The mouse muscle creatine kinase promoter faithfully drives reporter gene expression in transgenicXenopus laevis

2004 ◽  
Vol 18 (1) ◽  
pp. 79-86 ◽  
Author(s):  
Wayland Lim ◽  
Eric S. Neff ◽  
J. David Furlow
Keyword(s):  
Gene Expression ◽  
Creatine Kinase ◽  
Transgene Expression ◽  
Muscle Development ◽  
Fluorescent Protein ◽  
Regulatory Sequences ◽  
Gene Promoters ◽  
Muscle Creatine Kinase ◽  
Mouse Muscle ◽  
Muscle Creatine

Developing Xenopus laevis experience two periods of muscle differentiation, once during embryogenesis and again at metamorphosis. During metamorphosis, thyroid hormone induces both muscle growth in the limbs and muscle death in the tail. In mammals, the muscle creatine kinase (MCK) gene is activated during the differentiation from myoblasts to myocytes and has served as both a marker for muscle development and to drive transgene expression in transgenic mice. Transcriptional control elements are generally highly conserved throughout evolution, potentially allowing mouse promoter use in transgenic X. laevis. This paper compares endogenous X. laevis MCK gene expression and the mouse MCK (mMCK) promoter driving a green fluorescent protein reporter in transgenic X. laevis. The mMCK promoter demonstrated strong skeletal muscle-specific transgene expression in both the juvenile tadpole and adult frog. Therefore, our results clearly demonstrate the functional conservation of regulatory sequences in vertebrate muscle gene promoters and illustrate the utility of using X. laevis transgenesis for detailed comparative study of mammalian promoter activity in vivo.

scholarly journals E-box sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice.

1996 ◽  
Vol 16 (9) ◽  
pp. 5058-5068 ◽  
Author(s):  
M A Shield ◽  
H S Haugen ◽  
C H Clegg ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Skeletal Muscles ◽  
Transgene Expression ◽  
Regulatory Region ◽  
Regulatory Sequences ◽  
Striated Muscles ◽  
Muscle Creatine Kinase ◽  
E Box ◽  
Muscle Creatine ◽  
E Boxes

Previous analysis of the muscle creatine kinase (MCK) gene indicated that control elements required for transcription in adult mouse muscle differed from those required in cell culture, suggesting that distinct modes of muscle gene regulation occur in vivo. To examine this further, we measured the activity of MCK transgenes containing E-box and promoter deletions in a variety of striated muscles. Simultaneous mutation of three E boxes in the 1,256-bp MCK 5' region, which abolished transcription in muscle cultures, had strikingly different effects in mice. The mutations abolished transgene expression in cardiac and tongue muscle and caused a reduction in expression in the soleus muscle (a muscle with many slow fibers) but did not affect expression in predominantly fast muscles: quadriceps, abdominals, and extensor digitorum longus. Other regulatory sequences with muscle-type-specific activities were found within the 358-bp 5'-flanking region. This proximal region conferred relatively strong expression in limb and abdominal skeletal muscles but was inactive in cardiac and tongue muscles. However, when the 206-bp 5' enhancer was ligated to the 358-bp region, high levels of tissue-specific expression were restored in all muscle types. These results indicate that E boxes and a proximal regulatory region are differentially required for maximal MCK transgene expression in different striated muscles. The overall results also imply that within skeletal muscles, the steady-state expression of the MCK gene and possibly other muscle genes depends on transcriptional mechanisms that differ between fast and slow fibers as well as between the anatomical and physiological attributes of each specific muscle.

Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene

1989 ◽  
Vol 9 (6) ◽  
pp. 2627-2640
Author(s):  
J N Buskin ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Muscle Creatine Kinase ◽  
Mouse Muscle ◽  
Nuclear Extracts ◽  
Muscle Creatine

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

scholarly journals Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene.

1989 ◽  
Vol 9 (6) ◽  
pp. 2627-2640 ◽  
Author(s):  
J N Buskin ◽  
S D Hauschka
Keyword(s):  
Creatine Kinase ◽  
Regulatory Elements ◽  
Site Directed Mutagenesis ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Muscle Creatine Kinase ◽  
Mouse Muscle ◽  
Nuclear Extracts ◽  
Muscle Creatine

The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.

scholarly journals Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

1996 ◽  
Vol 16 (4) ◽  
pp. 1649-1658 ◽  
Author(s):  
D B Donoviel ◽  
M A Shield ◽  
J N Buskin ◽  
H S Haugen ◽  
C H Clegg ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Transgenic Mice ◽  
Cardiac Muscle ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Muscle Creatine Kinase ◽  
Regulatory Regions ◽  
Mouse Muscle ◽  
Muscle Creatine

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.

M-creatine kinase gene expression in mechanically overloaded skeletal muscle of transgenic mice

1995 ◽  
Vol 269 (3) ◽  
pp. C665-C674 ◽  
Author(s):  
R. W. Tsika ◽  
S. D. Hauschka ◽  
L. Gao
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Muscle Creatine Kinase ◽  
Kinase Gene ◽  
Mouse Muscle ◽  
Average Decrease ◽  
Mechanical Overload ◽  
Mouse Lines ◽  
Specific Mrna

The molecular pathways and regulatory molecules that underlie changes in gene transcription during mechanical overload of skeletal muscle remain obscure. To better understand this process, we have examined mouse muscle creatine kinase (MCK) gene expression in mechanically overloaded plantaris (OP) muscle of transgenic and nontransgenic mice. Northern blot analysis revealed that endogenous MCK-specific mRNA transcripts were decreased 150% in the OP muscles after 6 wk. To identify the MCK gene regions involved in the response to mechanical overload, three different mouse MCKCAT transgenes were studied by measuring chloramphenicol acetyltransferase (CAT assays) activity in OP and sham-operated (control plantaris) muscles. Mouse lines carrying (+enh206)117MCKCAT and -1256MCKCAT transgenes exhibited 30 and 40% lower CAT levels, whereas two mouse lines carrying -3300MCKCAT transgenes exhibited average decreases of 430%. Nearly identical results, including measurements of exogenous CAT mRNA, were obtained 2 days postoverload. Six weeks or 2 days of mechanical overload led to an average decrease in MM-CK isoprotein of 140%. These data provide evidence that mechanical overload induces changes in MCK gene expression that appear to be regulated by at least two portions of the MCK gene: the 206 base pair 5' enhancer and the -3,300 to -1,257 region.

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