Colony Assessment

It is essential for accurate phenotypic characterization that the integrity of the genetic alteration of a specific line be maintained over several generations of backcrossing using sound breeding practices and accurate record keeping. In addition, basic observational assessments at the colony level to detect any unusual characteristics of newly generated mouse lines are mandatory in research involving genetically engineered mice.


An important strategy in validating and maintaining the integrity of genetically altered mutant mouse lines for distribution is to be able to accurately and efficiently identify the specific transgene or mutation. Because homozygosity may instill lethality or infertility, many imported transgenic and gene-targeted mutant lines are maintained as heterozygotes so must be individually genotyped. It is essential to obtain genotyping protocols from the scientific group originally characterizing the mouse line. Even then, a significant amount of time and resources may be necessary to optimize PCR protocols (or Southern blotting if necessary) for each mouse line. Oligonucleotide primers can be custom produced by a reliable commercial company. Genomic DNA for these assays is isolated from tissue biopsy samples using standard DNA isolation procedures. DNA probes for Southern blots can be labeled with 32P-dCTP using the random primer method. PCR and Southern blot analysis should be performed on tail or ear punch biopsies obtained from 2- to 4-week-old potential founder pups as a source of DNA for testing transgene integration or deletion mutation. Once mice integrating the transgene or exhibiting the deletion mutation have been identified, these mice are set up in breeding pairs. The goals of breeding the founder mice are to observe the inheritance and expression patterns of transgenic lines through several generations, to expand the colony to provide mice for experimental study, and to produce transgenic homozygosity for comparison with the heterozygous state. Breeding directly to homozygosity means +/- brother to +/-sister mating of offspring.

Accurate and user-friendly record keeping programs are vital for optimizing the expression of the mutation, maximizing the number of viable offspring, and minimizing the potential confounding influence of background genes from breeder parents. Record keeping systems should incorporate a combination of written and computer-based records. Either a commercial colony management software system or standard Excel spreadsheets can be used to maintain a master list of every mouse that is ear tagged or otherwise marked for identification using tattoos, ear punches, subcutaneous transponders, or toe clipping. Items recorded in the master list include birth date, transgene name, generation, line, sex, parents, phenotype and genotype, and information about the specific project. Establishing a pedigree for each founder mouse that is mated is a vital part of the recording system. Customized cage cards for breeder cages provide a summary of the breeding activity for each cage. Customized weaning cards record the line and ear tag numbers so weanlings can be readily located and tracked. A separate file on the Excel spreadsheet or within the database system for breeding records should contain birth dates of litters, number of pups born and weaned per litter, ear tag numbers of pups, breeder setup date, breeder retirement date, strain, generation, line, and comments.

Because many transgenic mice are developed utilizing strains that are not desirable genetically for use as a background strain, backcrossing to the desired congenic strain is necessary. As each consecutive generation is backcrossed to the desired strain, the proportion of the genome originating from that strain increases until at the tenth generation, the desired background strain is 99.9% of the genome and considered congenic. As backcrossing for 10 generations can take upwards of 2.5 to 3 years, a method to shorten this time can be highly cost-effective. In speed congenics, offspring are screened for those with higher percentages of the background strain-specific markers, using single sequence polymorphisms.7 These tests can be performed by commercial laboratories. Those mice exhibiting higher percentages of the background strain in their genome are then used as breeders to produce the next generation. Using speed congenics can reduce the number of generations required to display a transgene or deleted mutation on a known, desired strain by one half to only five generations. In addition, embryo transfer can be used in an attempt to further shorten the time to have inbred mutant congenics to within a year.

Selection of controls for genetically altered mice is essential for valid scientific experiments. For stocks of mixed or segregated genetic background, littermate controls are best. In cases where lines are maintained by homozygous sibling matings and, therefore, do not have wild-type littermates available, determination of the appropriate controls can be based on Jackson Laboratory recommendations.

When transgenic or wild-type control or breeder mice are shipped into and received by a barrier facility, maintained in a specific pathogen-free environment, quarantine is essential to eliminate viruses or other pathogens that imported mice may be harboring. For example, it has been reported that up to 48% of the strains imported to the Jackson Laboratory in 1997 had evidence of prior infection with one or more viruses.55 Genetic and microbiologic status of mouse lines should be assessed by the health surveillance staff (as described below).

100 Pregnancy Tips

100 Pregnancy Tips

Prior to planning pregnancy, you should learn more about the things involved in getting pregnant. It involves carrying a baby inside you for nine months, caring for a child for a number of years, and many more. Consider these things, so that you can properly assess if you are ready for pregnancy. Get all these very important tips about pregnancy that you need to know.

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