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Extended abstract from Scand-LAS 2024

Non-invasive sampling methods for genotyping: Improvements towards the 3Rs principle

By , , , ,
1Charles River Laboratories, Research Models and Services (RMS), Germany

Abstract

Genetically modified mice are usually genotyped using invasive ear or tail biopsies. In accordance with the 4R principle (Replacement, Reduction, Refinement and Responsibility) we have developed non-invasive sampling methods to prevent stress and pain for mice. DNA could be extracted from oral swabs and hair follicles using conventional PCR and real-time PCR. Signals could be detected up to 25 days after sampling, if the swabs were shipped and stored at +4°C. In a pilot study, oral swabs, hair and biopsies were compared, showing consistent genotyping results in all three sampling methods with hair being more susceptible to contamination. Oral swab and hair follicle sampling provide an alternative that can be used for large-scale routine genotyping, especially if no invasive biopsy is allowed (e.g., animals with ear tags or toe tattoos) or no second biopsy is possible.

Introduction

The Charles River 3Rs Mission strives to advance sci-ence by improving laboratory techniques in line with the 3Rs principle, introduced by Russell and Burch (1959), and focuses on enhancing animal well-being. The 3Rs are: Replacement - avoiding or replacing the use of animals, Reduction – minimizing number of animals and Refinement – minimizing pain, stress and suffering. In research with genetically modified mice, which involves genotyping using invasive ear or tail biopsies in 92% of cases (Mazlan et al. 2014), there is a growing shift towards non-invasive sampling techniques such as oral swabs, or collecting hair or feces. Our EU Charles River genetic testing laboratory has successfully tested and proven the efficacy of genotyping using oral swabs and hair from rodents, aligning with the 3Rs principle and European regulations. The advantages of using non-invasive methods, like oral swabs and hair instead of biopsies are shown in Figure 1 below.

Benefits and applications of non-invasive sampling methods for genotyping
Figure 1. Benefits and applications of non-invasive sampling methods for genotyping

Methods

Housing and husbandry: The genetically modified mice were kept in stable groups within an isolator, under a 12:12-hour light/dark cycle, with temperatures ranging from 20-24°C and humidity levels between 45-65%. The mice were bred and raised under micro-biologically defined conditions (specific-pathogen-free(SPF) status according to FELASA standard) and pro-vided with sterilized food pellets and water ad libitum.Cage enrichment included cardboard rodent houses orplay tunnels, wooden gnaw sticks and paper tissue. Vet-erinarians and animal technicians ensured animal welfare daily in accordance with national and internationallaws and guidelines for the care and use of laboratoryanimals.
Research Conditions: For the data represented here, both male and female mice from different trans-genic lines were used. Due to animal well-being and the size of the swab head, all animals were at least 16 days old before oral swab and hair follicle samples were taken. Several swab-types were tested in terms of the cotton head size and surface structure. Based on the initial testing results, we identified the most effective swab type for achieving optimal genotyping results. A thorough sampling procedure is important to acquire sufficient animal tissue for further processing.
Sampling: Oral swab samples were taken from mice, as shown in Figure 2. The swabs were autoclaved and brought into the animal barrier facility according to standard procedures. Mice were securely scruffed and the swab was twirled around for 5-19 seconds to collect samples from the inside of the cheek. Swabbing was done carefully to avoid hurting the mice. Hair folli-cle samples were obtained by carefully plucking a small amount of hair (10-20 hairs). After sampling, mice were placed back into their cages. The oral swabs were left to dry before placing each swab and hair sample into in-dividual tubes. Surplus tissue (ear) from individual ani-mal identification was used for routine genotyping and as a control. Finally, the samples were shipped to the genotyping facility. All samples were taken at AAALAC accredited CRL sites according to animal welfare rules and guidelines.

The mouse was securely scruffed to prevent it from moving its head. The autoclaved oral swab was gently inserted at an angle into the oral cavity of the animal to collect the sample from the inside of the cheek
Figure 2. The mouse was securely scruffed to prevent it from moving its head. The autoclaved oral swab was gently inserted at an angle into the oral cavity of the animal to collect the sample from the inside of the cheek

Processing: Throughout the whole processing work-flow, samples were kept in a 96-well format to avoid potential mix up and to enable the processing of large number of samples.
Lysis and DNA extraction: The oral swabs were incubated in lysis buffer for 2h at 56°C. Hair and ear biopsies were incubated under the same conditions but overnight. DNA was extracted using Solid Phase Reversible Immobilization (SPRI) technology. Purified DNA was stored at +4°C (short term) until PCR analyses.
Polymerase Chain Reaction (PCR) and analysis of results: DNA extracted from the samples (oral swabs, hair and biopsies) was subjected to either conventional PCR and analysis using capillary gel electrophoresis (CE) (LabChip GX Touch, Perkin Elmer) or real-time PCR (quantitative PCR and endpoint analysis) using StepOne Cycler (ThermoFisher Scientific). Slight-ly adapted conditions were established if needed e.g. increased number of PCR cycles, template or primer concentration. Results from conventional and real-time PCR were analyzed and compared among the different sample types.

Results

1. Oral swab genotyping - Suitable for every kindof PCR: Oral swabs taken from transgenic lines (KO,KI, etc.) were subjected to conventional PCR with am-plicons ranging from 100 to 1500 bp in length (Fig-ure 3A) and to real-time Endpoint analysis (Figure 3B)and zygosity testing (qPCR) for transgenic lines. In ourstudy we could show that >98% of oral swab samplesled to clear results. Furthermore, >99% of the resultsfrom oral swabs matched those from correspondingear biopsies.
2. Shipment and Storage conditions for oralswab – (RT (+ 20°C), + 4°C and – 20°C): The ro-bustness of oral swabs genotyping in terms of ship-ment and storage time/condition were tested for up to 25 days using capillary gel electrophoresis. The percentage ratio of the PCR amplicon in ng/μl for bi-opsies versus oral swabs is shown in Figure 4. Signals could be detected up to 18 days after sampling if the oral swabs were shipped and stored at RT (+20°C). The best results were obtained when the samples were stored and shipped at -20°C. Visible signals and eval-uable results were also detected at +4°C up to 25 days after sampling.
3. Swabs versus Hair: In this study we developeda simple, economic and efficient strategy to extract DNA from hair follicles of mice which are suitable for genotyping. When comparing oral swab and hair fol-licle samples, we were able to demonstrate consistent genotyping results from hair follicles.

Conclusion

In accordance with the 3Rs principle, we have opti-mized and expanded non-invasive genotyping meth-ods for mice within our automated workflow. This approach involves the collection of oral swabs or hair follicles, and provides an alternative to invasive biop-sies, particularly in cases where biopsies are prohibited (e.g., animals with ear tags or tattoos) or a secondary bi-opsy is not feasible. Non-invasive sampling serves as an appropriate substitute for invasive biopsies, significant-ly reducing stress and pain. These techniques are appli-cable to mice aged over 16 days. In addition, these sam-ple types, like standard biopsies, can be processed on a large scale in an automated workflow and are therefore an equivalent alternative for routine genotyping.

References

  1. Mazlan, N.H.B., Salesansky, N.L., Burn, C.C., Wells, D.J., (2014). Mouse identification methods and potentialwelfare issues: a survey of current practice in the UK.Animal Technology and Welfare. 13(1), 1-10
  2. Russell, W.M.S., Burch, R.L., (1959). The principles of humane experimental technique. London: Methuen.