Gene transfer into individual muscle fibers and conditional gene expression in living animals.
Pressure injection of DNA directly into individual fibers of surgically exposed soleus muscle leads to efficient and reliable expression of the transgene. Conditionally regulated gene expression in a single muscle fiber was analyzed in vivo by co-injecting a tetracycline-regulated lacZ reporter construct and a transactivator (rtTA) expression vector. The tetracycline-responsive element revealed significant basal transcriptional activity that was further increased by rtTA even in the absence of the effector doxycycline (dox). The high basal activity of the simple two-component system precludes tight gene regulation in muscle. Concomitant expression of the silencer tTS(Kid), however, reduced the basal activity to low or undetectable levels. This allowed the specific activation of the tetracycline-responsive element by the application of dox. Direct gene transfer can thus be employed to express transgenic proteins in distinct muscle fibers at spatially defined regions and to regulate gene expression conditionally.
Sander
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A
Abteilung Zellphysiologie, Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Güth
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A
Brenner
H R
HR
Witzemann
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V
D000818:Animals; D000900:Anti-Bacterial Agents; D004318:Doxycycline; D005786:Gene Expression Regulation / Q000187:drug effects*; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007763:Lac Operon; D008164:Luminescent Proteins / Q000235:genetics; D008845:Microinjections; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology; D010957:Plasmids / Q000494:pharmacology*; D051381:Rats; D015533:Transcriptional Activation / Q000235:genetics; D019076:Transgenes / Q000235:genetics
D000900:Anti-Bacterial Agents; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004318:Doxycycline