Na Li*, Pak Kin Wong*, James Lin**, and Chih-Ming Ho*. *Department of Mechanical & Aerospace Engineering,. University of California, Los Angeles, USA.
SINGLE CELL TRANSFECTION DYNAMICS IN A MICROFLUIDIC SYSTEM Na Li*, Pak Kin Wong*, James Lin**, and Chih-Ming Ho* *Department of Mechanical & Aerospace Engineering, University of California, Los Angeles, USA **Department of Pediatrics, David Geffen School of Medicine, University of California, Los Angeles, USA
Abstract In this study, molecular beacons have been successfully transfected into living cells with high efficiency (80%~90%) inside a microfluidic system. This will enable a sensitive and specific method to monitor gene expression dynamics in single living cells. Fluorescence conjugated transfection reagent was adopted to investigate the transfection dynamics of molecular beacons. The development of microfluidic systems has facilitated real-time monitoring of individual cells, which revealed the transfection dynamics of molecular beacons at the single cell level. Keywords: molecular beacon, transfection, microfluidics, gene expression dynamics, single cell 1. Introduction Study of cell responses to various stimuli has important implications for biology and medicine. Compared to traditional cell culture systems, microfluidic cell culture systems have demonstrated advantages in generating the desired physical or chemical stimulations in space and time. However, their potentials to facilitate real-time screening of cellular responses have not been fully exploited. In this paper, we propose to use molecular beacons to monitor real-time gene expression dynamics of individual living cells inside a microfluidic cell culture system. Molecular beacons (Figure 1) have a stem-and-loop structure and undergo a spontaneous fluorogenic conformational change upon hybridization to the complementary nucleic acid target [1]. They have been shown to be extremely specific and sensitive in various applications. In this paper, transfection of molecular beacons was successfully demonstrated with high transfection efficiency inside a microfluidic system. Furthermore, single cell transfection dynamics were also investigated.
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Figure 1. Schematic of specific RNA/DNA detection with molecular beacon 2. Experimental We developed a microfluidic cell culture system (Figure 2). A PDMS microchannel (30um high, 1mm wide) was loaded into a heating chamber that is mounted on an inverted optical microscope. The entire setup was inside of a vertical clean bench, thus minimizing the possibility of contamination. Adoption of CO2-independent cell culture
media (Invitrogen) has eliminated the need to control the CO2 level inside the close chamber. Human embryonic kidney (HEK) 293T and Hela cells have been successfully cultured for up to one week in this system. (a)
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Figure 2. Microfluidic cell culture system. (a) Setup (b) Scheme of microfluidic device (c) Assembly of microfluidic device
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3. Results and discussion A molecular beacon that targets human beta-actin mRNA, was designed based on previous literature [2] and characterized. JetPEITM (Polyplus transfection) was used to deliver the molecular beacon into 293T cells. As shown in Figure 3a, using concentrations that were optimal for 96-well plates resulted in lower transfection efficiency in the microchannel. This decrease may be attributable to the much greater surface-to-volume ratio at the scale of the microchannel, compared to a traditional cell culture system. After optimization for the microfluidic cell culture system, a high transfection efficiency of 80%~90% has been achieved (Figure 3b and c). For our molecular beacon, 2’-O-methyl modification was used to improve resistance to cellular ribonucleases. As shown in Figure 3d, transfection efficiency and mean signal-to-noise ratio (SNR) were similar between day I and day II, which suggests that the 2’-O-Me modified molecular beacons remains intact after 2 days. (b) (c) (a) 100 u-channel (d)1.5 1.0
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Figure 3. Transfection of beta-actin molecular beacons into 293T cells. (a) Comparison between microchannel and 96-well plate. Bright field (b) and fluorescence (c) images illustrating high transfection efficiency in microchannel at optimized condition. (d) Stability of molecular beacon inside of living cells In the dynamics experiments, jetPEI-Fluor, a red fluorescent derivative of jetPEI, was used to transfect the green fluorescent labeled molecular beacons. This allows us to track jetPEI™/molecular beacon complexes. Data from a typical cell (Figure 4a) showed that the molecular beacon SNR began to increase significantly after 1 hour and
approached steady state in around 2hrs, whereas the SNR of jetPEI had minimal variation during the same time period. This implies that the jetPEI™/molecular beacon complex is taken up by cells within 2hr and that the additional time for the molecular beacon to reach the steady state may be attributed to other processes like the escape of jetPEI™/molecular beacon complex from the endosome, dissociation of the complex, and hybridization of molecular beacon to its specific mRNA target. This observation agrees with the proposed transfection mechanism in the literature [3]. Data from additional cells were plotted in Figure 4 b. Data of two individual cells and the average from the group of cells were highlighted. It was noted that the dynamics were similar between different cells. However, there was a relatively large difference in characteristic time to onset of transfection. This demonstrates that single cell studies provide information on cellular dynamics that may not be captured in aggregate data. (a)8 (b) 1.2 Molecular beacon
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Figure 4. Transfection dynamics of molecular beacons with rhodamine –labeled jetPEI in a single cell (a) and group of individual cells (b). 4.
Conclusions The paper demonstrated that transfection of molecular beacons can be achieved inside a microfluidic system with a high efficiency. Results of single cell transfection dynamics showed that a microfluidic system can potentially be a powerful tool to explore the distinct mechanisms of various cellular internalization methods. Acknowledgements The work is supported by CMISE through NASA URETI program and NIH NIDCR. References 1. S. Tyagi and F.R. Kramer FR, Molecular beacons: probes that fluoresce upon hybridization, Nat Biotechnol, 14, pp. 303-308, (1996). 2. D.L. Sokol, X. Zhang X, P. Lu, and A.M. Gewirtz, Real time detection of DNA.RNA hybridization in living cells, Proc Natl Acad Sci USA, 95(20), pp. 11538-11543, (1998). 3. O. Boussif, F. Lezoualc'h, M.A. Zanta, M.D. Mergny, D. Scherman, B. Demeneix, and J.P. Behr, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc Natl Acad Sci USA, 92(16), pp. 7297-7301, (1995).
