NDLI: Highly parallel introduction of nucleic acids into mammalian cells grown in microwell arrays

Content Provider	Royal Society of Chemistry (RSC)
Author	Head, Steven Jain, Tilak Saez, Enrique McBride, Ryan
Copyright Year	2009
Abstract	High-throughput cell-based screens of genome-size collections of cDNAs and siRNAs have become a powerful tool to annotate the mammalian genome, enabling the discovery of novel genes associated with normal cellular processes and pathogenic states, and the unravelling of genetic networks and signaling pathways in a systems biology approach. However, the capital expenses and the cost of reagents necessary to perform such large screens have limited application of this technology. Efforts to miniaturize the screening process have centered on the development of cellular microarrays created on microscope slides that use chemical means to introduce exogenous genetic material into mammalian cells. While this work has demonstrated the feasibility of screening in very small formats, the use of chemical transfection reagents (effective only in a subset of cell lines and not on primary cells) and the lack of defined borders between cells grown in adjacent microspots containing different genetic material (to prevent cell migration and to aid spot location recognition during imaging and phenotype deconvolution) have hampered the spread of this screening technology. Here, we describe proof-of-principles experiments to circumvent these drawbacks. We have created microwell arrays on an electroporation-ready transparent substrate and established procedures to achieve highly efficient parallel introduction of exogenous molecules into human cell lines and primary mouse macrophages. The microwells confine cells and offer multiple advantages during imaging and phenotype analysis. We have also developed a simple method to load this 484-microwell array with libraries of nucleic acids using a standard microarrayer. These advances can be elaborated upon to form the basis of a miniaturized high-throughput functional genomics screening platform to carry out genome-size screens in a variety of mammalian cells that may eventually become a mainstream tool for life science research.
Starting Page	3557
Ending Page	3566
Page Count	10
File Format	HTM / HTML PDF
ISSN	14730197
Volume Number	9
Issue Number	24
Journal	Lab on a Chip
DOI	10.1039/b913794g
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Phenotype Deconvolution Transfection Microscopy Mouse Systems biology Mammalian Genome Functional genomics Macrophage
Content Type	Text
Resource Type	Article
Subject	Chemistry Nanoscience and Nanotechnology Biochemistry Bioengineering Biomedical Engineering

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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