An essential step in mouse breeding is genotyping them to determine the genotype of every mouse in the litter. It is also useful to differentiate between various groups of experimental mice if any confusion arises. When genotyping, you will be hunting for the specific gene that you want your mice to have or a genetic marker identifying a particular group of mice.
Even though mouse genotyping is a simple enough technique, once standardized, here are a few specific tips to a hassle free genotyping experience:
1. Know What you are Looking For
If you have detailed information about the genomic modification, you can identify the gene sequence under investigation. What kind of genetic profile or modification are you trying to identify? Is it a gene knock-out, knock-in, cre-lox or Flp-FRT deletion? Did you create the genetic modification yourself or acquire the mice from commercial vendors or a collaborator’s labs?
With this information, design primers and an appropriate mouse genotyping assay. Look for reliable genotyping protocols through commercial vendors or labs that work on the same mouse lines. But even if you are starting from scratch, it is easy enough to work through a genotyping protocol when you know what gene you want to probe.
2. Pick the Correct Technique for your Mouse Genotyping
Genotyping is straightforward, and a few standard techniques exist to carry it out. Once you initially standardize your technique, the setup can be used for all lab generations to come.
Southern blotting analysis is considered the gold standard genotyping technique for establishing founder animals, because it has a low false positive rate and provides more reliable information. For all further characterizations of offspring from established mouse lines, use PCR, because it’s quick and easy. You can use a conventional end-point PCR with an agarose gel readout for most situations. If you need higher sensitivity and reliability, use a quantitative PCR with a melting curve analysis. In rare cases, such as for a thorough mapping of the genetic profile, you can sequence the region of interest.
3. Design the Right Primers
The most important component of any PCR reaction is a perfect set of primers. Spend the most time and effort at this step.
If your mice came from a trusted source with a set of genotyping primers, then great. You’re all ready to test them in a PCR. If not, design primer sets like you would for a regular PCR reaction. Design them around the targeted region to either detect the knocked out/in region directly or additional gene sequences (like a neomycin resistance cassette that was added to facilitate the genetic modifications). This serves to identify gene insertions and deletions, but also distinguishes between wild type, heterozygous, and homozygous mutant animals.
Make sure you also include internal control primers to an unrelated genomic region to exclude any faults with DNA quality or PCR amplification.
4. Material for DNA Isolation
There are several ways to obtain DNA for mouse genotyping. The one you use depends on the established practice in your lab and the quantity of DNA required for the assay.
You can collect DNA from ear tissue (0.5 to 2 mm) using an ear punch. This yields a larger quantity of quality DNA. Ear tissue is easy to collect when mice are ear punched for identification. Alternatively, you can take tail biopsies (less than 5 mm of the tail tip) from young mice to obtain enough material for good genomic DNA isolation. Other sources include blood, fecal pellets, buccal swabs or fur, although they generally give poorer yields. Whichever method you choose, keep in mind that younger mice and fresh tissue give better quality genomic DNA.
5. Isolating Genomic DNA for Genotyping
Once you have your sample tissue in hand, it is time to isolate genomic DNA from the cells.
Use a ‘dirty’ DNA prep for most mouse genotyping PCR protocols. They are quick, easy, and inexpensive. An example is the HotSHOT technique of DNA preparation: boil mouse tissue samples in an alkaline lysis solution containing NaOH and EDTA, then neutralize them with Tris-Cl.1
If a dirty DNA prep doesn’t work, then your protocol may require purer DNA. In that case, you’ll need to look into a more time-consuming phenol-chloroform extraction or a comparatively expensive DNA isolation kit.
Standardize your genotyping PCR the same way you would for any PCR. Some points to consider:
Optimize your PCR primers using positive controls
Titrate the amount of genomic DNA to find the optimum template concentration
Use touchdown PCR in those annoying cases in which the wild type and homozygous mutant DNA behave differently under the same PCR conditions
Always include wild type and mutant control samples to compare bands on gel or curves in a melt curve analysis
Often, the difference in the band sizes of the wild type and mutant bands will be very small – make sure you get good separation in the gel by using a longer run
Finally, remember to genotype the offspring at regular intervals, even from established homozygous lines, to rule out any mix-ups.
These tips should help you establish a reliable mouse genotyping protocol to identify your mutant mice.
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