SB-Transposition: MAR & SV40 Excision Assay
2009-04-08
Loading...
View/Download File
Persistent link to this item
Statistics
View StatisticsJournal Title
Journal ISSN
Volume Title
Title
SB-Transposition: MAR & SV40 Excision Assay
Authors
Published Date
2009-04-08
Publisher
Type
Presentation
Abstract
Sleeping Beauty transposon (SB-Tn) is an effective system that incorporates genes into vertebrate (human) chromosomes without using viruses. The Excision Assay is part of a project that determines the transposition efficiency using vector backbone elements hypothesized to increase the probability of successful gene insertion. The DNA elements used are the matrix-attachment regions (MAR) and SV40 enhancer. MAR contributes to retention of the plasmid in the host nucleus by interacting with the host nuclear proteins; SV40 enhancer promotes a high translocation rate of the SB-Tn vector into the host nucleus. The vector backbone also expresses the obligate SB transposase that must be supplied in mammalian cells to cut and paste the SB-Tn from the plasmid vector into the host chromosome. Following transfection of the vector series into a human hepatocellular carcinoma cell line (huh7), the total DNA is isolated to determine the ratio of excision product plasmids (resealed vector backbone following the cutting and pasting of the SB-Tn into the host genome to the original SB-Tn vectors transfected). This approach addresses if vector backbone modifications influence persistence of the original vector and/or excision product. If the excision plasmid, containing the SB transposase is retained in the nucleus of the host cell, the continued expression might potentially lead to harmful transposition activity remobilizing the SB-Tn integrated in the host genome.
Description
Additional contributors: Lucas Sjeklocha; Phil Wong; Betsy Kren and Clifford Steer (faculty mentors)
Related to
Replaces
License
Series/Report Number
Funding information
Isbn identifier
Doi identifier
Previously Published Citation
Other identifiers
Suggested citation
Goh, Diane. (2009). SB-Transposition: MAR & SV40 Excision Assay. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/51741.
Content distributed via the University Digital Conservancy may be subject to additional license and use restrictions applied by the depositor. By using these files, users agree to the Terms of Use. Materials in the UDC may contain content that is disturbing and/or harmful. For more information, please see our statement on harmful content in digital repositories.