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Cell-Based Microarrays: Recent Advances for Gene Function Analyses

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Cell-Based Microarrays

Part of the book series: SpringerBriefs in Cell Biology ((SBCB))

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Abstract

Reverse transfected cell microarrays (RTCM) are a powerful tool for the systematic analyses of gene functions. With this technology more than a thousand different nucleic acids can be transfected into eukaryotic cells in parallel on a single glass slide. This allows high-throughput analyses of gene functions using gain-of-function, loss-of-function, and mutation approaches. RTCM paved the way for genome-wide gene function analyses in order to determine gene functions involved in the molecular regulation of cell phenotypes in physiologic and pathophysiologic processes. Since RTCM was first introduced in 2001, the technique has been well established. Themethod was successfully used in several genome-wide and large-scale screenings, and novel analysis methods to detect gene functions have been developed. This chapter will summarize the most recent technological developments in the usage of RTCM, including optimization of (1) transfection efficiency, (2) reporter systems and automated data acquisition, (3) spotting density with decreased cross-contamination, and (4) the new development of assays to screen for paracrine gene effects.

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Abbreviations

A549:

Human alveolar basal epithelial cells

cDNA:

Complementary DNA

CFP:

Cyan fluorescent protein

COPI/COPII:

Coat protein I/Coat protein II

COX-2:

Cyclooxygenase 2

cPARP:

Cleaved poly ADP ribose polymerase

CRE:

cAMP response element

DNA:

Deoxyribonucleic acid

dsRNA:

Double-stranded ribonucleic acid

ECM:

Extracellular matrix protein

EdU:

5-ethynyl-2′-deoxyuridine

ER:

Endoplasmic reticulum

GBP-1:

Guanylate-binding protein 1

(E)GFP:

(Enhanced) green fluorescent protein

GPCR:

G protein-coupled receptor

GPR160:

G protein-coupled receptor 160

HCT116:

Colon carcinoma cell line

HEK293:

Human embryonic kidney cell line

HEK239T:

Human embryonic kidney cells expressing SV40 large T antigen

HeLa:

Cervix carcinoma cell line

HFIB:

Human fibroblasts

HIV:

Human immunodeficiency virus

HT29:

Colon adenocarcinoma cell line

HUVEC:

Human umbilical vein endothelial cells

ICAM-1:

Intercellular adhesion molecule 1

IFN-α:

Interferon-α

IFN-γ:

Interferon-γ

JNK:

c-Jun N-terminal kinase

K562:

Human erythroleukemia cell line

KPL-4:

Breast cancer cell line

LICM:

Lentivirus-infected cell microarray

LMP:

Low melting point

MAPK:

Mitogen-activated protein kinase

MCF-7:

Breast cancer cell line

MicroSCALE:

Microarrays of spatially confined adhesive lentiviral features

miRNA:

Micro ribonucleic acid

NF-κB:

Nuclear factor-kappa B

NLS:

Nuclear localization signal

NPY:

Neuropeptide Y

PC3:

Prostate cancer cell line

PEST:

Proline–glutamic acid–serine–threonine–protein sequence

PLL:

Poly-l-lysine

RFP:

Red fluorescent protein

RNAi:

Ribonucleic acid interference

RTCM:

Reverse transfected cell microarray

S2R+:

Schneider S2 embryonic drosophila cell line

SHARPIN:

SHANK-associated RH domain interactor

shRNA:

Short hairpin ribonucleic acid

siRNA:

Small interfering ribonucleic acid

SW480:

Colon adenocarcinoma cell line

TNF-α:

Tumor necrosis factor-α

TORC1:

Target of rapamycin complex 1

tsO45G:

Temperature-sensitive CFP-coupled viral membrane protein

TUNEL:

Terminal deoxynucleotidyl transferase dUTP nick end label

U2OS:

Human osteosarcoma cell line

VCAM-1:

Vascular cell adhesion molecule 1

WiDr:

Colon adenocarcinoma cell line

YFP:

Yellow fluorescent protein

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Acknowledgement

We thank Mahimaidos Manoharan for valuable technical help and Alexander Jakin for help with the graphics.

Funding 

This work was supported by grants of the German Research Foundation (DFG-GK 1071, STU 238/6-1, SFB796), the German Federal Ministry of Education and Research (BMBF, Polyprobe-Study), the FAU Emerging Fields Initiative to M.S., of the German Cancer Aid (109510) to M.S. and E.N., and of the Interdisciplinary Center for Clinical Research (IZKF) of the University Hospital of the University of Erlangen-Nuremberg to M.S. and R.J.

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Authors and Affiliations

  1. Division of Molecular and Experimental Surgery, University Medical Center Erlangen, Friedrich-Alexander University of Erlangen-Nuremberg, Schwabachanlage 10, 91054, Erlangen, Germany

    Dominika Gaus, Elisabeth Kuhn, Andreas Konrad, Ramona Jochmann, Priya Chudasama, Elisabeth Naschberger & Michael Stürzl

Authors
  1. Dominika Gaus
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  2. Elisabeth Kuhn
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  3. Andreas Konrad
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  4. Ramona Jochmann
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  5. Priya Chudasama
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  6. Elisabeth Naschberger
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  7. Michael Stürzl
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Corresponding author

Correspondence to Michael Stürzl .

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© 2014 Ella Palmer

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Gaus, D. et al. (2014). Cell-Based Microarrays: Recent Advances for Gene Function Analyses. In: Cell-Based Microarrays. SpringerBriefs in Cell Biology. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-0594-2_1

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