Transgene expression and silencing in a tick cell line

A model system for functional tick genomics

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

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)963-968
Number of pages6
JournalInsect Biochemistry and Molecular Biology
Volume38
Issue number10
DOIs
StatePublished - Oct 1 2008

Fingerprint

Beauty
Ixodes scapularis
Ticks
Genomics
Transgenes
Ixodes
transgenes
ticks
Genes
Cells
cell lines
genomics
Cell Line
double-stranded RNA
transposons
Kanamycin Kinase
Genome
Inverted Repeat Sequences
cells
Transposases

Keywords

  • Cell line
  • Ixodes scapularis
  • RNAi
  • Sleeping Beauty
  • Transformation

Cite this

Transgene expression and silencing in a tick cell line : A model system for functional tick genomics. / Kurtti, Timothy J.; Mattila, Joshua T.; Herron, Michael J.; Felsheim, Roderick F.; Baldridge, Gerald D.; Burkhardt, Nicole Y.; Blazar, Bruce R.; Hackett, Perry B.; Meyer, Jason M.; Munderloh, Ulrike G.

In: Insect Biochemistry and Molecular Biology, Vol. 38, No. 10, 01.10.2008, p. 963-968.

Research output: Contribution to journalArticle

Kurtti, Timothy J. ; Mattila, Joshua T. ; Herron, Michael J. ; Felsheim, Roderick F. ; Baldridge, Gerald D. ; Burkhardt, Nicole Y. ; Blazar, Bruce R. ; Hackett, Perry B. ; Meyer, Jason M. ; Munderloh, Ulrike G. / Transgene expression and silencing in a tick cell line : A model system for functional tick genomics. In: Insect Biochemistry and Molecular Biology. 2008 ; Vol. 38, No. 10. pp. 963-968.
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