Preparation of PCR-Quality Mouse Genomic DNA with Hot Sodium Hydroxide and Tris (HotSHOT)

Truett, Gary E.; Heeger, Peter S.; Mynatt, Randall L.; Truett, Alycia A.; Walker, Jerilyn A.; Warman, Matthew L.

doi:10.2144/00291bm09

Cited by 1,410 publications

(1,079 citation statements)

References 3 publications

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“…DNA was prepared from toe biopsy tissues by heating the sample in 1 ml of 50 mM NaOH for 1 hour. The DNA solution was neutralized with 100 l of 1M Tris buffer and used directly in the polymerase chain reaction (PCR) (28). Primer sequences for rat microsatellite markers defined as DxMity, DxMghy, DxRaty, and DxGoty were obtained from Research Genetics (Huntsville, AL), and those for markers defined as DxWoxy were obtained from the Wellcome Institute for Human Genetics (Oxford, UK).…”

Section: Methodsmentioning

confidence: 99%

A comparative genetic analysis between collagen‐induced arthritis and pristane‐induced arthritis

Olofsson¹,

Lu²,

Holmberg³

et al. 2003

Arthritis & Rheumatism

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Objective. To compare the genetic regulation of collagen-induced arthritis (CIA) with that of pristaneinduced arthritis (PIA) in rats.Methods. A genome-wide linkage analysis of an (E3 ؋ DA)DA backcross of rats with CIA (n ‫؍‬ 364 male rats; the same strain combinations as previously used to determine the genetic control of PIA) was performed. The strongest loci in both CIA and PIA (i.e., Cia12/Pia4 and Cia13/Pia7) were isolated in congenic strains. Susceptibility in both congenic strains was tested in rats with CIA and in rats with PIA.Results. We found a striking, although not complete, similarity of the arthritis-controlling loci in CIA and in PIA, as well as the previously defined loci associated with cartilage destruction, antibody production, and the acute-phase response. All major PIA quantitative trait loci (QTLs) identified in early severe arthritis were also strong regulators of CIA. The 2 strongest QTLs, Cia12/Pia4 on chromosome 12 and Cia13/Pia7 on chromosome 4, were also analyzed in congenic strains with DA or E3 as the background genome. Consistent with the results of linkage analysis, the congenic strain experiments showed that the chromosome 4 locus was more penetrant in CIA than in PIA, while the chromosome 12 locus almost completely dominated the control of PIA severity.Conclusion. The underlying genetic control of CIA was found to have many, but not all, pathogenic mechanisms in common with PIA, despite the use of a cartilage-specific antigen (type II collagen) to induce CIA but not PIA.

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Section: Methodsmentioning

confidence: 99%

A comparative genetic analysis between collagen‐induced arthritis and pristane‐induced arthritis

Olofsson¹,

Lu²,

Holmberg³

et al. 2003

Arthritis & Rheumatism

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“…mPGES-1 knockout mice (n = 8), heterozygous (n = 8), and wildtype DBA/1lac J (n = 8) littermates arise from the mPGES-1 +/2 matings. Genomic DNA from mice tail fragment was prepared following the Hot-SHOT technique (32) and was used to perform the genotyping PCR. The PCR primer sequences designed to amplify a native mPGES-1 locus fragment were (sense) 59-TCCCAGGTGTTGGGATTTAGACG-39 and (antisense) 59-AGGTGGCTGTACTGTTTGTTGC-39, and those designed to amplify a neomycin-resistance gene fragment were (sense) 59-CT-CCAGTACTGAGCCAGCTGC-39 and (antisense) 59-TGCTACTTCCAT-TTGTCACGTCC-39.…”

Section: Animalsmentioning

confidence: 99%

Inhibition of Matrix Metalloproteinase-3 and -13 Synthesis Induced by IL-1β in Chondrocytes from Mice Lacking Microsomal Prostaglandin E Synthase-1

Gosset

Pigenet

Salvat

et al. 2010

The Journal of Immunology

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Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1β is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE2 remains controversial. The goal of this study was to determine the role of PGE2 on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE2 synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1β–induced PGE2 synthesis was dramatically reduced in mPGES-1−/− and mPGES-1+/− compared with mPGES-1+/+ chondrocytes. A total of 10 ng/ml IL-1β increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1+/+ chondrocytes in a time-dependent manner. IL-1β–induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1−/− and mPGES-1+/− chondrocytes compared with mPGES-1+/+ chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE2 in mPGES-1−/− chondrocytes. Finally, in mPGES-1−/− chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE2 regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE2 plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.

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“…In brief, cells or tissue pieces to be genotyped were lysed by being boiled in lysis solution (25 mM NaOH [pH 12] and 0.2 mM EDTA) for 20-30 min for the extraction of genomic DNA. 23 Alkaline pH was neutralized by the addition of an equal volume of neutralization buffer (40 mM Tris-HCl [pH 5]). 1 mL of the resultant genomic DNA solution was used as a template in 20 mL of PCR reaction with 0.5 units of DreamTaq polymerase (Bioline).…”

Section: Genotypingmentioning

confidence: 99%

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

Windpassinger

Piard

Bonnard

et al. 2017

The American Journal of Human Genetics

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In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

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Preparation of PCR-Quality Mouse Genomic DNA with Hot Sodium Hydroxide and Tris (HotSHOT)

Cited by 1,410 publications

References 3 publications

A comparative genetic analysis between collagen‐induced arthritis and pristane‐induced arthritis

A comparative genetic analysis between collagen‐induced arthritis and pristane‐induced arthritis

Inhibition of Matrix Metalloproteinase-3 and -13 Synthesis Induced by IL-1β in Chondrocytes from Mice Lacking Microsomal Prostaglandin E Synthase-1

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

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