Setup and use of the Murine Pathology Database (MPD)

• What is the MPD?
• Will the MPD work for me?
• Installing and launching the database
• Initial Setup
• Initial Entry of Founder Mice
• Entering genotypes of new pups
• Viewing Mouse ID information and creating Inventories
• Removing Mice from the colony (with no specimens)
• Necropsies and tissue specimens
• Histology
• Freezer Records
• Images
• Plasmids
• Oligonucleotides and PCR protocols

Will the MPD work for me?

The MPD is a laboratory relational database that allows users to cross-reference mouse inventory data, breeding records, pedigrees, pathology data, histology images, autoradiographs, PCR genotyping protocols, freezer archives, and reagents (specifically plasmids, oligonucleotides, and antibodies). It is comprised of a set of FileMaker templates which can be used on a single computer or on a server with multiple clients. The Database is modular with the major components in separate files so undesired parts can simply be discarded with only minor modifications to the Directory (to prevent error messages).

Will the MPD work for me?

To use MPD templates you must have Filemaker Pro software, either a Mac or PC version, (v.7+). The mouse database requires unique identification numbers for each mouse. If your animal colony is identified only by cage or rack this solution will not work for you. The numbering system is specific, so if you want to use a different format, some changes may be necessary to the MPD layouts and some scripts. You must also be willing to enter the identification numbers and information on your mouse colony manually, at least at the outset. You must also be prepared to keep the database updated whenever you do something to an animal, whether that is breeding, weaning, necropsy, etc. However, it is not necessary to keep a parallel set of paper records to track your colony. Hard copies of MPD inventories may be printed for notebooks, and regular backups of the MPD onto a CD or a second hard disk is recommended. Handwritten notes on preprinted forms at the time of necropsies or breeding/weaning are also useful as temporary records until the data can be entered into the MPD. Likewise the MPD uses a specific system for uniquely identifying frozen samples and histology cassettes.

Before using the MPD templates you should first familiarize yourself with this ReadMe document and test out the templates. It is probably better to make basic structural changes (such as a modified numbering system) before entering large volumes of data.

Table 1: Summary of ID conventions used by the MPD. The items in parentheses give the format of the IDs and a typical starting value. Values in bold type are those that are supplied by the user. Many IDs combine the use of a prefix and suffix to allow numbers to be recycled amongst different users or groups. Some of the conventions used are optional (as listed in the Notes column).

A. Installing and launching the database

• Copy the "MPD" folder to your hard drive and rename it as you wish
• Make an alias to the "Directory" file. Place this alias in a convenient location.
• Launch the MPD by double clicking the "Directory" alias.
• If the database is being hosted on a remote machine then use the File>Open Remote command to access the MPD Directory.
• Enter your user name and password (if not set the user name is admin and password is blank).

B. Initial Setup

From the Directory page open the "Global Value Lists" file.

Select "Show User Dependent Lists"

1. Add entries with a User name (e.g. "Matthew"). Create new records as necessary to generate new users.
2. Add Mouse ID prefixes with the format "MF 0" (two initials <space> number)
3. Add Breeder Groups for each User. Breeder groups will be used as a prefix when sorting cages in inventory lists. It is our convention to use an abbreviation of the parents transgenes as Breeder Groups separated by periods. For example if the parents of a mouse harbored both Kip1 and Myc transgenes the mouse belongs to the "K.M" Breeder Group.

The breeder group name is used as a prefix when sorting and listing cages. For Breeder cages our convention is to use cage letters, but stock cages have cage numbers (e.g. K.M-A is a breeder cage, but K.M-1 is a non-breeder cage).

1. Add Gene names for each user. The Gene name is what the gene will be called in genotype lists. A gene name refers to the gene locus and not to the specific mutation of the gene. Genotype is listing of possible combinations maternal and paternal alleles for the gene locus (e.g. +/+, +/-, ?/+, etc.) Thus a p27 knockout has the gene name "p27" and the corresponding genotype for this gene name is "-/-"
2. Add genotypes for each user. Only one genotype can be displayed per gene name (locus). Thus if multiple versions of the gene locus is possible then multiple genotypes must be entered in the global value lists, e.g. +/+, +/-, -/-, a/+, a/a, a/b, etc. In order to maintain uniform sorting and Gene IDs each genotype should have a single alphanumeric prefix: i.e. +/+ is actually listed as "0) +/+" (no quotes), and +/- is entered as "1) +/-"
   

Table 2. This table shows examples of how Breeder Groups, genotypes, Gene IDs and cages are classified in the database. The appropriate User names, Breeder Groups, Gene names, and Genotypes should be listed in the Global Value Lists file to allow auto entry by pop-up menus. If transgenes at two different loci are bred into a single mouse then each is given a gene name which is locus specific. A unique genotype should be listed separately for each gene locus reflecting both parental alleles. If heterozygotes can not be differentiated from homozygotes then indicate this with a question mark (e.g. ?/?) The Breeder Group is an abbreviation of the of the names of the parents transgenes. It also is used as a prefix to sort cages into groups.

1. Enter the names of the Breeder Groups for this user. Breeder groups should be an abbreviated description of the gene names that will intercrossed. For example p27 knockouts are "K" and c-Myc transgenics are "Myc". If p27 knockouts are bred to Myc transgenics then the pups form a new group called "K.Myc" (see C.3 below). If multiple versions of the same allele will be created they should be readily identifiable in the breeder group name (e.g. "K", vs "Kt" to distinguish the simple null from the T187 point mutant.
2. Add Strain names for the mice that this User will use. Strain names should be consistent across users to avoid confusion.
3. If the user will breed wildtype stocks (mice without ID numbers) add wildtype stock names. These names will be used to identify cages with in the Wildtype Stocks file.
4. Optional: Enter Experiment names for the Users mice (this can be done later)
   
   Select "Show User Independent Lists"
5. Genotypes is currently not used but contains example genotypes
6. Note the values that are entered here. They can be modified in the future if necessary.
7. Enter the IR#s that will be used.
   
   Select "Show Freezer Lists"
8. Note the values that are entered here. They can be modified in the future if necessary.
9. Enter new freezer names if necessary. (These correspond to the Engineering numbers on each freezer).

C. Initial Entry of Founder Mice

The identification of founder mice in the initial colony must be entered manually. The easiest way to do this is to enter the founder information in the Mouse ID records directly. Only founder mice should be entered this way. All mice generated by breeding within the colony should be entered using the Breeding table to record IDs and genotypes. This is much less error prone than manually creating Mouse IDs and will ensure that the pedigree linkage and strain ratios are maintained.

1. From the Directory select the Breeding Records button to open the Breeding file.
2. Change to the "Weddings" layout, and create a new record.
3. Fill out all of the information in this layout starting with the User name. This will activate the user specific pop up lists. If there is no mouse information in the database on the mouse the red fields will needed to be filled out manually for both the male (Dad) and the female (Mom) mice. Check "Founder" for each mouse. Use the "Dad" and "Mom" buttons to view the new Mouse ID records that you have just created and fill in extra details.
   br> The Pups Breeder Group should be a combined abbreviation of the transgenes of both parents. Thus the Breeder Group of the pups is "K.Myc" if a male Kip +/- is bred to a female Myc ?/+ mouse.
4. When each record is complete select the button "Auto-complete Parent ID cards".
5. Repeat (2-4) for each new breeder pair.

D. Entering genotypes of new pups.

1. Perform a "Find" with the Mom and Dad's prefix and Toe IDs.
2. If only one record is found and it is blank then you can use this to fill the IDs of the new pups. If a litter has already been entered for this pair then use the "Add Litter" button to created a new litter entry for this pair.
3. Enter the Pup's "DOB", "Num", "Prefix", and range of "Toe #'s". (Num refers to the number that were born, and Toe #'s refer to the IDs given to mice when tail or toe biopsies were taken for genotyping.
4. Select the "Create Litter ID cards" to automatically generate ID cards for all of the pups. (It is important not to bypass this step)
5. Select the "Pups" button to view the new ID cards and be sure that you made no entries. Return to the Breeder Records file and use the "Delete Litter ID cards" if you made a mistake and need to recreate the pups ID cards.
6. When genotype information is available for this litter then return to this Breeder Record and enter the information here.
7. When the mice are weaned return to this record and indicate the sex and new cage number of the pups.
8. If some pups are sac'd without being weaned then a DOD should be entered for each of these pups on the Breeder Record. Also select "Not used" as an Out category.
   br>Note: To sort properly Breeder cages should be designated with cage "Letters" but non-breeder cages are designated with cage "numbers". Mice that are sac'd after weaning are entered in either the "Out Mice" list or the "Necropsy Notes".

E. Viewing Mouse ID information and creating Inventory Lists.

1. Enter a User name in the Directory. Select the "ID cards" button.
2. This will take you to the Mouse ID file and will display the current Users alive mice.
3. To sort the Users mice by or cage select the button:"Sort live non-breeders by group, cage, ID#."
4. To sort the Users mice by genotype:
• Change to the Gene List layout
• Select the "Gene ID" button to create Gene IDs
• Select the button "Sort live non-breeders by group, genotype, age."
5. To view more detailed information on an individual mouse change to the "Mouse ID" layout.
6. To create an inventory list for a single experiment:
• Select "Sort live non-breeders by group, cage, ID#."
• Select "Records > Modify last find"
• Enter the name of the experiment, and activate "find" button.
• Resort the records by the previous critieria ("Records > Sort").
• Print this experimental inventory. Check off the boxes to verify that the mice are present and accounted for in the colony and keep this hard copy inventory.

F. Removing Mice from the colony (with no specimens nor Necropsy).

If mice are removed from the colony this should be recorded with an "Out Mice" entry or a "Necropsy Note". This section describes how to make "Out Mice" entries. If a mouse was part of an experiment or if samples were taken then a Necropsy Note should be generated instead.

1. From the Directory select the "Out Mice" button.
2. Create an new record. Enter the dead mouse Prefix and Toe IDs. If the related information is correct for this mouse then enter a DOD (Date of death) and an Out Category.
3. If you make a mistake you should delete the DOD and the Out Category before deleting an Out Mice record.

Note: MIA (missing in action) is refers to mice that are absent at the time of an inventory and therefore do not have accurate DOD. If they are part of an observational study the most recent time at which they were noted to be alive should be entered in the Notes field based on the last experimental inventory. Regular inventories with complete documentation are a required to generate accurate survival data.

Note: If mice are part of an observational experiment it is also essential to note whether they died or were euthanized because they were sick. A Necropsy Note should be used instead of an Out Mice note.

G. Necropsies and tissue specimens.
The Necropsy Notes file should be used to record gross observations at the time of necropsy and the IDs of specimens that are obtained. Embryos should first have litter IDs entered in the Breeding records. The alternative transplant layout should be used for used in transplant studies, in order to track the identity of both the donor tissue and recipient mouse.

1. From the Directory select the "Necropsy Notes" button.
2. Create a new Necropsy Notes record.
3. Enter the Prefix, Toe ID and DOD of the necropsied mouse. The related information regarding the mouse genotype and strain should appear.
4. If the mouse looked sick then describe the appearance and behavior of the mouse. If the mouse did not appear sick then state the reason that it is being necropsied.
5. When the mouse is dissected record the visual appearance of any abnormal organs under "gross findings". Also list which areas of the mouse were inspected and whether any organs were inspected under a stereomicroscope.
6. Record the weight of the mouse and the weight of organs and the numbers of tumors per organ as relevant.
7. Assign prefix and sample ID numbers to specimens that are taken during the necropsy. Use the "DOD" button to use the date of death as the sample prefix. Also indicate the type tissue and a description (e.g. lung, normal).
8. If appropriate indicate whether the sample was fixed, frozen or explanted for culture. Also enter the type of fixative used.
9. Specimens which are frozen should have an entry listed for the name of the Freezer, Box, Row and Column. Select the "Record" button to transfer this information to the Freezer records.
10. If a photograph is taken at the time of Necropsy then this can also be recorded under a specimen record. Select the "Note Photo" button and enter the photo ID in the dialogue box. Select the "Go to Images" button to view the new Images record and to import the photo (or a reference to its location).

The DOD is routinely used as a prefix for all of the specimens that are obtained on a given day. If more than one user will be performing necropsies in a single day they should use different specimen numbers.

Specimen records are permanent but Freezer vial records should be deleted if a sample is removed from the freezer.

H. Recording and Summarizing Histological Observations.

1. From the Directory open the "Specimens" file.
2. Find the specimen by performing a search for the DOD and specimen #.
3. Indicate the type of stain used. Change the status from "pending" to "done".
4. Record your observations under Microscopic Observations.
5. Return to the Necropsy Notes for this mouse and enter an opinion about the ultimate cause of death in the field "Final Diagnosis".
6. To view a list of all of the specimens taken for a given experiment then perform a search in the "Specimens" file and change to a list layout (e.g. "B&W Short Form"). To view an organized list of all of the animals necropsied for an experiment then perform a search for the experiment in the "Necropsy Notes" file and select the "Show Summary" button in one of the list layouts. (Gene IDs must first be calculated in the Mouse ID records (see E.6).

I. Recording and Viewing Freezer Records.

The Freezer records is a list of the individual boxes kept in freezers and the individual samples within those boxes. Selecting "Frozen Samples" in the Directory will take you "Frozen Stocks Home" layout. This serves as a central navigation point if you use the Freezer templates as a stand alone solution.

Create Freezer Box records
The Freezer Overview is listing of boxes in various freezers. Start here and identify the boxes in a freezer before entering specific frozen samples.

1. From the Directory or the Frozen Stocks Home layout select "Freezer Overview" button.
2. The Freezer Overview is a listing of individual boxes in a freezer.
3. Create a new record for each freezer box. Enter the name of the freezer, Box ID, and box size. (User, sample type, and experiment are optional).

Freezer Overview This Freezer overview must be used to create new freezer boxes.
It also has links to view boxes with the correct configuration of tubes.

Create individual Vial or Sample records:

1. From the Directory select the "Frozen Samples" button.
2. From the Frozen Stocks Home layout select whether this is a sample for an Ultra low (Bacteria, Tissue, or Any) or for a Cryo freezer (Frozen Cells).
3. Enter the Freezer, Box, Row, and Column information for the sample.
   

Tip: Use Necropsy Notes to automatically create frozen sample records if this is a specimen from a necropsy.
Note: Boxes should be named with the format "Letter Number" (with no space) e.g. A3.
The letter and the number represent the rack and box number, respectively.

Rows and columns may either be listed either as letters or numbers, e.g. (A,5), (1,5), and (1,E) are all equivalent.

Specific layouts exist for Bacteria Stocks, Cryopreserved cells, Necropsy Tissues, Human Tissues, and other reagents. Using the correctl layout will ensure that you enter the correct type of data.

To view an overview of all of the samples in a box

1. Return to the Freezer Overview to see a preview of the contents of a freezer box by selecting "View Freezer Box" from any one of the Frozen Samples layouts.
2. If you have have navigated to the Freezer Overview from the Directory you will need to select which box to view and then the "View Box Layout" button to shift to the correct layout.
   br>Note: The box record will display correctly (buttons will link to the correct layout) only if the correct box dimensions were specified.
   br>Tip: Clicking on a slot within a freezer box will call up a script that allows you to change the location of a box or a vial, but you can only edit or delete a sample from the Frozen Samples records.
3. To edit the sample information for the contents of a box then select use the "Edit Box Contents" button to navigate back to the "Frozen Samples" file.
   

J. Adding Images to the MPD

Photographs can be recorded directly at the time of Necropsy (see Necropsy Notes).  The following instructions are for adding images directly to the Images database.

To add images directly to the Images database:

1. From the Directory select the "Image Files" button.
2. Enter a unique Photo ID (our convention is to use the names assigned by the digital camera)
3. Change to the "Gel Photo" layout to add photos (from gels or autorads), or to the "Histo photo" layout to add photomicrographs or photographs of gross specimens.
4. For the "Gel photo" layout the Mouse ID for each lane of the gel is entered separately. For the "Histo Photo" layout a single Mouse ID is entered for the image.
5. You may view a listing of all of the images for a given mouse in the Mouse ID. Necropsy Notes, and Specimen files and navigate between the related records using the buttons provided in each file.

1. Photos stored in a shared iPhoto lilbrary can be used without making extra copies of them in the MPD. If the library is on a remote volume you will need to logon to this computer first. Select the "Link to iPhoto Library" button. You will be prompted for the Photo ID number and the Date that the photo was taken. If not entered previously you will also need to enter the "Photo Volume" (i.e. Hard disk name) and library path (e.g. Users/Shared/iPhoto Library/ ) The photos will then appear in the Image record without having been copied to the MPD.

Digital gel photographs or autoradiographs should be viewed in the "Gel Photo" layout. This provides cross references to the samples loaded in each lane.

K. Plasmid Database

The plasmid database records the identity of plasmids and notes on their construction. It contains links to freezer stocks and plasmid maps.

1. From the Directory select the "Plasmids" button.
   br> The convention for plasmid IDs are "AB123", where "AB is the initial of tihe creator, and 123 is an ID number. Each creator thus may have their own number series. Any other type of name for the plasmid should be listed under "aliases".
2. Enter the IDs of your vectors and their vendors. Vectors should be assigned unique plasmid numbers IDs, and should not be given duplicate IDs by different users.
3. Enter the IDs of custom made plasmids with the IDs of their vectors and inserts. Make a note of the restriction enzymes used to cut the inserts or whether inserts were PCR amplified.
4. To add map data switch to the "Details" layout. An image file can be pasted into the map container. This is useful for commonly used vectors, but it will increase the size of the database.

To add a link to GCK Maps
A link to GCK (Gene Construction Kit) or other types of external plasmid map files is automated for computers running Mac OS X. If the map files are on a remote computer then the user must first establish an active connection to that computer. (Go > Connect to Server). Select the "GCK Map" checkbox in the Plasmid Database and enter the filename of the GCK map.

Plasmid maps can still be cut and pasted directly in the container field in the "Plasmid Details" layout. Use the "GCK Map" button to link to separate plasmid map files.

Before users can link to external files the GCK folder path must first be specified in the (hidden) Plasmids Setup layout. Pathnames should follow Applescript conventions, e.g. VolumeName:Users: Shared:GCK Maps.

L. Oligonucleotide Library and PCR protocols

The Oligos link in the Directory navigates to the MPDs oligonucleotide reference library. The initial layout displays a complete listing of oligos. To see more details about an oligo change to the Details layout. To enter a new oligo first create a new record and then select the "Edit Oligo" button. Oligos should be identified by a prefix (e.g. the creators initials or an abbreviation for the gene name) and a consecutive number. Fill in the other relevant information. Melting temperatures and molecular weights of oligos are calculated automatically. If the Genbank UI is known then the "Go to Genbank" script will provide a shortcut to the sequence via a web browser. Alternatively the "Blast alignment" button can be used to perform a Blast search with the sequence provided.

The PCR Protocols link in the Directory provides a listing of established protocols for PCR based genotyping of animals. The initial layout is a listing of all protocols. The "Protocol" button navigates to a layout where details about a PCR protocol should be entered. Toggle the check boxes to indicate whether one or two reactions are to be listed in a given protocol (e.g. one for the wildtype and one for a mutant allele.). Links to the Oligo database are active once the Primer names are entered.

Use the "Setup" button to create a master mix print out. Enter the number of reactions (Rxn number) and the recipe will automatically be calculated. A space is provided for a photograph of the PCR gel on a printout, or a digital image can be pasted into the PCR record itself.