producers, and each laboratory group will determine which it prefers. We use Kodak (Eastman Kodak, Rochester, NY) color slide (Ektachrome EPY 64T and EPT 160T) and black-and-white negative (TMX 100 and Tri-X Pan) films. Color slide films need to match the temperature of the lights used to illuminate the specimen. If tungsten lights are used, films labeled with a T are used without color correction filters. Daylight films will have a...

K Positional Cloning of the Mutation

If no candidate gene has been found, or if all of them have been discarded, the cloning of the mutation requires constructing a physical map of the chromosomal region and looking for all possible expressed DNA sequences in this interval. Because this is a very tedious process, it is recommended that a very high-resolution, accurate, genetic map first be established. The closest flanking genetic markers that show recombination with the mutation are used to isolate YAC (yeast artificial...

Fixatives

Fixatives are used to preserve the natural morphology and ultrastructure of a specimen. The most widely used fixatives for EM are the noncoagulant fixatives, such as glutaraldehyde, paraformaldehyde, acrolein, and osmium tetroxide. They are known as noncoagulant fixatives because they crosslink proteins and make them less likely to become extracted by solvents during processing, but they do not coagulate or denature the proteins. They are also called additive fixatives because they work by...

S

Salivary glands, 76, 83 Sample tab-page, 44 Scanning electron microscopy (SEM), 121-122, 125 heavy metals, 125-126 routine protocols, 126-127 Screening programs, spontaneous mutations, 180 SEM, see Scanning electron microscopy Seminal vesicles, 74 Sensitivity digoxigenin-UTP probes for in situ hybridization, 174 radiolabeled cRNA, 145 Serum, 62 (SSLP), 24 Single-reflex cameras, 92 Single-strand conformation polymorphisms (SSCPs), 26, 30 in situ hybridization with nonradiolabeled probes, 168...

The Jackson Laboratory

Sundberg, DVM, Ph.D., Diplomate ACVP Pathology Program, 600 Main St., Bar Harbor, ME 04609-1500 From Sundberg Strain C57BL 6J Genotype + Pedigree 1234.567 Other ID sample project Date Born 06 10 97 of animals 1 Sex F Mating Pair Richard S. Smith, MD, D.Med. Sei., Diplomate AAO Items recorded include Color photographs B & W photographs Photomicrographs Electron micrographs Radiographs In Situ Prep. Histology Frozen Tissue Photo CD History and Clinical Signs Gross Description Variable...

ASelection of Mutants

There has been an explosion in the rate of production of transgenic and targeted mutation mice since the two technologies were developed (Figure 13.1). It is not possible (or necessary) for TJL to import and distribute every genetically engineered mouse produced. Therefore, TJL's Genetic Resource Committee has established criteria for selecting mutant strains (1) the immediate need for use in biomedical research, (2) the number of requests for mice being received by the investigator(s) who...

Acknowledgments

The MMR has been continuously supported by the National Science Foundation (NSF) since 1960 (currently DBI 95-0222) and by the National Center for Research Resources (NCRR) at the National Institutes of Health (NIH) since 1970 (P40 RR01183). It also receives funding from the National Institute of Child Health and Human Development (NICHD HD53230), the National Eye Institute (NEI R01 E05578), the National Institute for Deafness and Communicative Disorders (NIDCD DC62108), and the Foundation...

A Putting it All Together

First, some of the controls used in the application need to be understood An application's window is the area on the computer screen that displays the functionality of the application. A pop-up is a small window with a few controls or a question that needs to be addressed before the program operation can continue. A save button will usually be complemented by a cancel button or a popup window that allows the save to be cancelled. A tab control is something like the window version of a file...

Ovarian transplantation

Ovarian transplantation is another strategy to successfully breed certain kinds of mutant, transgenic, or knock-out mice. Ovarian transplantation should be considered when (1) the mutant ovary contains relatively normal numbers of functional oocytes that are capable of normal maturation and (2) gonadotrophin deficiency, physical abnormality, or early mortality prevent the mutant mouse from conceiving, mating, or sustaining a pregnancy. Ovary transplantation will not repair absence of germ cells...

HNuclear Track Emulsions and Autoradiography

The remaining steps are often problematic for investigators who have never worked with nuclear track emulsions. The first decision is which emulsion to use. The properties of Ilford and Kodak nuclear track emulsions are quite different, and protocols for their use are not interchangeable. If you select an Ilford emulsion, consult Hogan et al.1 The following protocols are for Kodak NTB emulsions. Select NTB-2 (Eastman Kodak No. 165 4433) for 35S cRNA probes. This is probably also the best choice...

Examples of a working computerized system

Unique Identifier Large scale data collection operations, such as diagnostic laboratories, clinics in hospitals, or drug safety studies in industry, assign a specific identifier for each individual animal. This is often referred to as a case or accession number and is utilized to organize all the materials generated by the case. In a relational database, this unique identifying number becomes what is called the primary key and is used to tie all the database information...

Inbred vs outbred

Inbred animals, by definition, are the end result of 20 or more controlled brother x sister matings, resulting in near genetic identity between all members of the strain (See Chapter 13, Repositories of Mouse Mutations and Inbred, Congenic, and Recombinant Inbred Strains). Because inbred mice can be raised in a controlled environment with a defined pathogen status (specific pathogen free colony), most of the mice in that colony will maintain a remarkably similar phenotype.83637 There may be...

BControl Tissues

With each immunohistochemical run, a control tissue should be included to help determine the success of the reaction (see Table 10.2) A known positive control is used to prove that the reaction worked. It may be a normal tissue known to express TABLE 10.1 VENDERS THAT PROVIDE ANTIBODIES AND KITS American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, Tel 800 638-6597, Fax 301 816-4379, E-mail tech atcc.org, www.atcc.org Antibody Resource Page Online, Berkeley...

Photographing live mice

Photographing free-ranging, live mice requires patience. It is best to work out the distances for depth of focus, background material (color and texture), flash intensity, and film type well in advance by practicing with a dead mouse or simply a wad of paper. Work through several f-stops, distances, etc. Record every detail and condition of the test photographs while they are being taken. This part is tedious but necessary until the capabilities of the camera system are well understood. For...

Necropsy procedure

Once the mouse has been euthanized it should be superficially disinfected by submersion in a dilute solution of a germicidal detergent such as Calgon Vestal Process NPD One Step Germicidal Detergent (ConvaTec, St. Louis, MO), or a solution of 95 ethanol. When necropsying a mouse with an abnormality of the hair coat, it is important to collect samples of the hair before the mouse is dipped in the disinfectant. The hair should be plucked manually using the thumb and forefinger. Do not use...

Detection of the nonradiolabeled probe

Immunological detection of the DIG-labeled probe is performed using the Genius system developed by Boehringer-Mannheim (Indianapolis, IN) (see Table 12.2). Following posthybridization washes, the sections are first equilibrated in Buffer 1 0.1 M maleic acid 0.15 M NaCl (pH 7.5) , blocked for one hour in Buffer 2 (1 blocking powder in Buffer 1), and then incubated with an anti-DIG antibody (ranging from a 1 1000 to a 1 4000 dilution) in Buffer 2 for one hour at room temperature. The sections are...

Copy stands and camera supports

A hand-held camera with a fast film is adequate for photographing live mice moving around, especially when an electronic flash is used (see below). However, a support for the camera is useful so that the mouse can be allowed to run around, and yet you can follow it easily without fear of dropping the camera. A tripod is often adequate for this purpose. A more stable platform is useful for necropsy specimens, particularly when close-up work is required. A variety of commercially available copy...

C How to Choose the Breeding Scheme

The breeding scheme, i.e., the genotypes of the parents to be mated, depends on the mode of inheritance of the mutation. The possibilities are illustrated in Figure 2.1 (dominant mutation) and Figure 2.2 (recessive mutation). It should be noted that, in several instances with recessive mutations, it is necessary to produce four times more progeny than will be useful for the mapping because it is not always possible to deduce the genotype at the disease locus based on the phenotype of the mice...

Immunohistochemical Staining of Frozen Tissue specifically

Encircle sections with PAP pen (Kiyota International, Elk Grove Village, IL cat K-500) 4. Hydrate in PBS* (for any amount of time) 5. Fix in Morpho-Save (Ventana Medical Supplies, Tucson, AZ cat 250-010) 15 min 7. Incubate with Peroxo-Block (Zymed, So. San Francisco, CA cat 00-2015) 30 sec 9. Apply a few drops of reagent A** and incubate 10 min 11. Apply a few drops of reagent B** and incubate 10 min 13. Block with TNB blocking buffer*** (0.5 gm blocking reagent 100 ml PBS) 30 min 14. Blot...

Clinical evaluation

Ear Notching Mice

Live mice should be carefully examined for both behavioral and physical abnormalities. Most homozygous recessive mutations (m m) are available with heterozygotes (+ m) or wild-type (+ +) age- and sex-matched controls on the same genetic background (where m the mutant gene being studied, and + the normal, or wildtype, gene). Controls should be examined side by side with mutants as a basis for comparison. Familiarity with the normal phenotype of the background strain is essential in assessing the...

Prehybridization and hybridization considerations

Various steps are taken to reduce nonspecific binding of a probe to the slide and target tissue (see Table 12.1). These steps involve an incubation in 0.1 M triethano-lamine containing 0.25 acetic anhydride, in order to reduce nonspecific binding of either radiolabeled or DIG-labeled probes to the slide or to the tissues. Reagents also commonly added to the prehybridization buffer to reduce background binding to tissue sections include carrier DNA, tRNA, Denhardt's, and bovine serum albumin...

Photographing necropsy specimens

Live mice move around, so many frames will have to be taken in the hope of obtaining an aesthetically pleasing, focused image that illustrates the lesions in question. However, multiple views are often useful because, until the image is selected, it can be difficult to determine which view is most useful. Also, if multiple views are taken, they can be used in subsequent papers or book chapters without copyright infringement concerns. The specimen should be carefully prepared. Excess blood...

Tissue preparation

Regardless of the signal detection method employed, precautions mentioned in the previous chapter should still be exercised when collecting tissues for nonradiolabeled ISH studies. Again, one should take steps to eliminate possible contamination by RNases by baking glassware, autoclaving solutions, and using RNase-free solutions (i.e., diethylpyrocarbonate (DEPC)-treated water, RNase-free DNase, etc.). The steps in preparing the target tissue for in situ hybridization i.e., fixation and...

Is the genetic control simple monogenic trait or complex polygenic trait

Once it has been established that the trait is genetically determined (even though some environmental parameters may play a role), one should ask whether the trait is controlled by a single gene or by multiple genes and, in the first case, whether it is dominant or recessive, autosomal, or sex-linked. These questions can be answered from the results of a few simple crosses. When possible, it is desirable to perform two reciprocal crosses, where affected females males are mated with unrelated,...

Trimming tissues for histology

After the tissues you have collected have been fixed for a sufficient amount of time (see section on fixatives for different guidelines), they must be trimmed before being delivered to the histology laboratory for embedding. Proper trimming of the tissues will ensure that they are presented on the slide in an orientation that will allow appropriate interpretation of their cellular structure by the pathologist. Presentation is critical when trying to identify any variation from normal or any...

E Phenotyping the Progeny

Once the type and the size of the cross has been decided, the appropriate number of matings should be set up to produce the progeny. It should be remembered that, with interspecific or intersubspecific crosses, not all matings will be fertile and assuming that 10 to 20 of them will not yield progeny provides a reasonable safety margin. In the case of interspecific backcrosses, only F1 females will be useful to generate the second generation because all F1 males are sterile. Each BC or...