A novel reverse transduction adenoviral array for the functional analysis of shRNA libraries.
BACKGROUND
The identification of novel drug targets by assessing gene functions is most conveniently achieved by high-throughput loss-of-function RNA interference screening. There is a growing need to employ primary cells in such screenings, since they reflect the physiological situation more closely than transformed cell lines do. Highly miniaturized and parallelized approaches as exemplified by reverse transfection or transduction arrays meet these requirements, hence we verified the applicability of an adenoviral microarray for the elucidation of gene functions in primary cells.


RESULTS
Here, we present microarrays of infectious adenoviruses encoding short hairpin RNA (shRNA) as a new tool for gene function analysis. As an example to demonstrate its application, we chose shRNAs directed against seven selected human protein kinases, and we have performed quantitative analysis of phenotypical responses in primary human umbilical vein cells (HUVEC). These microarrays enabled us to infect the target cells in a parallelized and miniaturized procedure without significant cross-contamination: Viruses were reversibly immobilized in spots in such a way that the seeded cells were confined to the area of the viral spots, thus simplifying the subsequent addressing of genetically modified cells for analysis. Computer-assisted image analysis of fluorescence images was applied to analyze the cellular response after shRNA expression. Both the expression level of knock-down target proteins as well as the functional output as measured by caspase 3 activity and DNA fractionation (TUNEL) were quantified.


CONCLUSIONS
We have developed an adenoviral microarray technique suitable for miniaturized and parallelized analysis of gene function. The practicability of this technique was demonstrated by the analysis of several kinases involved in the activation of programmed cell death, both in tumor cells and in primary cells.
Oehmig
Angelika
A
NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany. aoehmig@yahoo.com
Klotzbücher
Andrea
A
Thomas
Maria
M
Weise
Frank
F
Hagner
Ursula
U
Brundiers
Ralf
R
Waldherr
Dirk
D
Lingnau
Andreas
A
Knappik
Achim
A
Kubbutat
Michael H G
MH
Joos
Thomas O
TO
Volkmer
Hansjürgen
H
D000256:Adenoviridae / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D020868:Gene Silencing; D005822:Genetic Vectors / Q000737:chemistry; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D022661:RNA, Untranslated / Q000032:analysis*
D022661:RNA, Untranslated