Mouth cell collection device for newborn mice

Zhang, Y.-H.; Huang, Bing-Ling; Eastman, K.; McCabe, Linda L.; MacLennan, Nicole K.; McCabe, Erb

doi:10.1016/j.ymgme.2006.03.014

Cited by 9 publications

(9 citation statements)

References 8 publications

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“…Actually, after necessary adjustments all tested samples worked even with primers producing amplicons up to 800 bp, which is higher than in other published protocols (Mulot et al 2005, Zhang et al 2006. The advantage of the presented protocol is that only 1 -3 µL of DNA solution is required for PCR, compared with e.g.…”

Section: Discussionmentioning

confidence: 85%

“…As an alternative, the procedure based on lysis with NaOH and subsequent boiling on 98°C was also tested (Zhang et al 2006). Instead of using the cell collecting device as required by the original protocol, the samples were collected using cotton swabs.…”

Section: Resultsmentioning

confidence: 99%

“…The presented protocol was based on collection of cells in physiological solution followed by a simple alkaline lysis of cells at 3 temperatures. Since our procedure sampled a sufficient amount of cells, no special scoop device or additional tissue damage was necessary (Zhang et al 2006). …”

Section: Discussionmentioning

confidence: 99%

“…Therefore, further modifications were tested: in order to get better rate of amplification, the number of PCR cycles was increased, and in order to get higher concentration of DNA, it was precipitated with isopropanol or Na-acetate. However, results were not improved.As an alternative, the procedure based on lysis with NaOH and subsequent boiling on 98°C was also tested (Zhang et al 2006). Instead of using the cell collecting device as required by the original protocol, the samples were collected using cotton swabs.…”

mentioning

confidence: 99%

“…After swabbing of buccal mucosa, DNA could be isolated using commercially available kits (Mulot et al 2005) or extensive procedures which involve collecting devices (Zhang et al 2006), proteinase K and/or phenol/chloroform extraction (Cao et al 2003). In addition, simple procedures based on alcaline lysis were reported, both in humans (Rudbeck et al 1998) and mice (Meldgaard et al 2004).…”

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confidence: 99%

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Mice Genotyping Using Buccal Swab Samples: An Improved Method

et al. 2008

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Routine methods used to genotype mice involve isolation of DNA from partially amputated neonate's tail, toe or ear. Inevitable drawback of such techniques is animal`s pain response and increased spending of time and funds because of obligatory DNA purification. In order to implement a noninvasive and simple protocol for mouse DNA isolation, we have improved the method based on samples collected by swabbing of the inner cheek. Combining alkaline and temperature lysis it was possible to isolate DNA solution ready for PCR in less than an hour. Testing the method on three different mouse lines showed that it is highly efficient, the volume of the PCR samples could be reduced to 25 µl and fragments up to 800 bp were successfully amplified. This protocol reduces animal discomfort, shortens the time for DNA isolation, and enables amplification of larger DNA fragments with optimal success rate, thus considerably facilitating large-scale genotyping of different mouse lines.Key words: DNA isolation, buccal swabs, genotyping, PCR, mouse Transgenic mice are an indispensable experimental model in biomedical research and their successful breeding requires a simple and reliable genotyping procedure, which is typically performed using PCR (polymerase chain reaction). Obtaining high quality genomic DNA is a critical for successful PCR amplification. Although large quantities of DNA can be isolated from a variety of samples, the routinely used methods for mouse genotyping involve partial amputation of a neonate's tail, ear or toe (Hanley et al. 1991, Ren et al. 2001, Malumbres et al. 1997. Although efficient, the procedures are invasive, mutilating and the animals regularly exhibit pain response. Moreover, additional step of DNA purification after tissue lysis represents a significant burden, especially in laboratories which need to genotype a large number of different samples. Therefore, every simplification of DNA isolation procedure is welcomed, as it follows the trend in refinement of animal handling procedures, and also saves both time and money (Council of Europe, 2006.).Buccal swabs as a source of cells for DNA isolation was used to avoid the invasiveness of previously applied procedures. After swabbing of buccal mucosa, DNA could be isolated using commercially Here we report an improved simple method (Meldgaard et al. 2004) for mouse genotyping using buccal swab samples. By modifying the alkaline lysis and boiling method it was possible to reliably isolate the DNA from mouse buccal mucosa using simple cotton sticks and the basic, easily affordable chemicals. The PCR was performed in minimal volume (25 µL) and modified in order to achieve amplification of PCR products up to 800 bp. MATERIALS AND METHODS Animals and housing Sampling of buccal epithelial cells and isolation of DNAThe sampling of the buccal mucosa required some skilfulness and routine, as the sample should contain cells of buccal mucosa and not those of tongue. The mice were firmly held in the hand with their neck skin fixed between the thumb and the...

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Section: Discussionmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Mice Genotyping Using Buccal Swab Samples: An Improved Method

et al. 2008

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The sacroiliac joint: An original and highly sensitive tool to highlight altered bone phenotype in murine models of skeletal disorders

Hilliquin,

Zhukouskaya,

Fogel

et al. 2024

Bone

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The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes

2008

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In mammals, mitochondrial DNA (mtDNA) sequence variants are observed to segregate rapidly between generations despite the high mtDNA copy number in the oocyte. This has led to the concept of a genetic bottleneck for the transmission of mtDNA, but the mechanism remains contentious. Several studies have suggested that the bottleneck occurs during embryonic development, as a result of a marked reduction in germline mtDNA copy number. Mitotic segregation of mtDNAs during preimplantation, or during the expansion of primordial germ cells (PGCs) before they colonize the gonad, is thought to account for the increase in genotypic variance observed among mature oocytes from heteroplasmic mothers. This view has, however, been challenged by studies suggesting that the bottleneck occurs without a reduction in germline mtDNA content. To resolve this controversy, we measured mtDNA heteroplasmy and copy number in single germ cells isolated from heteroplasmic mice. By directly tracking the evolution of mtDNA genotypic variance during oogenesis, we show that the genetic bottleneck occurs during postnatal folliculogenesis and not during embryonic oogenesis.

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Mouth cell collection device for newborn mice

Cited by 9 publications

References 8 publications

Mice Genotyping Using Buccal Swab Samples: An Improved Method

Mice Genotyping Using Buccal Swab Samples: An Improved Method

The sacroiliac joint: An original and highly sensitive tool to highlight altered bone phenotype in murine models of skeletal disorders

The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes

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