Overview: Engineering Transgenic Constructs and Mice

Haruyama, Naoto; Cho, Andrew; Kulkarni, Ashok B.

doi:10.1002/0471143030.cb1910s42

Cited by 69 publications

(53 citation statements)

References 19 publications

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“…Targeted insertion of a mutation by homologous recombination, instead, may generate mouse models that reflect the clinical phenotypes more precisely. 23 Thus, by homologous recombination, we generated a knock-in mouse model of the Sgcd gene p.S151A missense mutation to investigate whether this variant can cause cardiomyopathy. Heterozygous S151A knock-in mice developed a rather mild phenotype of cardiomyopathy.…”

Section: Discussionmentioning

confidence: 99%

S151A δ-sarcoglycan mutation causes a mild phenotype of cardiomyopathy in mice

et al. 2013

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So far, the role of mutations in the d-sarcogylcan (Sgcd) gene in causing autosomal dominant dilated cardiomyopathy (DCM) remains inconclusive. A p.S151A missense mutation in exon 6 of the Sgcd gene was reported to cause severe isolated autosomal dominant DCM without affecting skeletal muscle. This is controversial to our previous findings in a large consanguineous family where this p.S151A mutation showed no relevance for cardiac disease. In this study, the potential of the p.S151A mutation to cause DCM was investigated by using two different approaches: (1) engineering and characterization of heterozygous knock-in (S151A-) mice carrying the p.S151A mutation and (2) evaluation of the potential of adeno-associated virus (AAV) 9-based cardiac-specific transfer of p.S151A-mutated Sgcd cDNA to rescue the cardiac phenotype in Sgcd-deficient (Sgcd-null) mice as it has been demonstrated for intact, wild-type Sgcd cDNA. Heterozygous S151A knock-in mice developed a rather mild phenotype of cardiomyopathy. Increased heart to body weight suggests cardiac enlargement in 1-year-old S151A knock-in mice. However, at this age cardiac function, assessed by echocardiography, is maintained and histopathology completely absent. Myocardial expression of p.S151A cDNA, similar to intact Sgcd cDNA, restores cardiac function, although not being able to prevent myocardial histopathology in Sgcd-null mice completely. Our results suggest that the p.S151A mutation causes a mild, subclinical phenotype of cardiomyopathy, which is prone to be overseen in patients carrying such sequence variants. Furthermore, this study shows the suitability of an AAV-mediated cardiac gene transfer approach to analyze whether a sequence variant is a disease-causing mutation.

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Section: Discussionmentioning

confidence: 99%

S151A δ-sarcoglycan mutation causes a mild phenotype of cardiomyopathy in mice

et al. 2013

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“…Lack of transcription of the transgene or misexpression of the transgenic RNA can result from the generation of transgenic mice (Haruyama et al 2009;Su et al 2004). This is precluded in the case of the Pou4f3-α10 line, since transgenic RNA was present in the cochlea as assessed by RT-PCR and it was localized to the IHC region as demonstrated by in situ hybridization.…”

Section: Discussionmentioning

confidence: 99%

Constitutive Expression of the α10 Nicotinic Acetylcholine Receptor Subunit Fails to Maintain Cholinergic Responses in Inner Hair Cells After the Onset of Hearing

Taranda

Ballestero

Hiel

et al. 2009

JARO

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Efferent inhibition of cochlear hair cells is mediated by α9α10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express α10 into adulthood by expressing the α10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the α10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 α10 transgenic mice backcrossed to a Chrna10 −/− background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the α10 transgene restored nAChR function. However, in the α10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental downregulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.

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“…In addition, any selection cassette that is used is normally floxed and placed in an intron so that it can be excised and cause minimal effects on gene expression (Hacking 2008). The generation of transgenic models utilises a targeting construct which usually contains the cDNA carrying the mutation, together with an appropriate promoter and poly(A) sequence, which is injected into the pronucleus of fertilised mouse eggs (Haruyama et al 2009). The transgene then undergoes random insertion into the genome, and several copies are often inserted together, which generates an overexpression model.…”

Section: Targeted Strategiesmentioning

confidence: 99%

Mouse models for inherited endocrine and metabolic disorders

Piret

Thakker²

2011

Journal of Endocrinology

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In vivo models represent important resources for investigating the physiological mechanisms underlying endocrine and metabolic disorders, and for pre-clinical translational studies that may include the assessments of new treatments. In the study of endocrine diseases, which affect multiple organs, in vivo models provide specific advantages over in vitro models, which are limited to investigation of isolated systems. In recent years, the mouse has become the popular choice for developing such in vivo mammalian models, as it has a genome that shares w85% identity to that of man, and has many physiological systems that are similar to those in man. Moreover, methods have been developed to alter the expression of genes in the mouse, thereby generating models for human diseases, which may be due to loss-or gainof-function mutations. The methods used to generate mutations in the mouse genome include: chemical mutagenesis; conventional, conditional and inducible knockout models; knockin models and transgenic models, and these strategies are often complementary. This review describes some of the different strategies that are utilised for generating mouse models. In addition, some mouse models that have been successfully generated by these methods for some human hereditary endocrine and metabolic disorders are reviewed. In particular, the mouse models generated for parathyroid disorders, which include: the multiple endocrine neoplasias; hyperparathyroidism-jaw tumour syndrome; disorders of the calcium-sensing receptor and forms of inherited hypoparathyroidism are discussed. The advances that have been made in our understanding of the mechanisms of these human diseases by investigations of these mouse models are described.

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Overview: Engineering Transgenic Constructs and Mice

Cited by 69 publications

References 19 publications

S151A δ-sarcoglycan mutation causes a mild phenotype of cardiomyopathy in mice

S151A δ-sarcoglycan mutation causes a mild phenotype of cardiomyopathy in mice

Constitutive Expression of the α10 Nicotinic Acetylcholine Receptor Subunit Fails to Maintain Cholinergic Responses in Inner Hair Cells After the Onset of Hearing

Mouse models for inherited endocrine and metabolic disorders

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