Transgene expression and silencing in a tick cell line: A model system for functional tick genomics

详细信息    查看全文

• 作者：Timothy J. Kurtti ; Joshua T. Mattila ; Michael J. Herron ; Roderick F. Felsheim ; Gerald D. Baldridge ; Nicole Y. Burkhardt ; Bruce R. Blazar ; Perry B. Hackett ; Jason M. Meyer ; Ulrike G. Munderloh
• 关键词：Ixodes scapularis ; Transformation ; Cell line ; RNAi ; Sleeping Beauty
• 刊名：Insect Biochemistry and Molecular Biology
• 出版年：2008
• 出版时间：October 2008
• 年：2008
• 卷：38
• 期：10
• 页码：963-968
• 全文大小：712 K

文摘

The genome project of the blacklegged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6. Plasmid DNA and dsRNA were effectively transfected into ISE6 cells and they were successfully transformed to express a red fluorescent protein (DsRed2) and a selectable marker, neomycin phosphotransferase (NEO). Frequency of transformation was estimated as 1 transformant per 5000–10,000 cells and cultures were incubated for 2–3 months in medium containing the neomycin analog G418 in order to isolate transformants. Genomic integration of the DsRed2 transgene was confirmed by inverse PCR and sequencing that demonstrated a TA nucleotide pair inserted between SB inverted/direct repeat sequences and tick genomic sequences, indicating that insertion of the DsRed2 gene into the tick cell genome occurred through the activity of SB transposase. RNAi using dsRNA transcribed from the DsRed2 gene silenced expression of red fluorescent protein in transformed ISE6 cells. SB transposition in cell line ISE6 provides an effective means to explore the functional genomics of I. scapularis.