DNA from tissues of young mice is optimal for genotyping

Picazo, María G.; García‐Olmo, Dolores C.

doi:10.1016/j.ejbt.2014.12.002

Cited by 12 publications

(12 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Another noninvasive method is the use of fecal pellets but this requires special animal husbandry with one animal per cage and this is not in accordance with the principles of the replacement, reduction and refinement (3Rs). Blood sample collection for genotyping is highly invasive and is not recommended. Finally, ear punching allows genotyping of the animals but also allows their individual marking with one manipulation only (in accordance with the 3Rs).…”

Section: Discussionmentioning

confidence: 99%

One‐hour universal protocol for mouse genotyping

2020

View full text Add to dashboard Cite

Background: Transgenic animals are widely used for research and for most of them, genotyping is unavoidable. Published protocols may be powerful but may also present disadvantages such as their cost or the requirement of additional steps/equipment. Moreover, if more than one strain must be genotyped, several protocols may need to be developed.Methods: we adapted the existing amplification-resistant mutation protocol to develop the 1-h universal genotyping protocol (1-HUG), which allows the robust genotyping of genetically modified mice in 1 h from sample isolation to polymerase chain reaction gel running.Results: This protocol allows the genotyping of different mouse models including mdx mouse, and FLExDUX4 and HSA-MerCreMer alone or in combination. It can be applied to different types of genomic modifications and to sexing. Conclusions:The 1-HUG protocol can be used routinely in any laboratory using mouse models for neuromuscular diseases. K E Y W O R D S DMD, FSHD, genotyping, mouse model, neuromuscular diseases 1 | INTRODUCTION Genetically modified mice are routinely used in many laboratories across the world and the most popular modifications are deletion, insertion in a known or unknown location and point mutation. Many methods have been proposed to genotype these mice, and those developed for the mdx mouse perfectly illustrate the difficulty in finding the best one. This mouse is the animal model for Duchenne muscular dystrophy (DMD) and carries a nonsense point mutation (C-to-A mutation) in exon 23, leading to a truncated and nonfunctional protein. 1These methods include an allele-specific oligonucleotide (ASO)based hybridization method, 2 a SNaPshot assay, 3 a high resolution melting polymerase chain reaction (PCR), 4 a restriction fragment length polymorphism (RFLP), 5 a dideoxy fingerprinting (ddF), 6 and an amplification resistant mutation system (ARMS) method. 7,8 These methods may be powerful but many of them also present disadvantages such as their cost, the requirement of additional steps, or the purchase of specific expensive equipment. For example, in the ARMS protocol, the PCR reaction is performed with three primers in the same tube: one forward primer and two reverse primers that differ only in the last nucleotides. To be able to distinguish the WT and mdx allele, the WT primer is 17 bp longer than the mdx one. 7 A 3% agarose gel and a long running time are required to allow the correct discrimination between mdx and WT samples. Moreover, numerous laboratories that use the mdx mouse also need to genotype other genetically modified mouse models and having one general method for different kinds of modification, including sexing, is valuable.

show abstract

Section: Discussionmentioning

confidence: 99%

One‐hour universal protocol for mouse genotyping

2020

View full text Add to dashboard Cite

show abstract

“…Table shows the age(s) at which each tissue sampling method can be used. Sampling should, where possible, be done on young animals for the reasons listed below: DNA from tissues of young mice is more optimal for genotyping than that from older animals (Picazo & García‐Olmo, ). In a newborn mouse (particularly before 12 days of age), discomfort due to the sampling is reduced because the sampled tissue is not fully ossified and because the nociceptive stimulus may not result in the conscious perception of pain due to the lack of a competent pain pathway at this age (Hankenson, Garzel, Fischer, Nolan, & Hankenson, ; Silverman & Hendricks, ; Wever, Geessink, Brouwer, Tillema, & Ritskes‐Hoitinga, ). If genotyping is completed before weaning, extra animals or those of nondesired genotype can be sacrificed before separation of young animals into different cages. …”

Section: Commentarymentioning

confidence: 99%

“…Table 2 shows the age(s) at which each tissue sampling method can be used. Sampling should, where possible, be done on young animals for the reasons listed below: r DNA from tissues of young mice is more optimal for genotyping than that from older animals (Picazo & García-Olmo, 2015).…”

Section: Decontaminationmentioning

confidence: 99%

“…Ear clipping (also ear notching or ear punching) should not be carried out on mice <2 weeks of age because the ear is not yet fully developed and the removal of even a small piece of tissue can represent a significant proportion of the pinna. Ear clipping is the preferred sampling method for animals >4 weeks of age because it produces little discomfort in older mice (Picazo & García‐Olmo, ) and therefore does not require anesthesia. During sampling, care should be taken not to accidentally drop or lose the very small ear tissue sample.…”

Section: Commentarymentioning

confidence: 99%

“…These data are consistent with the finding that over 15% of lines deposited to public repositories, such as the Mutant Mouse Resource & Research Centers (MMRRCs) and the Jackson Laboratory (JAX), do not carry the mutation specified by the depositor (Lloyd, Franklin, Lutz, & Magnuson, ). Inconclusive genotyping is one factor that can impact preclinical studies and basic research reproducibility, contributing to the “reproducibility crisis” (Cinelli, Rettich, Seifert, Bürki, & Arras, ; Picazo & García‐Olmo, ). Moreover, genotyping errors can lead to genetic contamination of stocks and even to the extinction of a genetically unique mouse line (Lloyd et al., ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High‐Throughput Genotyping Protocol for Any Type of Mutation

et al. 2019

View full text Add to dashboard Cite

Genotyping consists of searching for a DNA sequence variation localized at a well‐defined locus in the genome. It is an essential step in animal research because it allows the identification of animals that will be bred to generate and maintain a colony, euthanized to control the available space in the animal facility, or used in experiment protocols. Here we describe polymerase chain reaction (PCR) genotyping protocols for fast, sensitive, easy, and cost‐effective characterization of mouse genotype. We discuss optimization of parameters to improve the reliability of each assay and propose recommendations for enhancing reproducibility and reducing the occurrence of inconclusive genotyping. All steps required for efficient genotyping are presented: tissue collection; sample verification and direct DNA lysis; establishment of a robust genotyping strategy with reliable, rapid, and cost‐effective assays; and finally, transition to high‐throughput automatized PCR, including mix miniaturization and automation. © 2019 The Authors. Basic Protocol 1: Tissue sampling methods and procedure Basic Protocol 2: Sample verification and DNA lysis Basic Protocol 3: Design of a genotyping strategy Basic Protocol 4: Moving to high‐throughput genotyping

show abstract

Iron from nanostructured ferric phosphate: absorption and biodistribution in mice and bioavailability in iron deficient anemic women

Baumgartner

Winkler

Zandberg

et al. 2022

Sci Rep

View full text Add to dashboard Cite

Food fortification with iron nanoparticles (NPs) could help prevent iron deficiency anemia, but the absorption pathway and biodistribution of iron-NPs and their bioavailability in humans is unclear. Dietary non-heme iron is physiologically absorbed via the divalent metal transporter-1 (DMT1) pathway. Using radio- iron isotope labelling in mice with a partial knockdown of intestine-specific DMT1, we assessed oral absorption and tissue biodistribution of nanostructured ferric phosphate (FePO4-NP; specific surface area [SSA] 98 m2g-1) compared to to ferrous sulfate (FeSO4), the reference compound. We show that absorption of iron from FePO4-NP appears to be largely DMT1 dependent and that its biodistribution after absorption is similar to that from FeSO4, without abnormal deposition of iron in the reticuloendothelial system. Furthermore, we demonstrate high bioavailability from iron NPs in iron deficient anemic women in a randomized, cross-over study using stable-isotope labelling: absorption and subsequent erythrocyte iron utilization from two 57Fe-labeled FePO4-NP with SSAs of 98 m2g−1 and 188 m2g−1 was 2.8-fold and 5.4-fold higher than from bulk FePO4 with an SSA of 25 m2g−1 (P < 0.001) when added to a rice and vegetable meal consumed by iron deficient anemic women. The FePO4-NP 188 m2g-1 achieved 72% relative bioavailability compared to FeSO4. These data suggest FePO4-NPs may be useful for nutritional applications.

show abstract

DNA from tissues of young mice is optimal for genotyping

Cited by 12 publications

References 17 publications

One‐hour universal protocol for mouse genotyping

One‐hour universal protocol for mouse genotyping

Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High‐Throughput Genotyping Protocol for Any Type of Mutation

Iron from nanostructured ferric phosphate: absorption and biodistribution in mice and bioavailability in iron deficient anemic women

Contact Info

Product

Resources

About