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Minimal piggyBac vectors for chromatin integration (1138.12)
Author(s) -
Solodushko Victor,
Troyanovsky Boris,
Bitko Vira,
Fouty Brian
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1138.12
Subject(s) - plasmid , transposable element , transgene , biology , transposition (logic) , chromatin , genetics , transformation (genetics) , gene delivery , gene , genome , microbiology and biotechnology , computational biology , transfection , computer science , artificial intelligence
We describe novel transposon piggyBac vectors engineered to deliver transgenes as efficiently as currently available piggyBac systems, but with significantly less helper DNA co‐delivered into the host genome. To generate these plasmids, we identified a previously unreported aspect of transposon biology, that the full‐length terminal domains required for successful plasmid‐to‐chromatin transgene delivery can be repositioned from the transgene delivery cassette to the other (helper) parts of the plasmid. This design decreased the size of the required terminal domains within the delivered gene cassette of the piggyBac vector from about 1,500 to just 98 base pairs. Herein we tested the transposition efficiency of these minimal piggyBac transposons on different cell types: HEK293, HeLa, L929, and primary pulmonary artery smooth muscle cells. Transposition efficiency was defined as transgene (RFP) positivity at 28 days. The specificity of integration was determined by PCR and qPCR. Conclusion: Relocating most of the long terminal‐repeat sequences from the delivered gene cassette of minimal piggyBac vectors into the helper region of the same plasmid decreased the amount of non‐essential (helper) DNA incorporated into the host cell genome without a significant decrease in transposition efficiency. This may reduce the risk of target cell transformation. Grant Funding Source : Supported by the American Heart Association to V.S. (12GRNT12070291)