Functional screening for proapoptotic genes by reverse transfection cell array technology

被引：45

作者：

Mannherz, Otto ^{[1
]}

Mertens, Daniel ^{[1
]}

Hahn, Meinhard ^{[1
]}

Lichter, Peter ^{[1
]}

机构：

[1] Deutsch Krebsforschungszentrum, Div Mol Genet B060, D-69120 Heidelberg, Germany

来源：

GENOMICS | 2006年 / 87卷 / 05期

关键词：

reverse transfection; cell array technology; apoptosis; caspase; nuclear morphology;

D O I：

10.1016/j.ygeno.2005.12.009

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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. (c) 2006 Elsevier Inc. All rights reserved.

引用

页码：665 / 672

页数：8

共 20 条

[1] Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis [J].

Bauer, MKA ;

Schubert, A ;

Rocks, O ;

Grimm, S .

JOURNAL OF CELL BIOLOGY, 1999, 147 (07) :1493-1501

[2] Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses [J].

Bhattacharjee, A ;

Richards, WG ;

Staunton, J ;

Li, C ;

Monti, S ;

Vasa, P ;

Ladd, C ;

Beheshti, J ;

Bueno, R ;

Gillette, M ;

Loda, M ;

Weber, G ;

Mark, EJ ;

Lander, ES ;

Wong, W ;

Johnson, BE ;

Golub, TR ;

Sugarbaker, DJ ;

Meyerson, M .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (24) :13790-13795

[3] The gene ontology annotation (GOA) project: Implementation of GO in SWISS-PROT, TrEMBL, and InterPro [J].

Camon, E ;

Magrane, M ;

Barrell, D ;

Binns, D ;

Fleischmann, W ;

Kersey, P ;

Mulder, N ;

Oinn, T ;

Maslen, J ;

Cox, A ;

Apweiler, R .

GENOME RESEARCH, 2003, 13 (04) :662-672

[4] Gene expression patterns in human liver cancers [J].

Chen, X ;

Cheung, ST ;

So, S ;

Fan, ST ;

Barry, C ;

Higgins, J ;

Lai, KM ;

Ji, JF ;

Dudoit, S ;

Ng, IOL ;

van de Rijn, M ;

Botstein, D ;

Brown, PO .

MOLECULAR BIOLOGY OF THE CELL, 2002, 13 (06) :1929-1939

[5] Major DNA fragmentation is a late event in apoptosis [J].

Collins, JA ;

Schandl, CA ;

Young, KK ;

Vesely, J ;

Willingham, MC .

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY, 1997, 45 (07) :923-934

[6] Automatic identification of subcellular phenotypes on human cell arrays [J].

Conrad, C ;

Erfle, H ;

Warnat, P ;

Daigle, N ;

Lörch, T ;

Ellenberg, J ;

Pepperkok, R ;

Eils, R .

GENOME RESEARCH, 2004, 14 (06) :1130-1136

[7] High-throughput protein analysis integrating bioinformatics and experimental assays [J].

del Val, C ;

Mehrle, A ;

Falkenhahn, M ;

Seiler, M ;

Glatting, KH ;

Poustka, A ;

Suhai, S ;

Wiemann, S .

NUCLEIC ACIDS RESEARCH, 2004, 32 (02) :742-748

[8] Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells [J].

Elbashir, SM ;

Harborth, J ;

Lendeckel, W ;

Yalcin, A ;

Weber, K ;

Tuschl, T .

NATURE, 2001, 411 (6836) :494-498

[9] Large scale molecular analysis identifies genes with altered expression in salivary adenoid cystic carcinoma [J].

Frierson, HF ;

Ei-Naggar, AK ;

Welsh, JB ;

Sapinoso, LM ;

Su, AI ;

Cheng, J ;

Saku, T ;

Moskaluk, CA ;

Hampton, GM .

AMERICAN JOURNAL OF PATHOLOGY, 2002, 161 (04) :1315-1323

[10] Diversity of gene expression in adenocarcinoma of the lung [J].

Garber, ME ;

Troyanskaya, OG ;

Schluens, K ;

Petersen, S ;

Thaesler, Z ;

Pacyna-Gengelbach, M ;

van de Rijn, M ;

Rosen, GD ;

Perou, CM ;

Whyte, RI ;

Altman, RB ;

Brown, PO ;

Botstein, D ;

Petersen, I .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2001, 98 (24) :13784-13789

← 1 2 →