Journal
BIOTECHNIQUES
Volume 69, Issue 4, Pages 281-288Publisher
FUTURE SCI LTD
DOI: 10.2144/btn-2020-0037
Keywords
alternative to molecular cloning; cell transfection; chemical DNA synthesis; protein translation; synthetic mRNA from DNA template
Funding
- NIH [R01AI44924, R21AI124766, R21AI144193]
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We present a method to synthesize mRNAs from synthetic DNA templates that produce biologically active proteins. To illustrate utility, we constructed five unique synthetic DNA templates, produced mRNAs and demonstrated biologic activity of their translated proteins. Examples include secreted luciferase, enhanced green fluorescence protein, IL-4, and IL-12A and IL-12B to form active IL-12. We propose that this method offers a cost- and time-saving alternative to plasmid-based cloning. METHOD SUMMARY In vitrotranscription reactions were performed starting with a double-stranded DNA fragment with the bacteriophage SP6 promoter. A 5 ' CAP and 3 ' poly (A) tail were added via enzymatic reactions to these single-stranded RNAs to produce functional mRNAs. Synthetic mRNAs were transfected into HeLa cells and expression of translated proteins determined.
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