Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression

Takeshita, Fumihiko; Takase, Keiko; Tozuka, Miyuki; Saha, Sukumar; Okuda, Kenji; Ishii, Norihisa; Sasaki, Shin

doi:10.3892/ijmm.19.2.309

Cited by 12 publications

(12 citation statements)

References 36 publications

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“…A expression construct was generated that induces ectopic expression of ADAR1 p110 by a muscle-specific muscle creatine kinase (MCK) promoter/enhancer, which is transcriptionally restricted to differentiated muscle cells. 31 Such exogenous construct resulted in higher expression of ADAR1 p110 in the late phase of differentiation course (48-96 h; Figure 3a), complementing the depleted pools of this protein at this stage. Quantitative examination of the extent of differentiation was done by measuring the extent of myotube fusion, defined as the percentage of total nuclei in myotubes that comprise multiple nuclei.…”

Section: Resultsmentioning

confidence: 85%

ADAR1 deaminase contributes to scheduled skeletal myogenesis progression via stage-specific functions

et al. 2014

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Adenosine deaminases acting on RNA 1 (ADAR1) catalyzes cellular RNA adenosine-to-inosine editing events on structured RNA molecules. In line with this critical role, ADAR1 exhibits ubiquitous expression and is essential for embryonic development. However, regulation and developmental significance of this RNA editor in a spatiotemporal context are largely elusive. Here we unveil a novel tissue-specific role of ADAR1 in skeletal myogenesis. ADAR1 expression displayed programmed alteration that is coordinated with differentiation cues, and mediated negatively by miRNA-1/206. Coincidently, ADAR1 exerts stage-dependent functions-suppression of apoptosis at the onset of differentiation and preservation of timely myotube formation through later phase. Furthermore, the post-transcriptional aspect of its myogenic role was illustrated by the spectrum of binding RNAs, as revealed by high-throughput approach, as well as by direct regulation of myogenesis-associated targets such as dynamin 1/2 (Dnm1/2) and annexin A4. Consequently, maintenance of target gene expression profiles likely contributes to a state of cytoskeleton and membrane dynamics that is amenable to myoblast morphogenesis. Collectively, these findings uncover a critical link of ADAR1 to myogenesis, and further highlight an epigenetic mechanism by which ADAR1 and miR-1/206 interplay to control scheduled myoblast-myotube transition.

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Section: Resultsmentioning

confidence: 85%

ADAR1 deaminase contributes to scheduled skeletal myogenesis progression via stage-specific functions

et al. 2014

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“…8,13 Recently, the MCK enhancer has been used to build a hybrid SV40 promoter for muscletargeted long-term transgene expression. 17 The MCK enhancer inserted in retrovirus long terminal repeat (LTR) also led to stable and specific expression of human factor IX in skeletal muscle. 18 Transgenic and tissue culture analyses showed that the MCK promoter was expressed at a high level only in differentiated skeletal and cardiac muscles.…”

Section: Instructionmentioning

confidence: 99%

Construction and analysis of compact muscle-specific promoters for AAV vectors

et al. 2008

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Adeno-associated viral (AAV) vectors have been broadly used for gene transfer in vivo for various applications. However, AAV precludes the use of most of the original large-sized tissue-specific promoters for expression of transgenes. Efforts are made to develop highly compact, active and yet tissue-specific promoters for use in AAV vectors. In this study, we further abbreviated the muscle creatine kinase (MCK) promoter by ligating a double or triple tandem of MCK enhancer (206-bp) to its 87-bp basal promoter, generating the dMCK (509-bp) and tMCK (720-bp) promoters. The dMCK promoter is shorter but stronger than some previously developed MCK-based promoters such as the enh358MCK (584-bp) and CK6 (589-bp) in vitro in C2C12 myotubes and in vivo in skeletal muscles. The tMCK promoter is the strongest that we tested here, more active than the promiscuous cytomegalovirus (CMV) promoter. Furthermore, both the dMCK and tMCK promoters are essentially inactive in nonmuscle cell lines as well as in the mouse liver (4200-fold weaker than the CMV promoter).The dMCK promoter was further tested in a few lines of transgenic mice. Expression of LacZ or minidystrophin gene was detected in skeletal muscles throughout the body, but was weak in the diaphragm, and undetectable in the heart and other tissues. Similar to other miniature MCK promoters, the dMCK promoter also shows preference for fast-twitch myofibers. As a result, we further examined a short, synthetic muscle promoter C5-12 (312-bp). It is active in both skeletal and cardiac muscles but lacks apparent preference on myofiber types. Combination of a MCK enhancer to promoter C5-12 has increased its strength in muscle by two-to threefold. The above-mentioned compact muscle-specific promoters are well suited for AAV vectors in muscle-directed gene therapy studies.

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“…This promoter has been well-documented in mouse experiments limiting transgene expression to skeletal muscle, even postnatal expression. 120,139,140,145,146 Therefore, this promoter was expected to also be efficient in driving muscle-specific expression in bovine cells.…”

Section: Discussionmentioning

confidence: 99%

308 Development of Transgenic Livestock With Reduced Myostatin Expression Using Rna Interference

Tessanne

Stroud

Long

et al. 2009

Reprod. Fertil. Dev.

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RNA interference (RNAi) is a means of regulating gene expression by targeting mRNA in a sequence-specific manner for degradation or translational inhibition. Short hairpin RNAs (shRNAs) and siRNAs have been extensively employed for manipulating gene expression in a wide range of species. However, the great majority of this work has involved in vitro studies with cells grown in culture. Our goal for this project is to produce transgenic livestock in which myostatin, a negative regulator of muscle growth, has been targeted for silencing by RNAi. In theory, livestock in which myostatin has been silenced should exhibit increased muscle growth and development. To that end, we designed shRNAs to target the bovine myostatin mRNA sequence. The shRNAs were cloned into a lentiviral vector that contains a cytomegalovirus promoter controlling green fluorescent protein and shRNA expression as well as neomycin resistance. Infective lentivirus was made in HEK293T cells through co-transfection of the lentiviral vector, a packaging plasmid, and a plasmid expressing the VSVG pseudotype. Bovine fetal fibroblasts were transduced, selected using Geneticin®, and nuclear transfer was utilized to produce cloned transgenic embryos. There were 186 fusion attempts resulting in 160 fused embryos (fusion rate = 86%). Of these, 54 reached the blastocyst stage (34%) and 10 embryos were transferred into 5 recipient females (2 embryos per recipient). At 40 days, ultrasound revealed 1 confirmed pregnancy. Current plans are to harvest this fetus at 90 days and analyze it for evidence of myostatin knockdown. The production of transgenic animals exhibiting myostatin knockdown through lentiviral-mediated RNAi will demonstrate the utility of RNAi in the study of gene function in large animal models without the need for homologous recombination techniques, which are currently inefficient in species other than mice.

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Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression

Cited by 12 publications

References 36 publications

ADAR1 deaminase contributes to scheduled skeletal myogenesis progression via stage-specific functions

ADAR1 deaminase contributes to scheduled skeletal myogenesis progression via stage-specific functions

Construction and analysis of compact muscle-specific promoters for AAV vectors

308 Development of Transgenic Livestock With Reduced Myostatin Expression Using Rna Interference

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