Functional screening for proapoptotic genes by reverse transfection cell array technology

Genomics
Otto MannherzPeter Lichter

Abstract

Application of mathematical algorithms to sequenced whole genomes revealed a large number of predicted genes, requiring functional assays for their characterization in a high-throughput manner. Here, we report on the development of a screening assay, which is based on reverse transfection of cellular arrays and subsequent analysis of cell morphology to identify novel proapoptotic genes. Expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pEYFP to screen for apoptotic body formation, based on EYFP fluorescence. The assay was validated and applied to 382 human sequence-verified full-length open reading frames, most of them of unknown function. In this initial screening, proapoptotic effects could be demonstrated for 10 of these genes. For 6 of them apoptosis induction could be confirmed both by TUNEL assay and by FACS analysis of cells stained according to Nicoletti: 1 gene was not yet annotated for an apoptotic function (ST6GAL2), while 5 genes were without annotated function (FLJ20551, CXorf12, FAM105A, TMEM66, C19orf4). Our study demonstrates the potential of this method to characterize functionally genes of unknown function in a highly parallel format.

References

Jul 1, 1997·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·J A CollinsM C Willingham
May 3, 2001·Nature·J Ziauddin, D M Sabatini
Nov 15, 2001·Proceedings of the National Academy of Sciences of the United States of America·A BhattacharjeeM Meyerson
Nov 15, 2001·Proceedings of the National Academy of Sciences of the United States of America·M E GarberI Petersen
Dec 14, 2001·Proceedings of the National Academy of Sciences of the United States of America·S RamaswamyT R Golub
Jun 12, 2002·Molecular Biology of the Cell·Xin ChenPatrick O Brown
Oct 9, 2002·The American Journal of Pathology·Henry F FriersonGarret M Hampton
Mar 26, 2003·The American Journal of Pathology·Christine A Iacobuzio-DonahueMichael Goggins
Oct 4, 2003·Genome Research·Rajeev KumarVivek Mittal
Jan 9, 2004·Journal of Biomolecular Screening·Brian L WebbFang Lai
Feb 6, 2004·Journal of Biochemistry and Molecular Biology·Ungsik YuSangsoo Kim
Feb 6, 2004·Nucleic Acids Research·Coral del ValStefan Wiemann
Apr 8, 2004·Neoplasia : an International Journal for Oncology Research·Daniel R RhodesArul M Chinnaiyan
Apr 16, 2004·Proceedings of the National Academy of Sciences of the United States of America·Jose M SilvaGregory J Hannon
Jun 3, 2004·Genome Research·Christian ConradRoland Eils

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Citations

Jul 17, 2008·Nucleic Acids Research·Christina Saeten FjeldboTorunn Bruland
Feb 23, 2012·Lab on a Chip·Elisabeth KuhnMichael Stürzl
Jul 19, 2012·BMC Research Notes·Sven FenglerPedro Roda-Navarro
Nov 8, 2008·Analytical and Bioanalytical Chemistry·Hiroyuki FujimotoHiroo Iwata
Aug 21, 2007·Expert Review of Proteomics·Vytaute StarkuvieneHolger Erfle
Oct 28, 2015·The FEBS Journal·Paul R Elliott, David Komander
Nov 21, 2007·Journal of Bioscience and Bioengineering·Satoshi FujitaJun Miyake
Sep 12, 2007·Journal of Microscopy·Z Pincus, J A Theriot
May 9, 2007·Biotechnology and Bioengineering·Angela K PannierLonnie D Shea
Aug 25, 2010·International Journal of Cancer. Journal International Du Cancer·Maria ShahmoradgoliPeter Lichter
Sep 8, 2016·Microarrays·Rebecca JonczykFrank Stahl
Jun 22, 2016·Molecular Neurobiology·Qiuyang ZhengYun-Wu Zhang
Oct 6, 2016·SLAS Discovery·Hi Chul KimYong-Jun Kwon
Mar 18, 2010·Journal of Biomolecular Screening·Richard H MaierKamil Onder
May 5, 2007·Journal of Biomolecular Screening·Mamatha SauermannStefan Wiemann
Jul 12, 2012·Oncology Letters·Rebeca González-FernándezPablo Martín-Vasallo

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