Mammalian Genome Size Project
Slide preparation protocols

Background:

         There are over 5,000 described species of mammals -- all united by a few key similarities, yet separated by a remarkable number of differences.  Mammals are a tremendously diverse group of organisms, with an incredible variety of body forms adapted to different environments and lifestyles.  Mammals have always been of considerable interest and they are of great economic importance in food and agriculture, disease, pest control, and eco-tourism.  While mammals have been relatively well studied in areas of physiology and ecology, with respect to genome size (i.e., the amount of DNA per haploid chromosome set), they have been relatively poorly represented.  After more than 50 years of continuous research into genome size variation, genome size measurements exist for only about 430 mammalian species.  Genome size data are increasingly important as genome size information is now required for complete genome sequencing proposals and other large-scale genomic work.  Genome size is also emerging as an important biological character with potential phenotypic consequences.  Even with the limited data available, genome size has been found to be positively associated with cell size and negatively correlated with metabolic rate.  There is also evidence in some groups that links genome size to physiological and development characteristics.  In order to investigate issues relating to genome size, additional data is needed from a wide variety of mammalian species.
         In the Gregory Lab at the University of Guelph, graduate student Jillian Smith seeks to greatly expand the mammalian genome size dataset and to characterize variation in DNA content and its potential consequences in this group.  To assess genome size variation, both flow cytometry (e.g., on frozen tissues) and Feulgen image analysis densitometry will be employed. For Feulgen image analysis, all that is required is an air-dried blood smear which can be easily prepared and shipped.  It is therefore possible for members of the scientific community to make a significant contribution to the study, and at the same time to obtain genome size data for their species of interest free of charge.

If you have access to large numbers of specimens, we will
be happy to provide you with kit that includes slides.

Please contact Jillian Smith at  if you would like to provide samples.

Data use policy:

• Collaborators who provide samples will be free to use the genome size data as they see fit, so long as we are cited as having performed the measurement(s).  We will only ask for coauthorship on papers using the data if the genome size estimates form a major component of the study in question.  We reserve the right to publish the data as part of the larger avian genome size project or subsets thereof, and will offer coauthorship as appropriate in cases where collaboration is extensive.  All genome size data will ultimately be deposited in the Animal Genome Size Database, which is freely accessible.
  

• An effective way to get blood from small mammals is with a needle and syringe, or collection tube with capillary action.  In small rodents the best site for sampling unanesthetized animals is the saphenous vein.  Saphenous vein sampling is described by Hem et al..  In larger animals such as rabbits, a sufficient blood sample can be taken from the ear.  A small syringe, or collection tube is sufficient – only a drop of blood is required.

• You may treat the syringes with heparin before use if clotting is a concern.  To do so, suck some heparin in and out of the syringe a few times to coat the inside.  Without treatment with an anticoagulant, the blood will clot quickly and will be very difficult to smear, but this is not a problem if you are preparing the smear immediately after sampling.
  

• Be sure to apply pressure to the needle site when removing the needle in order to prevent hemorrhaging below the skin.

Preparing blood smears:

• One individual per species is infinitetly better than none, but we will seek to analyze up to five individuals per species whenever possible.

• We use Fisherbrand Superfrost Plus slides (25 x 75 x 1.0mm, Fisher catalogue #12-550-15), but any standard microscope slide will suffice.   Slides are available in individual packages of ½ gross, or by the case of 10 gross.  Slide boxes are also needed for storage and shipping.  Plastic 100 slide capacity boxes are available from Fisher (and others) and cost on the order of $5 each.  Smaller slide boxes of 5, 25, and other capacities are also available (see below).
  

• Label the slide, in pencil, with the frosted side up.  Do not label in pen or marker (this will wash off during staining).  If you are providing samples from more than one individual, please take note of the individual number on each slide.
  

• Place a drop of blood on the slide near the label edge.

• Place a second slide at a roughly 45-degree angle to the first.  Gently back the angled slide into the blood drop.  When you touch the drop, the blood will expand to fill the space between the first and second slide.  Do not push the second slide through the droplet – you may damage the cells that way.  Instead, you will “pull” the cells along to smear them into a thin film.

• Gently move the second slide along toward the end of the first slide.  The blood will be pulled with it and will leave a thin film along the way.

• Allow the smear to air-dry, and store the slides in a slide box at room temperature before shipping.

Shipping slides:

• Please keep in mind that microscope slides are small pieces of glass and are very fragile.  Slide shipping containers are available in capacities of 2, 5, 25, 50, or 100 slides.  These are available from major suppliers such as Fisher at low cost and will ensure that your samples arrive in good shape.

• Pack the slides carefully in the slide box (put tissues in between them if you want), then pack the slide box in a cardboard box with some packing material (e.g., styrofoam “peanuts” or something of that nature).

• If you have to fill out a customs form, just call the contents “microscope slides -- non-hazardous” or something of that sort.

• Air-dried slides will keep for a long time, so they do not need to be shipped by express courier.

• Unfortunately, we are not able to cover the cost of shipping for small numbers of slides.  However, if financial constraints prevent you from sending material or if you intend to send a large number of samples, we will attempt to work something out.

Please contact Jillian Smith at  if you would like to provide samples.

Permits and animal care:

• Please take all steps to minimize the stress experienced by the animals and make sure that all sampling takes place in accordance with your institution's animal care regulations.  Refer to the Canadian Council on Animal Care guidelines on the care and use of wildlife for detailed guidelines.

• Sampling of mammals in the wild requires permits, often both state/provincial and federal.  Please consult with officials in your area and at your institution before collecting any specimens as only legally acquired material will be used in the study. 

• Shipping samples from some species out of your home country and/or into Canada may require special permits.  This will be particularly true for species that are listed in the International Convention on Trade in Endangered Species of Wild Fauna and Flora (CITES).  We can receive material from CITES-listed species through our direct affiliation with the Biodiversity Institute of Ontario, but you will also require export permits for these.

Links:

• The Gregory Lab, University of Guelph, Canada
• Jillian Smith -- M.Sc. Candidate in charge of the mammal genome size project
• Animal Genome Size Database
• Biodiversity Institute of Ontario
• Reprint of Hardie et al. (2002) with details of the image analysis methodology
• Avian Genome Size Project