An acetylcholine receptor alpha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice.

Klarsfeld, André; Bessereau, Jean-Louis; Salmon, Anne-Marie; Triller, Antoine; Babinet, C.; Changeux, Jean‐Pierre

doi:10.1002/j.1460-2075.1991.tb07990.x

Cited by 88 publications

(42 citation statements)

References 36 publications

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“…The rate of diffusion is affected by the rate of entrapment of the protein (i.e., targeting of the protein) when this is relevant (Ralston and Hall, 1989a;Klarsfeld et al, 1991). The results of the present study have caused us to hypothesize that the rate of the transcription of the nlsb-gal under the control of the MLC3F promoter is sufficiently high to overcome the fact that the protein is targeted to the nucleus.…”

Section: Discussionmentioning

confidence: 76%

Limitations of nlsβ‐galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer

Yang¹,

Ontell²,

Kelly³

et al. 1997

Anat. Rec.

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Background: Nuclear localizing b-galactosidase (nlsb-gal) is used as a marker for studying myoblast cell lineage and for evaluating myoblast survival after myoblast transfer, a procedure with potential use for gene complementation for muscular dystrophy. Usefulness of this construct depends on the establishment of the extent to which nlsb-gal or its mRNA may be translocated from the nucleus that encodes it to other non-coding myonuclei in hybrid myofibers and the ease with which the encoding and non-coding myonuclei can be distinguished. Previous in vitro studies (Ralston and Hall 1989. Science, 244:1066-1068) have suggested limited translocation of the fusion protein. We re-examined the extent to which nlsb-gal is translocated in hybrid myofibers, both in vitro and in vivo, and evaluated the extent to which one can rely on histochemistry to distinguish encoding from non-coding nuclei in these myofibers.Methods: Myotubes formed in co-cultures of a myoblast line (MM14 cells), stably transfected with a construct consisting of a nlsb-gal under the control of the myosin light chain 3F promoter and 38 enhancer (3FlacZ10 cells), and [3H]-thymidine-labeled parental MM14 cells (plated at ratios of 1:6 or 1:20, respectively) were reacted with X-gal. After autoradiography, the distance over which nlsb-gal was translocated in hybrid myotubes was determined. In vivo translocation of nlsb-gal was evaluated by injecting [3H]-thymidine-labeled 3FlacZ10 myoblasts into the regenerating extensor digitorum longus muscle of immunosuppressed normal and mdx (dystrophin deficient) mice. Sections stained with X-gal and subjected to autoradiography permitted determination of the extent of nlsb-gal translocation in hybrid myofibers.Results: In vitro: All nuclei in G92% of hybrid myotubes showed evidence of nlsb-gal after exposure to X-gal, suggesting extensive translocation. Within hybrid myotubes, MM14-derived myonuclei D350 mm from a 3FlacZ10-derived myonucleus showed evidence of nlsb-gal. In vivo: Similar translocation of nlsb-gal was observed in vivo. One week after myoblast transfer, donor-derived myonuclei were distinguishable from hostderived myonuclei containing nlsb-gal by the greater accumulation of reaction product in donor myonuclei after X-gal staining. However, 2 weeks after injection, host myonuclei often contained a significant amount of nlsb-gal, and accumulation of reaction product could not be used as the criterion for identification of donor myonuclei.Conclusions: Translocation of nlsb-gal (or its mRNA) is significantly greater than previously reported (Ralston and Hall 1989), resulting in large numbers of nlsb-gal positive non-coding myonuclei in hybrid myofi-

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Section: Discussionmentioning

confidence: 76%

Limitations of nlsβ‐galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer

Yang¹,

Ontell²,

Kelly³

et al. 1997

Anat. Rec.

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“…However, in innervated adult muscle, the expression of the nicotinic acetylcholine receptor is highly restricted to the subsynaptic nuclei located near the motor endplates [1,2]. In addition, in fused heterokaryons between muscle and non-muscle cells muscle gene products are localized in the vicinity of the nuclei which are responsible for their syntheses [3].…”

Section: Introduction 2 Materials and Methodsmentioning

confidence: 99%

Limited diffusibility of gene products directed by a single nucleus in the cytoplasm of multinucleated myofibres

Ono

Mizukawa

et al. 1994

FEBS Letters

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Two types of B-galactosidase genes, whose products are distributed in the nucleus (N/3-gal) or cytoplasm (Cp-gal), were injected with fructose intramuscularly into the quadriceps of adult mice. Regionally restricted and overlapped distributions of both gene products were observed in the myofibres. These findings indicate that NB-gal is incorporated into the nucleus responsible for its synthesis and that C&gal becomes located in the vicinity of the nucleus after its synthesis. This restricted location of C/3-gal in myofibres remained unchanged during the development of infant mouse muscle. Thus, the gene products directed by the nucleus of myofibres seem to show limited diffusibility, suggesting a universal localization of s&cellular domains in myofibres.

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“…For the construction of Tg2 mice, an XbaI-SalI restriction fragment containing the ␣ AChR3.3 subunit promoter sequence was excised from p ␣ ACh3.3CAT (17), and inserted into XbaI-SalI sites of pKSnlsLACZ (16). The NotI-KpnI restriction fragment was excised from p ␣ 3.3nlsLACZ, obtained and purified by using the Gene-Clean II system (Bio 101, La Jolla, CA).…”

Section: Generation Of Transgenic Micementioning

confidence: 99%

“…The first, Tg1, was constructed with an 842-bp fragment and the second, Tg2, with a 3300-bp fragment of the ␣ AChR promoter. In Tg1 transgenic mice (R ␣ NLZ2) ␤ -gal expression is primarily observed in developing skeletal muscle in embryonic and newborn mice (16) and at a high level in large myoid cells of the thymus of fetal and newborn mice. In the thymus of Tg2 mice, ␤ -gal was observed in large myoid dispersed cells (as in Tg1 mice) and also in epithelial cells, especially in cortical areas.…”

Section: Introductionmentioning

confidence: 99%

An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

Salmon

Bruand²,

Cardona³

et al. 1998

J. Clin. Invest.

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Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the ␤ -galactosidase ( ␤ -gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2)

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An acetylcholine receptor alpha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice.

Cited by 88 publications

References 36 publications

Limitations of nlsβ‐galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer

Limitations of nlsβ‐galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer

Limited diffusibility of gene products directed by a single nucleus in the cytoplasm of multinucleated myofibres

An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

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