A method for non-invasive genotyping of APCmin/+ mice using fecal samples
© Symonds and Fenech; licensee BioMed Central Ltd. 2012
Received: 3 November 2011
Accepted: 30 January 2012
Published: 30 January 2012
The APCmin/+ mouse is commonly used in cancer research and is just one of many genetically altered models that is currently being developed. With high numbers of breeding programs, it is important to have a simple method that can be used to genotype the mice non-invasively. Here we report a reproducible method for genotyping mice with DNA extracted from fecal samples. Comparison of fecal results with those obtained from intestinal tissue DNA and clinical outcome (presence/absence of tumors) showed this technique to have 100% accuracy. This non-invasive method of genotyping may be applied to other transgenic mouse models.
KeywordsAPCmin/+ feces genotyping cancer non-invasive
With advances in molecular genetics, there are large numbers of transgenic and knock-out mice being developed. The most common method for genotyping these mice is with DNA isolated from tail, ear or toe tissue, or from blood. These collection techniques however are invasive procedures and are not appropriate for repeat sampling. Recent publications have shown that DNA can be isolated from fecal samples, which allows for non-invasive genotyping [1, 2].
In this work we describe a method for genotyping APCmin/+ and wild-type mice with DNA isolated from fecal pellets. The APCmin/+ mouse is a model for familial adenomatous polyposis, with the mice highly susceptible to spontaneous intestinal adenoma formation at an early age, and anaemia as a secondary condition . This mouse model is therefore commonly used in cancer research. It has a chemically-induced dominant mutation (base substitution mutation) in the adenomatous polyposis coli (APC) gene which is considered a tumor suppressor gene . It has been shown that the main mechanism for tumor induction is loss of the wild-type (Apc+) allele (ie. loss of heterozygosity) [5, 6].
The aims of this study were to assess the sensitivity, specificity and reproducibility of genotyping APCmin/+ mice and their wild-type littermates with DNA isolated from their fecal samples.
Results and discussion
This study has shown that APCmin/+ mice and their wild-type litter mates (> 3 wk) can be genotyped using DNA isolated from whole fecal samples without the need for isolation of the colonic cells from the rest of the fecal mass. The collection technique is non-invasive to the mouse and therefore repeatable. Fecal pellet collection from mice aged greater than 3 wk was very simple, with the majority of mice providing a sample within 1 min. Collection of feces from mice younger than the weaning age (3 wk) was difficult due to their milk diet, and resulted in a poor DNA yield as the sample was small. DNA extraction from a mouse (> 3 wk) fecal pellet resulted in a yield of 20 - 350 ng/μL (average 75 ± 4 ng/μL (mean ± standard error)), and a purity (determined with the nanodrop OD260:OD280) of 2.02 ± 0.01. Following successful amplification of the DNA, running the PCR products on the gel allowed the determination of the genotype of the mouse.
Comparison of fecal genotyping result to other methods
Genotype determined from fecal DNA
Presence of intestinal tumors
Intestinal tissue genotype positive for wild-type
Intestinal tissue genotype positive for APCmin/+
The reproducibility of the genotyping with fecal samples was assessed by collecting and analyzing DNA from fecal pellets from mice on multiple occasions (with at least one week between collections). All samples gave the same genotype every time tested, showing the method to be reproducible (data not shown).
APCmin/+ and wild-type (APC+/+) mice were produced in a breeding program that involved mating male APCmin/+ (Jackson Laboratories, Bar Harbor, ME) with female C57BL/6 mice (ARC, Perth, Australia). All methods were approved by the CSIRO animal ethics committee (Adelaide, South Australia).
Fresh fecal samples were collected from the offspring (aged 3 - 13 wk) by placing the mouse in an empty cage until a pellet was passed. One fecal pellet per mouse was adequate for analysis. The DNA was isolated while the sample was fresh using the Qiagen Blood and Tissue Kit (Qiagen, Victoria, Australia). Briefly, 180 - 380 μL tissue lysis buffer was added to the fecal sample (depending on the size) along with 20 μL proteinase-K. The sample was incubated at 55°C for 2.5 h (vortexed every 45 min), followed by the addition of a further 200 μL lysis buffer. This resulted in the majority of the fecal pellet being dissolved and filtering of the sample was not performed. The DNA was precipitated with ethanol and purified with the Qiagen spin column according to the manufacturer's instructions. DNA was eluted in 100 μL AE buffer and quantified with a nanodrop (NanoDrop Technologies, Wilmington, DE). A sub-group of mice aged 13 wk were culled and small intestinal tissue was collected and stored at -80°C. DNA was isolated from the tissue following the above protocol, but the tissue was first lysed in 180 μL tissue lysis buffer with 20 μL proteinase-K overnight at 36°C.
Polymerase chain reaction primers 
5' GCC ATC CCT TCA CGT TAG 3'
5' TTC TGA GAA AGA CAG AAG TTA 3'
5' TTC CAC TTT GGC ATA AGG C 3'
Fecal genotyping results were assessed for sensitivity and specificity by comparing the results to those obtained from intestinal tissue DNA, and to the presence or absence of intestinal tumors in mice. The reproducibility of the fecal genotyping technique was assessed by collecting additional fecal pellets from mice, with at least one week between collections. Fecal samples were collected from wild-type (n = 21) and APCmin/+ mice (n = 29) on two to eleven occasions and analyzed for genotype.
In conclusion, this technique is suitable for genotyping APCmin/+ mice and their wild-type littermates. The genotype results using fecal DNA were consistent with that found from DNA isolated from small intestinal tissue, and also consistent with the presence or absence of intestinal tumors. It is a simple and reproducible test that can be developed for other genetically altered mouse models. Its non-invasive nature will reduce physical injury and anxiety of the mouse which will improve animal welfare.
adenomatous polyposis coli
Commonwealth Scientific and Industrial Research Organisation
polymerase chain reaction.
Erin Symonds was supported by a CJ Martin post-doctoral fellowship awarded by the NHMRC of Australia (ID 357702).
We would like to thank David Courage and Darien Sander for the care of the mice.
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