Bench Talk

Two Trending CRISPR-Cas9 Optimizations

Posted by Stephanie Sandor on Jan 24, 2019 1:20:02 PM

The spotlight continues to shine on an innovative technology, CRISPR-Cas9, due to its efficient site-directed mutagenesis. An example includes introducing variant Cas9 nucleases which affect the product of the studied protein. Also, it is utilized for RNA editing as opposed to DNA editing. The traditional use for Cas9 nuclease introduces double-strand DNA breaks that are repaired by nonhomologous end joining (NHEJ) or homology-directed repair (HDR) mechanics. These cause spontaneous insertions or deletions (InDels) which may give unwanted phenotypes.

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Tags: CRISPR-Cas9

5 customer questions about strains

Posted by Transnetyx Genetic Services Team on Dec 20, 2018 11:00:00 AM

At Transnetyx, our goal is to take care of all of your genotyping needs. Our Genetic Services team conducted a survey to collect the most common questions that our customers ask. The first topic we will cover are questions regarding strains.

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Tags: FAQs

Why does genetic background matter? (Pt. 2)

Posted by Nate Nowak on Dec 6, 2018 11:00:00 AM

 

So, we have discussed the impacts the genetic background of a mouse model can have on experimental data. Now, how can that impact be controlled and mitigated?

 

Obtain and Breed Proper Inbred and Congenic Strains

The first step in ensuring reliable, reproducible results is making sure that your colonies are not on mixed backgrounds, but rather have the background appropriate for your work. If you start out with the correct genetics, then you can monitor for changes. For all strains you should ensure the following during breeding:

  • Use brother x sister mating schemes
  • Periodically refresh inbred lines using pedigreed animals from a high-quality vendor or cryopreserved founders

For GEM lines add the following:

  • Back-cross to the parent strain every 10 generations to minimize drift.
  • Confirm the presence of mutant alleles with phenotyping and genetic testing.

For newly acquired strains make sure to validate both the background and mutations prior to breeding or beginning experiments.

Monitoring for Phenotypic Change

The experienced eyes of the animal husbandry team are a critical component of any genetic monitoring program. Institutional guidelines for colony management are the primary means used to establish and maintain inbred mouse colonies. They are also the principal means by which breeding errors that lead to genetic contamination are prevented. Initial and periodic training of animal husbandry personnel should provide the foundation for any genetic monitoring program. Any observed anomalies in phenotype are to be investigated, and mice exhibiting changes should promptly be removed from the breeding colony.

However, using this method alone may cause contamination to remain unidentified; not all evidence of genetic contamination is phenotypically obvious. Surveillance with laboratory methodologies should be part of genetic monitoring programs.

Genetic Monitoring by SNP Testing

Even with the best-trained animal care personnel and a robust genetic monitoring program, it is still necessary to complement this surveillance with laboratory methodologies that objectively identify genetic homogeneity within inbred strains of mice. SNPs offer a robust means of differentiating mouse strains, and SNP testing has been shown to be fast, efficient, and cost-effective genotyping method for genetic monitoring programs for both strains and substrains. SNP marker testing can be used in some or all of the following situations:

  • When adding new breeders.
  • When ensuring a lack of genetic contamination is critical (i.e., at the beginning and/or end of a study).
  • When used to spot check individual mice from each litter to ensure the genetic background is free from contamination.
  • When used to increase the speed of a backcross via marker-assisted breeding.

Genetic contamination can have wide-ranging, unexpected effects on research.

Undertaking strategies such as consistent breeding and husbandry practices, adherence to a clear colony management strategy, and routine genetic monitoring via SNP panels and allele-specific genotyping, are critical components of ensuring authentication of animal model resources.

Researchers should consider the impact of background strain and substrain in experimental design and analysis. By ensuring the use of the correct model, you are ensuring the quality and reproducibility of your results.

 

Citations

Fahey JR, Katoh H, Malcolm R, Perez AV. The case for genetic monitoring of mice and rats used in biomedical research. Mamm Genome. 2013 Apr;24(3–4):89–94

Guénet J-L, Benavides FJ. Mouse Strains and Genetic Nomenclature. In: Auwerx J, Brown SD, Justice M, Moore DD, Ackerman SL, Nadeau J, editors. Current Protocols in Mouse Biology [Internet]. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2011 [cited 2018 Sep 26]. Available from: http://doi.wiley.com/10.1002/9780470942390.mo100181

University of Michigan Animal Care & Use Program. My Strain Initiative | Animal Care [Internet]. [cited 2018 Sep 26]. Available from:https://animalcare.umich.edu/animal-use/my-strain-initiative.

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Tags: Genetic Monitoring, Genetic Background

Why does genetic background matter? (Pt. 1)

Posted by Nate Nowak on Nov 27, 2018 11:00:00 AM

Laboratory mice have played a long-standing and critical role in life sciences research. Small, easily housed and cared for, mice are inexpensive, breed quickly and have a short lifespan. All of the above, combined with genetic, biological and behavioral similarities to humans, have made mice indispensable to modern biomedical research. Add in the ease with which they can be genetically modified, and mice have become a powerful tool in research.

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Tags: Genetic Monitoring, Genetic Background

3 things to consider when working with mouse models

Posted by Scarlett Evans and Samira Lawton on Aug 1, 2018 8:58:30 AM

It’s far too common in the lab to be given a set of primers and a protocol and be asked to genotype without any clear specifics on the animal strain itself. While you may end up with bands at the right sizes, that doesn’t always guarantee that you have the exact mutation you think you do. Here are three things that we believe will be helpful to keep in mind when starting to genotype any line.

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Tags: Genotyping, Best Practices