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...