The Use of the Cre/<i>loxP</i>System to Study Oxidative Stress in Tissue-Specific Manganese Superoxide Dismutase Knockout Models

Marecki, John C.; Parajuli, Nirmala; Crow, John P.; MacMillan-Crow, Lee Ann

doi:10.1089/ars.2013.5293

Cited by 13 publications

(10 citation statements)

References 117 publications

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“…Thus, in some cases, removing the Neo gene from transgenic pigs at the final step is necessary. Traditionally, the Neo gene is inserted into the genome flanked by two loxP sites and removed by Cre 19 26 . Because PFF cell lines cannot sustain two rounds of selection, successive cloning is required to remove the Neo selection marker.…”

Section: Discussionmentioning

confidence: 99%

Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs

Ruan

et al. 2015

Sci Rep

118

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Transgenic pigs play an important role in producing higher quality food in agriculture and improving human health when used as animal models for various human diseases in biomedicine. Production of transgenic pigs, however, is a lengthy and inefficient process that hinders research using pig models. Recent applications of the CRISPR/Cas9 system for generating site-specific gene knockout/knockin models, including a knockout pig model, have significantly accelerated the animal model field. However, a knockin pig model containing a site-specific transgene insertion that can be passed on to its offspring remains lacking. Here, we describe for the first time the generation of a site-specific knockin pig model using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. We also report a new genomic “safe harbor” locus, named pH11, which enables stable and robust transgene expression. Our results indicate that our CRISPR/Cas9 knockin system allows highly efficient gene insertion at the pH11 locus of up to 54% using drug selection and 6% without drug selection. We successfully inserted a gene fragment larger than 9 kb at the pH11 locus using the CRISPR/Cas9 system. Our data also confirm that the gene inserted into the pH11 locus is highly expressed in cells, embryos and animals.

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Section: Discussionmentioning

confidence: 99%

Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs

Ruan

et al. 2015

Sci Rep

118

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“…A series of knockout mouse models for the Sod2 gene have been investigated mostly in context with research of the mitochondrial free radical theory of aging (Barja, 2013 ). Knock-out of both Sod2 alleles leads to lethality within 18 days of birth in different strain backgrounds (reviewed in Marecki et al, 2014 ). Mice with only one Sod2 -knock-out allele that express half levels of the SOD2 protein had normal life-span and no overt phenotype, but displayed increased oxidative damage as well as increased apoptosis tendency (Strassburger et al, 2005 ; Wenzel et al, 2008 ).…”

Section: Discussionmentioning

confidence: 99%

Effects of a Mutation in the HSPE1 Gene Encoding the Mitochondrial Co-chaperonin HSP10 and Its Potential Association with a Neurological and Developmental Disorder

Bie

Fernández‐Guerra

Birkler

et al. 2016

Front. Mol. Biosci.

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We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spasms at 3 months of age. Clinical exome sequencing revealed heterozygosity for a HSPE1 NM_002157.2:c.217C>T de novo mutation causing replacement of leucine with phenylalanine at position 73 of the HSP10 protein. This variation has never been observed in public exome sequencing databases or the literature. To evaluate whether the mutation may be disease-associated we investigated its effects by in vitro and ex vivo studies. Our in vitro studies indicated that the purified mutant protein was functional, yet its thermal stability, spontaneous refolding propensity, and resistance to proteolytic treatment were profoundly impaired. Mass spectrometric analysis of patient fibroblasts revealed barely detectable levels of HSP10-p.Leu73Phe protein resulting in an almost 2-fold decrease of the ratio of HSP10 to HSP60 subunits. Amounts of the mitochondrial superoxide dismutase SOD2, a protein whose folding is known to strongly depend on the HSP60/HSP10 complex, were decreased to approximately 20% in patient fibroblasts in spite of unchanged SOD2 transcript levels. As a likely consequence, mitochondrial superoxide levels were increased about 2-fold. Although, we cannot exclude other causative or contributing factors, our experimental data support the notion that the HSP10-p.Leu73Phe mutation could be the cause or a strong contributing factor for the disorder in the described patient.

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confidence: 99%

“…To study the physiologic importance of SOD2, several models of SOD2 deletion have been developed in different organisms (10). In mice, a constitutive deletion of SOD2 i sf o u n dt ob el e t h a l( 1 1 ,1 2 ) .T h et i m ep o i n to fd e a t h depends on the mouse strain and varies from late embryogenesis to early postnatal life (up to 18 days).…”

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Neuronal redox imbalance results in altered energy homeostasis and early postnatal lethality

et al. 2015

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Redox imbalance is believed to contribute to the development and progression of several neurodegenerative disorders. Our aim was to develop an animal model that exhibits neuron-specific oxidative stress in the CNS to study the consequences and eventually find clues regarding the pathomechanisms of oxidative insults in neuronal homeostasis. We therefore generated a novel neuron-specific superoxide dismutase 2 (SOD2)-deficient mouse by deleting exon 3 of the SOD2 gene using CamKIIα promoter-driven Cre expression. These neuron-specific SOD2 knockout (SOD2(nko)) mice, although born at normal frequencies, died at the age of 4 weeks with critical growth retardation, severe energy failure, and several neurologic phenotypes. In addition, SOD2(nko) mice exhibited severe neuronal alterations such as reactive astrogliosis, neuronal cell cycle inhibition, and induction of apoptosis. JNK activation and stabilization of p53, as a result of reactive oxygen species accumulation, are most likely the inducers of neuronal apoptosis in SOD2(nko) mice. It is remarkable that hypothalamic regulation of glucose metabolism was affected, which in turn induced necrotic brain lesions in SOD2(nko) mice. Taken together, our findings suggest that exclusive deficiency of SOD2 in neurons results in an impaired central regulation of energy homeostasis that leads to persistent hypoglycemia, hypoglycemia-related neuropathology, and an early lethality of the mutant mice.

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The Use of the Cre/loxPSystem to Study Oxidative Stress in Tissue-Specific Manganese Superoxide Dismutase Knockout Models

Cited by 13 publications

References 117 publications

Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs

Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs

Effects of a Mutation in the HSPE1 Gene Encoding the Mitochondrial Co-chaperonin HSP10 and Its Potential Association with a Neurological and Developmental Disorder

Neuronal redox imbalance results in altered energy homeostasis and early postnatal lethality

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