Elsevier

New Biotechnology

Volume 25, Issue 1, June 2008, Pages 55-67
New Biotechnology

Research paper
Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

https://doi.org/10.1016/j.nbt.2008.03.002Get rights and content

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.

Introduction

A major challenge of modern genetic research is to decipher how genes and their control elements within the mammalian genome operate together to perform physiological, developmental, and pathological responses in the living cell. Single-cell experiments represent a valuable work-bench to address the issue. Although many advances have been achieved in the past decades investigating cell populations as a whole by molecular biology techniques, one major new paradigm in gene expression is that it varies stochastically among different individual cells, thus implying that population analysis may be misleading 1, 2, 3. In fact, DNA, many mRNA molecules and some enzymes exist in low copy numbers and participate in stochastic reaction events that are often at non-equilibrium steady state, thus causing gene expression to vary from cell to cell even within very homogenous populations 4, 5, 6. Furthermore, when dealing with intact tissues or primary cultures from animals, cells of different type or differentiation stage coexist and interact within the same experimental sample. A textbook example is represented by neurons where gene expression events are related to the individual cell type and depend on complex signaling within neuronal networks [7]. On this basis, it is clear that a thorough understanding of gene expression mechanisms and cell behavior requires efficient methods for the investigation of molecular processes in single cells rather than in a population where averaging hides stochasticity and individual cell properties.

A comprehensive single-cell experimental approach should combine the possibility to both detect and manipulate genetic events in the target cell. Many recent studies have been focused on the development of new techniques for monitoring gene expression. Part of them draw upon the unique capabilities of molecular probes to track the fate of mRNAs and proteins in time-lapse fluorescence microscopy experiments 8, 9, 10, 11, 12, thus offering the possibility to investigate the dynamics of gene expression down to a single molecule resolution. Other approaches rely, instead, on the application to single cells of molecular biology and biochemical techniques such as the reverse transcription-polymerase chain reaction (RT-PCR) 6, 13, DNA-microarray analysis [14], and electrophoresis-based molecules separation [15]. On the contrary, the molecular tools to manipulate gene expression in living cells are in continuous development: DNA plasmid vectors to express exogenous genes; antisense molecules, such as oligodeoxyribonucleotides (ODNs), ribozymes, DNAzymes, and, most recently, interfering RNAs and peptide nucleic acids (PNAs), to knockdown gene expression [33]; decoy ODNs, to control nuclear transcription factors [16]; zinc finger nucleases (ZFNs) 17, 18, 19 or specifically engineered ODNs with locked nucleic acids [48], for site directed mutagenesis, and gene editing. Despite the availability of this rich molecular toolbox to monitor and manipulate gene expression in the living cell, single-cell delivery remains a major problem to tackle. Traditional delivery techniques, such as chemical or viral transfection, suffer, in this respect, major constraints: (i) they act on entire cell populations and not on individual cells; (ii) they work only with certain cell types and molecules. An alternative is represented by electroporation: the application of large voltages to the cells generates transient pores in the plasma membrane, thereby allowing the permeation of a large variety of molecules, from small ions to genetic constructs and proteins, to a broad range of cells, from cell lines to primary cells [20]. Although the method has the advantage to operate with different molecules and cell types, it is traditionally applied only to populations of cells. In fact, electroporation is usually carried out in cuvettes, thus sharing with other bulk-scale methods the averaging limitation and requiring cell harvesting and re-suspension for cells that are growing in adhesion, which destroys original cell–cell connections. Even in the case of ‘in situ’ electroporation, an advanced implementation where adherent cells are electroporated on millimeter-scale indium-tin-oxide (ITO)-coated glass slides [21], only large cell populations can be addressed. The development of new versatile single-cell molecular delivery approaches represents, therefore, a very useful and fundamental challenge.

Efficient single-cell delivery was achieved by using carbon fiber microelectrodes or glass micropipettes capable to produce membrane electroporation [22]. Such devices, however, are difficult to handle and can be hardly implemented into large-scale systems. To overcome these limitations, silicon microchips for single-cell electroporation have been recently developed, for example using a sort of miniaturized electroporation chamber or a microfluidic channel [22], thus paving the way to large-scale integration. In turn, these systems rely on a rather complex fabrication process and are working only on cells in suspension, which is a serious drawback for the treatment of most mammalian cells in culture that are typically growing in adhesion on a solid substrate.

In this work, we report on a new single-cell delivery technology based on a silicon chip featuring an array of cell-sized planar microelectrodes. The cells, growing in adhesion on the chip surface as on a conventional glass coverslip, were individually electroporated by imposing on the single microelectrodes appropriate voltage transients. In this way, impermeant molecules present in the extracellular medium were delivered to pre-selected target cells. Coupled to a PC driven control system capable to arbitrarily design the voltage protocols and to address independently the single electrodes, the chip was allowing a tunable delivery of molecules to individual cells with a fine control over the cell membrane permeability to the different compounds. Moreover, molecules could be delivered to cells located at different sites within the same culture (space-resolved), at arbitrarily chosen time points (time-resolved), and even sequentially to the same cell (serial delivery). The technique was successfully tested for delivery to CHO-K1 cells of several nucleic acids, ranging from ODNs to siRNAs and DNA plasmid vectors, thereby achieving the single-cell expression and silencing of fluorescent reporters. Thus, the approach is proposed as a new easy-to-use platform to perform space and time-resolved gene expression experiments in single cultured mammalian cells.

Section snippets

Single-cell electroporation array and control system

Figure 1a shows schematically the building blocks of the experimental setup. The key element of the system was a chip featuring an array of 60 cell-sized microelectrodes enabling single-cell electroporation. This silicon microchip was fabricated following a custom design and using the adaptation of the backend of a CMOS (complementary metal–oxide–semiconductor) process by ITC-irst, Povo-Trento, Italy. The array of 60 circular microelectrodes was integrated on the die. The metal lines connecting

Single-cell electroporation on cell-sized microelectrode

A single-cell growing in adhesion on a cell-sized microelectrode represents a well-defined system where voltages applied to the microelectrode and their transfer to the cell membrane can be precisely controlled. The tight adhesion contact between a cell and a solid semiconductor substrate, with a distance that is typically in the order of a few tens of nanometers, ensures a strong electrical coupling between the single cell and the microelectrode [25]. As ‘proof of principle’, we investigated

Discussion

We have developed a new on-chip single-cell electroporation technology enabling the delivery of impermeant compounds to pre-selected individual mammalian cells in culture. Controlled by an easy-to-use PC driven control system, a single-cell electroporation array based on cell-sized planar microelectrodes was used to perform a tunable, space- and time-resolved transfection of individual CHO-K1 cells. Target cells were directly addressed while growing in adhesion on the solid substrate of the

Acknowledgements

We thank Prof. C. Reggiani for critical reading of the manuscript and Prof. P. Fromherz for discussion and suggestions. This work was supported partly by a PRIN research grant from the “Ministero della Ricerca Scientifica e Tecnologica” of Italy. The manuscript is dedicated to the memory of Dr. Silvano Fumero, who has given this work a constant support, with enthusiasm and determination.

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