The Impact of Genetic Background and Genotyping Webinar: Questions and Answers

Refreshing breeder colonies is an important requirement and should be performed on regular basis every 5–10 generations of breeding. This will help to mitigate the risk posed by genetic drift and the occurrence of spontaneous mutations that may impact mouse phenotypes and experimental data bias.

Refreshing can be done in two ways. The first pertains to revitalizing cryostock. Cryopreservation should be done when we can be confident that the line has not already drifted. The second would be to breed a few generations (three are recommended) with wild-type animals purchased from genetically controlled colonies. Don't forget to breed transgenic females with WT males at first to refresh the Y chromosome.

At two weeks old, a mouse's tail is quite thin. It is preferable to have a sample of 0.5 cm.

How long can we keep DNA? This will depend on the type of preparation you have made. While purified DNA can be preserved for a very long time, especially if it is frozen at -20° C, the question arises for a crude lysate.

Having tested crude lysates genotyped fresh, after a 4° C storage or after freezing at -20 °C, our lab was able to obtain results after one month at 4° C (for some even longer). When you need to store crude lysates for later use, it is preferable to freeze them. Frozen crude lysates can still be used for PCR genotyping.

It is recommended to perform a second trial with a 1/10 dilution to reduce the potential inhibitor impact in the crude lysate.

Use of an appropriate wild-type and/or HE control mice will help you evaluate where exactly the change in phenotype came from. If the phenotype is induced by the mutant target gene, the controls should not show the phenotype.

What you have done so far has eliminated one major cause of experimental data bias. Mutations other than the NNT mutation could likely also impact glucose tolerance, so checking only the presence of an NNT mutation does not address all the risks. You also must avoid genetic drift in your colony that may impact other genes implicated in glucose tolerance. Refreshing your breeder colony is a crucial requirement and should be performed on a regular basis after 5–10 generations. This is intended to limit genetic drift and the occurrence of spontaneous mutations that may impact mouse phenotype and experimental bias.

Refreshing can be done in two ways. The first pertains to revitalizing cryostock. Cryopreservation should be done when we can be confident that the line has not already drifted. The second would be to breed a few generations (three are recommended) with wild-type animals purchased from genetically controlled colonies. Don't forget to breed transgenic females with WT males at first to refresh the Y chromosome.

Always backcross the first transgenic females with wild-type males to refresh/exchange the Y chromosome. In subsequent breedings, use transgenic males obtained from the first breeding round to mate with wild-type females.

This is correct if those controls are available. In certain instances, breeding schemes used for maintaining polygenic lines do not allow for the generation of control littermates. In that case, you're obliged to take controls from outside the colony. If your polygenic line is onto a well identified pure genetic background and your colony is refreshed on regular basis (e.g., revitalization from cryostock) then taking a control from an outside colony (known provider) can be done with minimum risk.

In addition to scissors or ear punching (sanitization required using an appropriate method [e.g., 70% ethanol]), you need a compress soaked in 70% diluted ethanol. Clean the scissors or ear puncher with the compress between each animal to avoid sample contamination (please pay attention to removing all tissue from the instruments after each animal). 

Further information can be found in this publication. 

Genetic background influences gene expression level. By consequence, Cre transgene expression may be impacted up or down by the genetic background of mice. The regulation cannot be anticipated. The issue with mixed backgrounds is that you will not be able to keep a 50/50 ratio, so after a while you will end up with a rather heterogenous distribution of each genetic background in your colony. This may lead to a higher variation in experimental data between individuals.

Depending on the test used, with STR markers, certain testing organizations advise to use at least three individuals. Most of the time, testing 3–5 individuals is enough to get informative indication on genetic background purity.

Theoretically speaking, brother/sister mating is the only way to propagate an inbred strain, especially for larger colonies. For smaller colonies, it is possible to do it differently but only if the colony is regularly refreshed following a well-designed strategy.

Yes, genetic background influences the engraftment ability of cells and tissues. Searching the literature for this topic will yield some good examples.

Yes. Please see the following:

• Phenotypic Effects of the Y Chromosome are Variable and Structures in Hybrids among House Mouse
• Recombinant Lines Sequence and Structural Diversity of Mouse Y Chromosomes
• Genetic Variation in Chromosome Y Regulates Susceptibility to Influenza A Virus Infection