Webinar: Mouse Study Reproducibility: The Impact of Genetic Background and Genotyping
This recorded webinar reviews pertinent topics for reproducibility in mouse studies, including genetic background and characterization, common inconsistencies/errors in genotyping, and best practices for establishing rapid and reliable testing protocols.
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Could you please speak to the issue of refreshing breeder colonies to maintain the overall genotype of a new mutant strain? We refresh every five generations to avoid creation of a substrain.
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.
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What is the optimal size of a two-week mouse sample for mouse genotyping? And how long can we keep DNA?
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.
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During times where you see weak bands, do you re-run tests again?
It is recommended to perform a second trial with a 1/10 dilution to reduce the potential inhibitor impact in the crude lysate.
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If an unexpected phenotype appears in a transgenic mouse colony, what should I do to evaluate whether this phenotype is from the mutant target gene or from the nonspecific background gene?
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.
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I have a question concerning my Cre/ loxP matings: I tested both lines for the NNT-mutation via PCR (due to C57BL/6J background). The PCR result was negative, but can I be 100% sure that males won't have impaired glucose tolerance?
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.
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I have created a new mouse line via CRISPR/Cas9 genetic engineering. How should I design my backcrossings? Should males carrying the mutation (the desired modification) mate with WT females or vice versa? ¬
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.
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Is it not correct that the proper controls are wild type (non-transgenic) from the breeding colony and not from commercial or other available stocks?
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.
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How do you clean your equipment between animals after tissue removal (i.e., razor, scissors, scalpel)?
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.
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Is it always recommended to use pure genetic backgrounds for Cre/ lox breeding, or can it be useful to mix backgrounds (e.g., 50/50%)? I heard that Cre recombinase expression can be reduced on a mixed background in comparison to pure BL/6J background.
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.
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How can I determine the correct sample size to control the genetic background of my colony?
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.
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Is brother/sister mating advisable to maintain inbred strains?
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.
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Have you ever heard about engraftment differences of xenografts in different backgrounds of immunodeficient mouse lines?
Yes, genetic background influences the engraftment ability of cells and tissues. Searching the literature for this topic will yield some good examples.
- Could you please suggest some literature regarding the impact of the Y choromosome from an error background?