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McEwan, Carolanne; Melton, David W.

doi:10.1023/a:1024273324890

Cited by 6 publications

(4 citation statements)

References 10 publications

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“…In our case, the ES cell clone used to generate our knockout mice was E14TG2a HM-1, which harbours an hprt allele containing a 55 kb-deletion in its 5′-flanking region [29]. Using a PCR protocol specifically designed to genotype this deletion [30], we found it was correlated with the reduction of HPRT expression (data not shown). The hprt and SAP102/ dlgh3 loci are separated by ∼48 Mb and since the hprt locus of 129S5 mice does not contain the deletion, these data suggested that the observed changes in hprt gene expression in SAP102 mutant mice was the result of a linked mutation originated in the ES cells.…”

Section: Resultsmentioning

confidence: 89%

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Clustered Gene Expression Changes Flank Targeted Gene Loci in Knockout Mice

Valor

Grant

2007

PLoS ONE

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BackgroundGene expression profiling using microarrays is a powerful technology widely used to study regulatory networks. Profiling of mRNA levels in mutant organisms has the potential to identify genes regulated by the mutated protein.Methodology/Principle FindingsUsing tissues from multiple lines of knockout mice we have examined genome-wide changes in gene expression. We report that a significant proportion of changed genes were found near the targeted gene.Conclusions/SignificanceThe apparent clustering of these genes was explained by the presence of flanking DNA from the parental ES cell. We provide recommendations for the analysis and reporting of microarray data from knockout mice

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Section: Resultsmentioning

confidence: 89%

“…For hprt alleles genotyping, the protocol described in [30] was used with few modifications. Reactions to detect wild-type and mutant alleles were set up in parallel for the same genomic DNA: 1 cycle of 94°C 5 min, 35 cycles of 94°C 30 s, 62°C 30 s for the wild-type allele or 67°C for the null allele, 72°C 60 s and 1 cycle of 72°C 5 min.…”

Section: Methodsmentioning

confidence: 99%

Clustered Gene Expression Changes Flank Targeted Gene Loci in Knockout Mice

Valor

Grant

2007

PLoS ONE

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“…In order to validate that the defects in germ cell differentiation observed in the chimeras are in tissue derived from the injected Taz Neo stem cells, we stained the tissue with an antibody to Hprt. The HM1 embryonic stem cell line used in this study has a deletion of the Hprt locus on the X-chromosome [ 40 ]. In wild-type mice, spermatogonia and early spermatocytes express Hprt ( Fig 2I ), but this is subsequently switched off in the differentiated cell types as a result of X-chromosome inactivation during meiosis.…”

Section: Resultsmentioning

confidence: 99%

Mouse Tafazzin Is Required for Male Germ Cell Meiosis and Spermatogenesis

et al. 2015

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Barth syndrome is an X-linked mitochondrial disease, symptoms of which include neutropenia and cardiac myopathy. These symptoms are the most significant clinical consequences of a disease, which is increasingly recognised to have a variable presentation. Mutation in the Taz gene in Xq28 is thought to be responsible for the condition, by altering mitochondrial lipid content and mitochondrial function. Male chimeras carrying a targeted mutation of Taz on their X-chromosome were infertile. Testes from the Taz knockout chimeras were smaller than their control counterparts and this was associated with a disruption of the progression of spermatocytes through meiosis to spermiogenesis. Taz knockout ES cells also showed a defect when differentiated to germ cells in vitro. Mutant spermatocytes failed to progress past the pachytene stage of meiosis and had higher levels of DNA double strand damage and increased levels of endogenous retrotransposon activity. Altogether these data revealed a novel role for Taz in helping to maintain genome integrity in meiosis and facilitating germ cell differentiation. We have unravelled a novel function for the Taz protein, which should contribute to an understanding of how a disruption of the Taz gene results in the complex symptoms underlying Barth Syndrome.

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“…To unequivocally demonstrate that the cells had replaced the “defective” endogenous chromosome with a normal one, we amplified the Hprt locus to discriminate the mutated from the wild type ( wt ) sequence [ 13 ]. In the presence of the E14TG2a deletion, which encompasses exons 1 and 2, a 628 bp-fragment was amplified, while the wt locus gave a 746 bp-amplicon.…”

Section: Resultsmentioning

confidence: 99%

Chromosome transplantation as a novel approach for correcting complex genomic disorders

et al. 2015

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Genomic disorders resulting from large rearrangements of the genome remain an important unsolved issue in gene therapy. Chromosome transplantation, defined as the perfect replacement of an endogenous chromosome with a homologous one, has the potential of curing this kind of disorders. Here we report the first successful case of chromosome transplantation by replacement of an endogenous X chromosome carrying a mutation in the Hprt gene with a normal one in mouse embryonic stem cells (ESCs), correcting the genetic defect. The defect was also corrected by replacing the Y chromosome with an X chromosome. Chromosome transplanted clones maintained in vitro and in vivo features of stemness and contributed to chimera formation. Genome integrity was confirmed by cytogenetic and molecular genome analysis. The approach here proposed, with some modifications, might be used to cure various disorders due to other X chromosome aberrations in induced pluripotent stem (iPS) cells derived from affected patients.

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Cited by 6 publications

References 10 publications

Clustered Gene Expression Changes Flank Targeted Gene Loci in Knockout Mice

Clustered Gene Expression Changes Flank Targeted Gene Loci in Knockout Mice

Mouse Tafazzin Is Required for Male Germ Cell Meiosis and Spermatogenesis

Chromosome transplantation as a novel approach for correcting complex genomic disorders

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