Large-scale gene trapping in C57BL/6N mouse embryonic stem cells

Hansen, Gwenn M.; Markesich, Diane C.; Burnett, Michael B.; Zhu, Qingfu; Dionne, Karen; Richter, Lizabeth J.; Finnell, Richard H.; Sands, Arthur; Zambrowicz, Brian; Abuin, Alejandro

doi:10.1101/gr.078352.108

Cited by 118 publications

(115 citation statements)

References 39 publications

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“…We have used the OmniBankII gene trap library containing mutated embryonic stem (ES) cell clones, which employed gene trapping with retroviral vectors in mouse C57BL/6N ES cells to generate the library. 23 The ES cell clone IST13504C3 was identified from the library to contain a retroviral insertion (of a gene trap vector Omnibank Vector 74) in the eIF2A gene intron 1 (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

The eIF2A knockout mouse

et al. 2016

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Eukaryotic initiation factor 2A (eIF2A) is a 65-kDa protein that was first identified in the early 1970s as a factor capable of stimulating initiator methionyl-tRNAi (Met-tRNA Met i ) binding to 40S ribosomal subunits in vitro. However, in contrast to the eIF2, which stimulates Met-tRNA Met i binding to 40S ribosomal subunits in a GTP-dependent manner, eIF2A didn't reveal any GTP-dependence, but instead was found to direct binding of the Met-tRNA Met i to 40S ribosomal subunits in a codon-dependent manner. eIF2A appears to be highly conserved across eukaryotic species, suggesting conservation of function in evolution. The yeast Saccharomyces cerevisae eIF2A null mutant revealed no apparent phenotype, however, it was found that in yeast eIF2A functions as a suppressor of internal ribosome entry site (IRES)-mediated translation. It was thus suggested that eIF2A my act by impinging on the expression of specific mRNAs. Subsequent studies in mammalian cell systems implicated eIF2A in non-canonical (non-AUG-dependent) translation initiation events involving near cognate UUG and CUG codons. Yet, the role of eIF2A in cellular functions remains largely enigmatic. As a first step toward characterization of the eIF2A function in mammalian systems in vivo, we have obtained homozygous eIF2A-total knockout (KO) mice, in which a gene trap cassette was inserted between eIF2A exons 1 and 2 disrupting expression of all exons downstream of the insertion. The KO mice strain is viable and to date displays no apparent phenotype. We believe that the eIF2A KO mice strain will serve as a valuable tool for researchers studying non-canonical initiation of translation in vivo.

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Section: Resultsmentioning

confidence: 99%

The eIF2A knockout mouse

et al. 2016

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“…S1D). Mice heterozygous for the mutation were generated on a mixed 129/Ola and C57BL/6 background using standard methods of host embryo microinjection, chimera production, and germ-line transmission (21).…”

Section: Resultsmentioning

confidence: 99%

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy

Pepe

Grubb

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Mitochondrial complex I (CI) deficiency is the most common mitochondrial enzyme defect in humans. Treatment of mitochondrial disorders is currently inadequate, emphasizing the need for experimental models. In humans, mutations in the NDUFS6 gene, encoding a CI subunit, cause severe CI deficiency and neonatal death. In this study, we generated a CI-deficient mouse model by knockdown of the Ndufs6 gene using a gene-trap embryonic stem cell line. Ndufs6 gt/gt mice have essentially complete knockout of the Ndufs6 subunit in heart, resulting in marked CI deficiency. Small amounts of wild-type Ndufs6 mRNA are present in other tissues, apparently due to tissue-specific mRNA splicing, resulting in milder CI defects. Ndufs6 gt/gt mice are born healthy, attain normal weight and maturity, and are fertile. However, after 4 mo in males and 8 mo in females, Ndufs6 gt/gt mice are at increased risk of cardiac failure and death. Before overt heart failure, Ndufs6 gt/gt hearts show decreased ATP synthesis, accumulation of hydroxyacylcarnitine, but not reactive oxygen species (ROS). Ndufs6 gt/gt mice develop biventricular enlargement by 1 mo, most pronounced in males, with scattered fibrosis and abnormal mitochondrial but normal myofibrillar ultrastructure. Ndufs6 gt/gt isolated working heart preparations show markedly reduced left ventricular systolic function, cardiac output, and functional work capacity. This reduced energetic and functional capacity is consistent with a known susceptibility of individuals with mitochondrial cardiomyopathy to metabolic crises precipitated by stresses. This model of CI deficiency will facilitate studies of pathogenesis, modifier genes, and testing of therapeutic approaches. mouse model | mitochondrial diseases M itochondria generate the majority of energy required for cellular function and survival. Defects in mitochondrial oxidative phosphorylation (OXPHOS) cause a wide range of diseases, collectively affecting ∼1/5,000 births (1). OXPHOS dysfunction was first identified in neuromuscular disorders but symptoms can potentially affect any organ, with any age of onset and any mode of inheritance (2). The heart, being highly energy dependent, is particularly vulnerable to OXPHOS defects, with cardiac involvement recognized in about a third of children and up to 80% of adults with OXPHOS disorders (3, 4). Complex I (CI) deficiency is the most common OXPHOS disorder and has a wide range of clinical presentations, including neurodegeneration, muscle weakness, cardiac failure, liver failure, and early death, often in childhood (5, 6). As identified by Cochrane review, "there is currently no clear evidence supporting the use of any intervention in OXPHOS disorders" (7). Thus, there is an urgent need for suitable animal models to study pathogenic mechanisms and novel therapeutic approaches. Such models can also shed light on the wide range of common, complex diseases to which mitochondrial dysfunction contributes (8).Despite the high prevalence of OXPHOS disorders, relatively few genetic models have been...

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“…The mutation in this clone was confirmed using inverse genomic polymerase chain reaction (PCR) as previously described. 35 Briefly, oligonucleotide primers complementary to the gene trap vector were used to amplify the vector insertion site from clone GST_4283_D8, which was then compared to mouse genome sequence assemblies to localize the insertion with respect to the exons and introns of the Fam20b gene.…”

Section: Tm1lexmentioning

confidence: 99%

“…Genotypes of offspring were determined by quantitative PCR as previously described. 35 Briefly, DNA isolated from tail biopsy samples was assayed by quantitative PCR for the neo gene, which is present in both the gene targeting and gene trapping vectors used to generate the mutations described in this study. …”

Section: Tm1lexmentioning

confidence: 99%

Amelogenesis Imperfecta and Other Biomineralization Defects in Fam20a and Fam20c Null Mice

et al. 2012

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The FAM20 family of secreted proteins consists of three members (FAM20A, FAM20B, and FAM20C) recently linked to developmental disorders suggesting roles for FAM20 proteins in modulating biomineralization processes. The authors report here findings in knockout mice having null mutations affecting each of the three FAM20 proteins. Both Fam20a and Fam20c null mice survived to adulthood and showed biomineralization defects. Fam20b -/-embryos showed severe stunting and increased mortality at E13.5, although early lethality precluded detailed investigations. Physiologic calcification or biomineralization of extracellular matrices is a normal process in the development and functioning of various tissues (eg, bones and teeth). The lesions that developed in teeth, bones, or blood vessels after functional deletion of either Fam20a or Fam20c support a significant role for their encoded proteins in modulating biomineralization processes. Severe amelogenesis imperfecta (AI) was present in both Fam20a and Fam20c null mice. In addition, Fam20a-/-mice developed disseminated calcifications of muscular arteries and intrapulmonary calcifications, similar to those of fetuin-A deficient mice, although they were normocalcemic and normophosphatemic, with normal dentin and bone. Fam20a gene expression was detected in ameloblasts, odontoblasts, and the parathyroid gland, with local and systemic effects suggesting both local and/or systemic effects for FAM20A. In contrast, Fam20c-/-mice lacked ectopic calcifications but were severely hypophosphatemic and developed notable lesions in both dentin and bone to accompany the AI. The bone and dentin lesions, plus the marked hypophosphatemia and elevated serum alkaline phosphatase and FGF23 levels, are indicative of autosomal recessive hypophosphatemic rickets/osteomalacia in Fam20c -/-mice.Keywords amelogenesis imperfecta, knockout, rickets, phosphatonin, ectopic mineralization, biomineralization, dentin, osteomalacia, osteosclerosisThe FAM20 family of secreted proteins in mammals consists of three members (FAM20A, FAM20B, and FAM20C) that were initially thought to have roles in regulating the differentiation and function of hematopoietic and other tissues. 65 Since then, there have been reports linking mutations in both FAM20A and FAM20C to developmental disorders involving bones and/or teeth, suggesting a role for FAM20 proteins in modulating biomineralization processes. In humans, a mutation in FAM20A was recently linked to the occurrence of amelogenesis imperfecta and gingival hyperplasia in a consanguineous family. 69 However, lesions were not reported in bones or teeth of transgenic mice with a partial deletion of Fam20a, and their stunted growth was attributed to malnutrition and an unspecified metabolic disorder. 4 To the best of our knowledge, there have been only two reports elucidating the function FAM20B, which was shown to be a kinase that phosphorylates glycosaminoglycans 50 and in vivo to be involved in cartilage matrix production and skeletal development in zebrafish. 2...

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Large-scale gene trapping in C57BL/6N mouse embryonic stem cells

Cited by 118 publications

References 39 publications

The eIF2A knockout mouse

The eIF2A knockout mouse

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy

Amelogenesis Imperfecta and Other Biomineralization Defects in Fam20a and Fam20c Null Mice

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