Volume 27 No. 3 2012

This edition of the BRL Bulletin is dedicated to and are maintained by mating non-related rodent genetic monitoring. Genetic monitoring is animals to retain maximum heterozygosity. essential when breeding inbred and transgenic Therefore, the goal with genetic monitoring of animals to verify that there is no genetic outbred stocks is to preserve the heterogeneity contamination that can adversely affect research and prevent formation of sublines. Strains are outcomes. It also will introduce Transnetyx Inc., considered inbred if they have been maintained a company that provides genotyping services by 20 or more sibling matings. The goals of with drop-off stations in the BRL and COMRB monitoring inbred strains are to ensure a facilities. More information about Transnetyx consistent genetic profile and to detect or Inc., will be provided below. eliminate mutations. The purpose of monitoring genetically manipulated rodents is to identify Genetic Monitoring the presence or absence of the gene of interest Genetic monitoring is the process of examining as well as to verify the background strain, molecular markers to identify an animal’s genetic which may influence gene expression. make-up. This technique is commonly performed in laboratory rodents, especially mice. Genetic Sources of Genetic Contamination monitoring is essential for quality control when Genetic contamination can occur in several breeding inbred and transgenic animals. Any ways. The most obvious source of genetic contamination or genetic drift over time contamination is the introduction of new genes can alter research results and interpretation. by inadvertent mixing and breeding of animals. Therefore, accurate genotyping is necessary for This can happen if animals or cages are not obtaining quality research data. properly identified, if animals are mixed during transport or housing, or by false genotyping Commercial rodent vendors adhere to strict results. Other sources of genetic contamination protocols and maintain detailed records to include residual heterozygosity and substrain prevent genetic contamination in their founder, divergence leading to genetic drift over time. expansion and production lines. Pedigrees are Although inbred strains are considered to be used to document the genotypes of the genetically stable, random gene mutations can foundation mating pairs as well as their progeny. still occur. Even a small source of Relatives can be easily identified and traced contamination can affect research data and back to their origin. Vendors use pedigrees to limit the ability to replicate the study. Routine document the breeders and maintain all lines. genetic screening can help to identify and Commercial vendors perform routine quality prevent contamination, which in turn can help control by systematically screening genotypes, save time and money in the future. verifying the genetic backgrounds of the animals, verifying mutant alleles in mutant Animal Identification stocks, and culling of any animals with In order to perform genotyping, it is important phenotypic changes. that animals are first properly identified. There are several types of identification methods that The goals of genetic monitoring vary depending have both advantages and disadvantages. on whether the animals are outbred stocks, Identification is often performed with ear inbred strains, and genetically manipulated punches or ear tags, while more permanent rodents. Outbred stocks are genetically diverse methods of identification can be accomplished Page 2 BRL BULLETIN with tattoos or microchips. rodents. A reader is placed over the location of the microchip which displays the unique Ear punches or notches are a common method identification code. These systems tend to be of identification in rodents. This technique is more expensive due to the initial cost of the relatively easy to perform and is inexpensive. A reader, software, and the cost of the universal numbering system can be used to microchips. Newer transponders can also achieve a unique number for each animal in the measure subcutaneous body temperature, colony. Using this universal numbering system which may be useful for certain studies. requires skill both in performing the ear punch as well as reading the ear punches. An alternative The Jackson Laboratory has recently released method is to use a combination of punches only a new identification system (JAXTag) that uses to identify the animal at the cage level. It is a modified ear tag. The tag contains a barcode important to consider that ears can be torn and that is read by commercially available punches can heal closed making identification scanners. Like microchips, they require larger difficult if using this method. initial costs associated with the scanner and software. The advantages of this new system Another common method of identification is to include that the tags are light-weight, non- apply a metal tag to the animal’s ear. Like ear irritating and secure for more than 18 months. punches, this is relatively easy to perform and inexpensive. This method can provide a unique Monitoring Methods number for each animal in the colony. There are several methods used to monitor Disadvantages of this method include ear tags genetic contamination including physical falling out, localized inflammation associated characteristics, biochemical and immunological with the ear tag, or being torn out by the animal techniques, and DNA-based techniques. or its cage mates. One helpful tip is to place Commercial vendors such as Charles River, even number tags in the right ear and odd Harlan, Jackson Labs, and Taconic offer number tags in the left ear so that in the event genetic monitoring services. Below is a brief that two animals in the same cage lose tags, description of available methods utilized to there is a better chance of identifying the correct screen for genetic variation. animal. Physical Characteristics A permanent method of identification is to tattoo It is important to be familiar with the physical a unique identifier on the tail of the animal. This characteristics of rodent research models in is often time consuming and more expensive order to identify changes over time. The most than ear punches or ear tags. There are obvious change that can occur with genetic automated commercial tattoo machines contamination is coat color. Other indications of available that can save time and produce reliably genetic contamination may include an readable results. Tattoo ink is available in a increased rate of abnormalities such as tail variety of colors as well as fluorescent ink that defects, eye defects, and seizures as well as can be visualized with a black light on pigmented changes in body size, weight, level of skin. The BRL has a tattoo device that is aggression, reproductive performance and available for rental and training can be provided lifespan. Being in tune with the normal variation by BRL staff. among the strains used can help quickly identify a problem if one arises. Microchips are small devices that contain a unique identification code and are implanted Biochemical Markers and Immunological under the skin. This technology is commonly Techniques used in larger species, but it is also available for Inbred mice and rats may have variations in Page 3 BRL BULLETIN specific biochemical and immunological markers methods to quantify the amount of nucleic acid that can be used to monitor genetic present. Absolute quantification determines an contamination. There are panels available to exact number by identifying specific target DNA evaluate strain-specific distribution patterns of molecules while relative quantification is based particular markers. Examples of biochemical and on the amount of DNA compared to internal immunological markers include erythrocytic reference genes. Both of these methods are antigens, hemolytic complement, isoenzymes, helpful for differentiating a hemizygote from a and the major histocompatibility complex. Most homozygote and in determining transgene of these methods can be performed using blood copy number, which can dramatically affect the or tissue samples. Another immunological phenotype. technique to determine genetic contamination is by performing skin grafts. This technique detects Another PCR technique involves the use of any genetic changes in histocompatibility. microsatellites, which are short repetitive DNA Animals that are genetically identical will not sequences that contain unique flanking reject a graft. A major change in regions. The number of repeats is highly histocompatibility causes rejection within 10 polymorphic and there are thousands days; however, minor changes can take months contained within the mouse and rat genomes. to detect. It is also important to keep in mind that Analysis of microsatellite locations can be used graft failure may occur for technical reasons. to characterize background strains as well as estimate genetic variation among animals. DNA-Based Molecular Techniques Panels have been developed for the most There are several types of DNA-based common inbred strains, but these panels can molecular techniques available to perform also be customized for other strains. genetic monitoring. The most common techniques include standard polymerase chain Tissue Collection for DNA-Based Methods reaction (PCR) or real time PCR. Each Tail snipping is a common method to collect technique has a specific use so it is important to tissue for genotyping. Using this technique, have an understanding of the different tests that 5mm of the distal tail tip is removed and are available. bleeding controlled by pressure, electric or chemical cautery methods. Tail snips can be PCR is one of the most common methods for performed in mice less than 28 days of age determining genetic variability. PCR has now without anesthesia. Mice older than 28 days replaced most of the biochemical and require general or local anesthesia. Repeated immunological tests described above. Current tail snips on the same animal is discouraged. PCR technology uses amplification of single Please see UIC Guidelines on Tail Snipping in nucleotide polymorphism (SNPs) markers. A Rodents for further details. Other samples that SNP is a variation of a single nucleotide in the can be used for DNA-based methods include genome that differs between members of a ear punches and oral swabs. These are often species. Typically, SNPs occur in the non-coding used as alternatives to tail snips in older regions of a DNA sequence. This method is rodents. helpful to evaluate multiple markers on different chromosomes, which can be used to monitor for Genetic Contamination Prevention changes in genetic background. This technique Strategies can also be used to verify that an induced point Accidental mismatings can lead to the mutation is still present. introduction of undesirable genetic material and can confound research. There are several Real time PCR is a method that quantifies the strategies that can be instituted to prevent reaction, unlike standard PCR. There are two genetic contamination. First, it is important to Page 4 BRL BULLETIN examine animals routinely for physical changes. information and assays, as well as view and Observation of any phenotypic or behavioral manage your genotyping results. There are two changes warrants investigation to identify pick up times: Wednesday by 2pm (results the accidental genetic contamination. Ideally, all following Tuesday AM) and Friday by 2pm animals undergoing experimentation should be (results the following Thursday AM). genotyped. Animals should be properly identified Transnetyx provides free sample submission with appropriate genotypes on cage cards and supplies as well as free shipping. Charges are any other records. Pedigrees are useful in billed through the BRL billing system. Before documenting and maintaining breeding lines. getting started, it may be helpful to view the Other management strategies include organizing online ordering system and fee structure at colony animals by genotype and separating http://www.mailyourtail.com/UIC/Default.aspx. cages with similar coat colors and genotypes to If you have any questions or would like to prevent inadvertent mixing of animals. consult a Transnetyx representative, please use the following contact information: All laboratories breeding rodent strains should have a genetic monitoring program in place. Mr. Ryan Yanase There are several items to consider prior to North American Sales Manager starting a program. First, it is important to 901-507-0476 develop baseline genetic information about each [email protected] strain that is being used. The availability of certain tests along with the goals of the specific Mrs. Kristin Uyl laboratory will determine the type of test best 847-814-2555 suited for routine genetic monitoring. Once the [email protected] appropriate tests are identified, the next step is to determine how many animals should be sampled, which depends on the size and scope References: of the animal colony. If breeding heterozygotes, Charles River Laboratories. Genetic Testing testing is typically performed on every litter but if Services breeders are homozygotes or inbred strains, a http://www.criver.com/en-us/prodserv/bytype/ minimum of testing one to two times per year is genetictesting/Pages/home.aspx recommended. Also, large breeding colonies Handbook of Laboratory Animal Science, 2nd may require more intense screening of the Edition. Volume 1: Essential Principles foundation stock than the production lines, which and Practices. CRC Press, 2003. can be randomly sampled. A well-devised genetic quality control program should not only The Jackson Laboratory. Quality Control encompass genotyping animals, but also Program. comprehensive training of staff and application http://jaxmice.jax.org/genetichealth/ of good colony management strategies. GQCprogram.html

The Mouse in Biomedical Research, 2nd Transnetyx Automated Genotyping (TAG) Edition. Academic Press, 2007. Transnetyx Inc. is a company that provides genotyping services with two drop-off stations. There is a TAG center located in the BRL (between rooms 123 and 125) and another TAG center located in COMRB (outside entrance) where samples are submitted and sent out for testing. The online ordering system allows you to place orders, manage your genetic strain