Quantitative genotyping of mouse brain-specific PEX13 gene disruption by real-time PCR

Müller, C. Catharina; Nourse, Jamie P.; Nguyen, Tam H.; Crane, Denis I.

doi:10.1016/j.jneumeth.2009.04.025

Cited by 4 publications

(7 citation statements)

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“…Semi-quantitative analysis of PEX13 exon 2 excision was carried out initially by Southern blot analysis on brain genomic DNA (Fig. 1B) and subsequently using a quantitative real-time PCR method (Müller et al, 2009). These analyses confirmed PEX13 disruption in brain of animals inheriting a PEX13 flox allele and the Cre transgene, but not, as expected, in PEX13 flox -positive animals lacking the Cre transgene.…”

Section: Resultsmentioning

confidence: 99%

“…EGL, external granule layer; IGL, internal granule layer. Note that A and B are reproduced in full with permission from Müller et al (Müller et al, 2009). …”

Section: Resultsmentioning

confidence: 99%

“…3A was determined using quantitative real-time PCR (Müller et al, 2009). A PEX13 excision range of 70.8–89.3% (average 81.1±0.7%; n =40) was established for the PEX13 brain mutant population (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For PEX13 brain mutants, genotyping of tail genomic DNA to determine the presence of the Cre transgene, the disrupted allele (inherited PEX13 Δ allele and Nes-Cre -generated PEX13 flox Δ allele) and the PEX13 flox allele, and Southern blot analysis and quantitative real-time PCR for determination and quantification of PEX13 exon 2 excision in brain, were performed as recently described (Müller et al, 2009). …”

Section: Methodsmentioning

confidence: 99%

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PEX13 deficiency in mouse brain as a model of Zellweger syndrome: abnormal cerebellum formation, reactive gliosis and oxidative stress

Müller

Nguyen

Ahlemeyer

et al. 2011

Disease Models &Amp; Mechanisms

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SUMMARYDelayed cerebellar development is a hallmark of Zellweger syndrome (ZS), a severe neonatal neurodegenerative disorder. ZS is caused by mutations in PEX genes, such as PEX13, which encodes a protein required for import of proteins into the peroxisome. The molecular basis of ZS pathogenesis is not known. We have created a conditional mouse mutant with brain-restricted deficiency of PEX13 that exhibits cerebellar morphological defects. PEX13 brain mutants survive into the postnatal period, with the majority dying by 35 days, and with survival inversely related to litter size and weaning body weight. The impact on peroxisomal metabolism in the mutant brain is mixed: plasmalogen content is reduced, but very-long-chain fatty acids are normal. PEX13 brain mutants exhibit defects in reflex and motor development that correlate with impaired cerebellar fissure and cortical layer formation, granule cell migration and Purkinje cell layer development. Astrogliosis and microgliosis are prominent features of the mutant cerebellum. At the molecular level, cultured cerebellar neurons from E19 PEX13-null mice exhibit elevated levels of reactive oxygen species and mitochondrial superoxide dismutase-2 (MnSOD), and show enhanced apoptosis together with mitochondrial dysfunction. PEX13 brain mutants show increased levels of MnSOD in cerebellum. Our findings suggest that PEX13 deficiency leads to mitochondria-mediated oxidative stress, neuronal cell death and impairment of cerebellar development. Thus, PEX13-deficient mice provide a valuable animal model for investigating the molecular basis and treatment of ZS cerebellar pathology.

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Section: Resultsmentioning

confidence: 99%

“…EGL, external granule layer; IGL, internal granule layer. Note that A and B are reproduced in full with permission from Müller et al (Müller et al, 2009). …”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

PEX13 deficiency in mouse brain as a model of Zellweger syndrome: abnormal cerebellum formation, reactive gliosis and oxidative stress

Müller

Nguyen

Ahlemeyer

et al. 2011

Disease Models &Amp; Mechanisms

Self Cite

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“…This is particularly useful when allele ratios have to be measured in small samples at several time points, such as in conditional deletion experiments involving hematopoetic stem cells [11]. Surprisingly, only one study formally evaluated this technical approach, however without testing the ability of the method to detect small changes in allele ratios [12]. …”

Section: Discussionmentioning

confidence: 99%

Analysis of conditional gene deletion using probe based Real-Time PCR

et al. 2010

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BackgroundConditional gene deletion using Cre-lox recombination is frequently used in mouse genetics; however recombination is frequently incomplete, resulting in a mixture of cells containing the functional (2lox) allele and the truncated (1lox) allele. Conventional analysis of 1lox/2lox allele ratios using Southern Blotting is time consuming, requires relatively large amounts of DNA and has a low sensitivity. We therefore evaluated the utility of Real-Time PCR to measure 1lox/2lox allele ratios.ResultsWe show that SYBR Green based Real-Time PCR analysis of 1lox/2lox allele ratios can generate erroneous peaks in the melting curve that are possibly caused by alternate hybridization products promoted by the palindromic loxP sequence motif. Since abnormal melting curves frequently contribute to dismissal of SYBR Green based data, we developed a convenient method with improved specificity that avoids such erroneous signals. Our data show that probe based Real-Time PCR, using a universal probe directed against the loxP site, can accurately detect small differences in 1lox/2lox allele ratios. We also validated this method in Fabpl4× at -132-Cre transgenic mice, measuring 1lox/2lox allele ratios that are in agreement with published data. Our Real-Time PCR protocol requires the use of one probe only for all reactions. Also the universal probe established in our assay is generally applicable to any experiment analyzing Cre-lox recombination efficiency, such that only primer sequences have to be adapted.ConclusionsOur data show that 1lox/2lox allele ratios are detected with high accuracy and high sensitivity with Real-Time PCR analysis using a probe directed against the loxP site. Due to the generally applicable probe the assay is conveniently adapted to all models of Cre-lox mediated gene deletion.

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FELASA guidelines for the refinement of methods for genotyping genetically-modified rodents

et al. 2013

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The use of genetically-modified (GM) animals as research models continues to grow. The completion of the mouse genome sequence, together with the high-throughput international effort to introduce mutations across the mouse genome in the embryonic stem (ES) cells (www.knockoutmouse.org) facilitates an efficient way to obtain mutated mouse strains as research models. The increasing number of available mutated mouse strains and their combinations, together with the increasing complexity in the targeting approaches used, reinforces the need for guidelines that will provide information about the mouse strains and the robust and reliable methods used for their genotyping. This information, however, should be obtained with a method causing minimal discomfort to the experimental animals. We have, therefore, compiled the present document which summarizes the currently available methods for obtaining genotype information. It provides updated guidelines concerning animal identification, DNA sampling and genotyping, and the information to be kept and distributed for any mutated rodent strain. As a starting point, we have decided to collect information on the genotyping practices applied in various European countries, in order to identify common problems and pitfalls faced and to better direct our recommendations. For this purpose, we have conducted a survey focused on genotyping-related procedures used. We have collected 158 responses from 25 countries (see Supplementary information http://lan.sagepub.com/ content/suppl/2013/05/28/0023677212473918.DC1/ LAN473918_Supplementaryd_data.pdf), which will be acknowledged in this report when appropriate. Only three replies were received with regard to rats, which seems to show that this species is still infrequently used for genetically-modified (GM) models. From the responses to this survey, we could conclude that the procedures are not harmonized among countries or even among different institutes within one country, and that the common practice is not always in accordance with the latest scientific findings.

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Quantitative genotyping of mouse brain-specific PEX13 gene disruption by real-time PCR

Cited by 4 publications

References 28 publications

PEX13 deficiency in mouse brain as a model of Zellweger syndrome: abnormal cerebellum formation, reactive gliosis and oxidative stress

PEX13 deficiency in mouse brain as a model of Zellweger syndrome: abnormal cerebellum formation, reactive gliosis and oxidative stress

Analysis of conditional gene deletion using probe based Real-Time PCR

FELASA guidelines for the refinement of methods for genotyping genetically-modified rodents

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