A highly efficient strategy to determine genotypes of genetically-engineered mice using genomic DNA purified from hair roots

Abstract

Genotyping of genetically-engineered mice is necessary for the effective design of breeding strategies and identification of mutant mice. This process relies on the identification of DNA markers introduced into genomic sequences of mice, a task usually performed using the polymerase chain reaction (PCR). Clearly, the limiting step in genotyping is isolating pure genomic DNA. Isolation of mouse DNA for genotyping typically involves painful procedures such as tail snip, digit removal, or ear punch. Although the harvesting of hair has previously been proposed as a source of genomic DNA, there has been a perceived complication and reluctance to use this non-painful technique because of low DNA yields and fear of contamination. In this study we developed a simple, economic, and efficient strategy using Chelex® resins to purify genomic DNA from hair roots of mice which are suitable for genotyping. Upon comparison with standard DNA purification methods using a commercially available kit, we demonstrate that Chelex® efficiently and consistently purifies high-quality DNA from hair roots, minimizing pain, shortening time and reducing costs associated with the determination of accurate genotypes. Therefore, the use of hair roots combined with Chelex® is a reliable and more humane alternative for DNA genotyping.

A Noninvasive Ocular (Tear) Sampling Method for Genetic Ascertainment of Transgenic Mice and Research Ethics Innovation

“…The sensitivity, repeatability, and reproducibility of our method compare favorably to that of the amplification using DNA from tail biopsies and other noninvasive methods (Broome et al, 1999;Malumbres et al, 1997;Meldgaard et al, 2004;Mitrečić et al, 2008;Otaño-Rivera et al, 2017;Ren et al, 2001;Schmitteckert et al, 1999), without the need of extra rounds of PCR and thus, our approach also compares favorably to the time one would spend performing such extra PCR analyses. Ocular sampling may be also carried out for the study of mutagenesis analyses, genetic monitoring of multiple loci, induced modification of transgene insertions, and others, and hence, versatility and robustness become evident.…”

“…As an alternative, noninvasive methods have far failed to meet analytical standards (sensitivity, precision, accuracy), as they suffer from cross-contamination (indicatively, hair harvesting), require a two-round PCR analysis or cannot be performed in mice of young age (Broome et al, 1999;Irwin et al, 1996;Lahm et al, 1998;Meldgaard et al, 2004;Mitrečić et al, 2008;Ohhara et al, 1994;Otaño-Rivera et al, 2017;Schmitteckert et al, 1999;Zimmermann et al, 2000).…”

Genotyping of mice lines for the TPH2 gene in routine practice: selection of a DNA extraction technique for PCR

“…Отбор тканей производится, как правило, биопсией хвоста. Так как хрящевая, костная ткань и эпителий являются сложными для выделения ДНК объектами [10], предлагаются различные варианты модификаций методик [7,9]. В качестве альтернативы предложено предварительное замораживание биопсий в жидком азоте [9].…”