SB-Transposition: MAR & SV40 Excision Assay

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https://hdl.handle.net/11299/51741

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SB-Transposition: MAR & SV40 Excision Assay

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Goh, Diane

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2009-04-08

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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.

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College of Biological Sciences

Department of Genetics, Cell Biology and Development

Academic Health Center

Department of Medicine

Division of Gastroenterology

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Additional contributors: Lucas Sjeklocha; Phil Wong; Betsy Kren and Clifford Steer (faculty mentors)

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UMTC Undergraduate Research Presentations and Papers (UROP)

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Goh, Diane. (2009). SB-Transposition: MAR & SV40 Excision Assay. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/51741.

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