We thank Lynette Henkel for her excellent support in performing the flow cytometric analysis. We thank Dr. Don Weldon and Dr. Victor Koong (Temecula, CA) for critical reading of the manuscript and helpful comments. This work was supported by grants from the German Research Foundation, KR-3770-7/1 and KR-3770-9/1 (M. K.), the Deutsche Stiftung f&#252;r Herzforschung, F/37/11 (M. K.), Dr. Rusche Research Grant 2014 from the Deutsche Stiftung f&#252;r Herzforschung and Deutsche Gesellschaft f&#252;r Thorax-, Herz- und Gef&#228;sschirurgie (M.-A. D.), and the Deutsches Zentrum f&#252;r Herz- und Kreislaufforschung S&#228;ule B Projekt, DZHK B 13-050A and DZHK B 14-013SE (M. K.).

1. Preparation of Media, Reagents and Cell Culture Conditions
<ol>
	<li>Human HEK 293 embryonic kidney cells
	<ol>
		<li>Culture HEK 293 cells (CRL-1573, ATCC) in DMEM/Ham&#8217;s F-12 medium supplemented with 5% FCS, L-glutamine (2 mM), and penicillin (100 U/ml)/ streptomycin (100 &#181;g/ml).</li>
		<li>To passage the cells, wash cells once with PBS, add 0.05% Trypsin/EDTA and incubate for 3 min at 37 &#176;C and 5% CO2. Transfer cells into a 15 ml tube, centrifuge for 5 min at 1,200 rpm (300 x g) and transfer approximately 5x105 cells into a fresh T25 culture flask.</li>
	</ol>
	</li>
	<li>Murine embryonic fibroblasts (MEFs)
	<ol>
		<li>Add 1 ml of a 0.1% gelatin solution to individual 6-wells and incubate at room temperature (RT) for 1 hr. Aspirate liquid and store at RT until use.</li>
		<li>Culture MEFs in DMEM supplemented with 10% FCS, L-glutamine (2 mM), L-glucose (4.5 g/l), sodium pyruvate (1 mM), and penicillin (100 U/ml)/streptomycin (100 &#181;g/ml) on gelatin-coated 6-well plates.</li>
		<li>Distribute MEFs into 6-well plates and let them grow until confluence. Add mitomycin (5 &#181;g/ml) for 2.5 hr to arrest cells. Aspirate medium, wash cells twice with complete MEF medium and add 3 ml MEF medium to each 6-well. These cells serve as a feeder-layer for murine and porcine iPS cells.</li>
	</ol>
	</li>
	<li>Culture of murine ES and iPS cells
	<ol>
		<li>To prepare mouse ES culture medium, supplement DMEM medium with 15% FCS, L-glutamine (2 mM), L-glucose (4.5 g/l), MEM non-essential amino acids (1X), &#946;-mercaptoethanol (0.1 mM) and leukemia inhibitory factor (1.000 U/ml).</li>
		<li>Rinse 6-wells with growth-arrested MEFs once with serum-free DMEM, add 2 ml mouse ES medium and murine iPS cells (20% of the cells of a confluent dish). Every day wash cells once with serum-free DMEM and add 2 ml fresh mouse ES medium.</li>
		<li>To passage the cells, wash each well once with serum-free DMEM, add 0.25% Trypsin/EDTA and incubate for 3 min at 37 &#176;C and 5% CO2. Transfer cells into a 15 ml tube, centrifuge for 5 min at 1,200 rpm (300 x g), resuspend cells in 500 &#181;l mouse ES medium and add 100 &#181;l of the suspension to 2 ml mouse ES medium in a fresh 6-well with a MEF feeder-layer.</li>
	</ol>
	</li>
	<li>Culture of pig iPS cells
	<ol>
		<li>To prepare pig iPS medium, supplement DMEM/Ham&#8217;s F-12 medium with 20% knockout serum, L-glutamine (2 mM), MEM non-essential amino acids (1X), &#946;-mercaptoethanol (0.1 mM), and penicillin (100 U/ml)/streptomycin (100 &#181;g/ml). Filter through a 0.22 &#181;M filter and store at 4 &#176;C for 2 to 3 weeks. Upon first use, add bFGF (10 ng/ml final concentration).</li>
		<li>Rinse 6-wells with growth-arrested MEFs once with serum-free DMEM/Ham&#8217;s F-12 and add 1.5 ml pig iPS medium and porcine iPS cells (10% of the cells of a confluent dish). Every day wash cells once with serum-free DMEM/Ham&#8217;s F-12 and add 1.5 ml fresh pig iPS medium.</li>
		<li>To prepare dissociation buffer add 2.5% trypsin, collagenase IV (10 mg/ml), knockout serum (20%) and CaCl2 (1 mM) to Ca2+-free PBS. Wash cells twice with PBS and incubate with dissociation buffer for 5 min at 37 &#176;C and 5% CO2. Transfer cells into a 15 ml tube, centrifuge for 5 min at 1,200 rpm (300 x g).</li>
		<li>Resuspend porcine iPS cells in 500 &#181;l fresh pig iPS medium and add 50 &#181;l of the suspension to 1.5 ml pig iPS medium in a fresh 6-well with a MEF feeder-layer. Every day wash cells once with serum-free DMEM/Ham&#8217;s F-12 and add 1.5 ml fresh pig iPS medium.</li>
	</ol>
	</li>
	<li>Feeder-free culture of human iPS cells
	<ol>
		<li>Thaw the original vial containing the basement membrane preparation at 4 &#176;C and dilute with ice-cold serum-free DMEM/Ham&#8217;s F-12 to 8 mg/ml, aliquot and store at -20 &#176;C until further use.</li>
		<li>Thaw an aliquot of the basement membrane preparation at 4 &#176;C and dilute with ice-cold serum-free DMEM/Ham&#8217;s F12 (90 &#181;g/ml). Pipet 1 ml of this solution into 3 cm culture dishes and swirl gently to ensure that the whole surface is covered. Seal dishes with parafilm and store at 4 &#176;C for up to one week. Prior to the use for cell culture incubate dishes for 30 min at 37 &#176;C or 3 hr at RT to allow polymerization of the basement membrane preparation.</li>
		<li>Aspirate medium from dishes coated with the basement membrane preparation and wash twice with serum-free DMEM/Ham&#8217;s F-12. Add 1.5 ml E8 medium to the culture dish and human iPS cells (10% of the cells of a confluent dish). Every day wash cells once with serum-free DMEM/Ham&#8217;s F-12 and add 1.5 ml fresh E8 medium.</li>
		<li>To passage the cells, rinse dishes twice with PBS and add 1 ml PBS/0.5 mM EDTA. Incubate the dishes for 5 min at RT. Monitor the cells under a microscope. Note: When the colonies start to round up and appear to have holes they are ready for transfer.</li>
		<li>Aspirate the liquid, add 1.5 ml E8 medium and detach cells by gently pipetting up and down. Transfer cells to a 15 ml tube, centrifuge for 5 min at 1,200 rpm (300 x g), resuspend in 500 &#181;l E8 medium and add 50 &#181;l of the suspension to 1.5 ml E8 medium in a fresh culture dish coated with the basement membrane preparation.</li>
	</ol>
	</li>
</ol>
2. Design of Target-sequences Across Species Borders
<ol>
	<li>Select reference human, murine and porcine cDNA sequences of the target genes (GAPDH, NANOG, GDF3) and perform a CLUSTAL multi-sequence alignment analysis. 
	Note: This will indicate the sequences homologous between species.
	<ol>
		<li>Use the following settings for multiple alignment: gap penalty (fixed) 10, gap penalty (varying) 5, transition weighting 0, gap separation penalty range 8, % identity for delay 40, maximum number of iterations to perform 3.</li>
	</ol>
	</li>
	<li>Submit desired target sequences to the manufacturer (see Table of Materials) for probe design and manufacturing. 
	Note: The manufacturer will then confirm compatibility of desired sequence with the technology. If desired sequence is not compatible, the manufacturer will suggest alternative sequences.
	<ol>
		<li>Ensure that homologous regions which can be used as potential capture strand sequences have a length of 27 bp. 
		Note: It is advisable to order several nanoparticles with different capture strands for a selected target gene as only the real cellular experiment will give a definite proof of the functionality of an individual sequence. Therefore, two different sequences for GAPDH and GDF3 were evaluated while three different nanoparticles were tested to evaluate NANOG expression. Their location and the exact sequences have been published elsewhere 17. The manufacturer commercially manufactures the nanoparticles with desired sequences and provides them in lyophilized format.</li>
	</ol>
	</li>
</ol>
3. Reconstitution of Nanoparticles and Addition to Cell Cultures
<ol>
	<li>Store lyophilized nanoparticles upon receipt at 4 &#176;C in the dark.</li>
	<li>Reconstitute nanoparticles by addition of 50 &#181;l double distilled water which gives a final concentration of 100 nM. Once reconstituted store nanoparticles at RT in the dark. Note: Under this condition they are stable for at least one year. Storage of reconstituted samples at 4 &#176;C may adversely affect their stability.</li>
	<li>Pre-dilute the nanoparticle stock solution with PBS to a concentration of 2 (HEK 293 cells) or 8 nM (iPS and ES cells) on the day of application. Note: This concentration is 20-fold higher than the final concentration in the culture dish. In ES cells and iPS cells, 400 pM of nanoparticles induced a fluorescent signal easily detectable under the microscope. For HEK 293 cells, a concentration of 100 pM is sufficient.</li>
	<li>Pipet 12.5 &#181;l of prediluted nanoparticles to a 24-well containing 237.5 &#181;l complete cell culture medium. For wells of different sizes, adjust the volumes accordingly. Swirl the plate carefully to ensure equal distribution of the nanoparticles. Incubate the cells overnight (O/N) at 37 &#176;C and 5% CO2 in a humidified atmosphere.</li>
	<li>The next day analyze cell cultures for gene-specific Cy3 fluorescence with an excitation wavelength of 546 nm (emission 575 &#8211; 640 nm) under a fluorescence microscope. Note: The period required to obtain a sufficiently strong fluorescence may depend on the cell type and the target gene. In these experiments, this was seen between 16 and 20 hr after the application of the probe.</li>
</ol>
4. Identification of Cy3 Positive Cell Populations by Flow Cytometry
<ol>
	<li>Grow HEK 293 or murine ES cells using the conditions specified in section 1.1 or 1.3, respectively. One day prior to the FACS analysis add GAPDH-specific nanoparticles at different concentrations to HEK 293 cells (see Figure 6A) and at 400 pM to murine ES cells. Incubate the cells O/N at 37 &#176;C and 5% CO2 in a humidified atmosphere.</li>
	<li>Analyze cell cultures for gene-specific Cy3-induced fluorescence under a fluorescence microscope.</li>
	<li>Detach HEK 293 cells as specified under point 1.1.2. Centrifuge, transfer into a 1.5 ml tube and resuspend in 200 &#181;l ice-cold PBS/0.5% BSA/2 mM EDTA (FACS buffer). Keep cells dark on ice until flow cytometric analysis.</li>
	<li>Detach murine ES cells as specified under point 1.3.3. Centrifuge, transfer into a 1.5 ml tube and resuspend in 200 &#181;l ice-cold PBS/0.5% BSA/2 mM EDTA. Keep cells dark on ice until flow cytometric analysis.</li>
	<li>Press the cells through a 30 &#181;M filter to prevent cell aggregation and add propidium iodide (2 &#181;g/ml final concentration) 2 min prior to the FACS analysis to exclude dead cells.</li>
	<li>To analyze Cy3-positive cells first set hierarchical gates on the FSC-A/SSC-A subset, the FSC-H/FSC-W-subset and the SSC-H/SSC-W subset. Use the forward and the side scatter to exclude cell debris and cells that are not scattered. Exclude dead cells by their propidium iodide staining displaying PE against PE-Texas Red. Perform the final sort gate on Cy3-PE positive cells (Figure 4).</li>
</ol>

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H.L. has received the nanoparticles from EMD Millipore Corporation free of charge. All other authors have nothing to disclose.

The present work describes the use of fluorescence labeled nanoparticles which allow the reliable evaluation of intracellular gene expression directly in live cells from different mammalian species with diverse histogenetic origin under a fluorescence microscope. This approach has a couple of advantages compared to other published methods. First of all the researcher is not limited with respect to either the target gene or cell type. All antibody based detection methods are restricted to a single epitope. Although fluorescent molecular beacons can also be designed for a broad spectrum of genes, this method requires the dissociation of cells and subsequent nucleofection 15. In contrast, the application of gene-specific nanoparticles does not require any manipulation of the target cell which engulfs the nanoparticles actively by endocytosis. During subsequent passaging the gold particles gradually leave the cells and the culture can be used in downstream experiments.
Nevertheless, the technology also still has some limitations. Some markers are obviously not detectable due to their carbohydrate nature such as SSEA-1 or TRA-1-81 20. At present, a large array of nanoparticles is commercially available. These particles have been pretested for their functionality in cellular experiments. The objective of the present work was to design customized probes which can be used across species borders. When following such an approach or when designing a probe for a novel target gene one has to be aware that the functionality of an in silico predicted probe needs to be thoroughly evaluated in vitro. The NANOG-SF3 probe which showed a 100% homology to the sequence homology did not work at all while NANOG-SF1 with one mismatched base in the murine and porcine sequence produced a nice fluorescence signal. Another question refers to the efficient uptake of the nanoparticles. The particles enter the cells by endocytosis, a process which is influenced by the size of the nanoparticles 21, the cell type and the differentiation status 22 and the cellular capability to perform phago- and macropinocytosis 23. The optimum concentration to obtain a satisfying fluorescence signal has to be tested for each individual application or cell line. To that end, the first experiment should always be the application of the uptake control to evaluate the compatibility of the probes within the cell types and culture conditions before moving on to target-specific detection of gene expression.
A critical point to obtain reliable results when applying this protocol to a novel cell line or cell population is the inclusion of proper controls. The ever fluorescing uptake control will provide a hint which concentrations of nanoparticles will induce a sufficient fluorescent signal in the target cell population. The fluorescence must be evaluated the next day as the signal will fade with time 17. At the same time the scramble control without a counterpart in the eukaryotic genome applied at an identical concentration must induce a negligible background signal. As a third control it is advisable to use a nanoparticle specific for a housekeeping gene (e.g. GAPDH, &#946;-Actin). These nanoparticles serve as a positive control that the approach can be used to detect specific gene expression in the selected target cell population. If these controls turn out satisfactory one can expect to obtain reliable specific fluorescent signals using nanoparticles specific for other target genes. The concentration of the applied nanoparticles may also be critical as they have been shown to down-regulate the target sequence 24. However, this effect requires much higher concentrations ( 5 nM) than the concentrations used in the experiments presented here (400 pM). At this concentration the nanoflares do not affect target gene mRNA or protein levels and concentrations as high as 2 nM do not impair the proliferation of HEK293 and murine ES cells 17. In addition, a whole genome expression analysis did not reveal any significant changes in gene expression 25. Therefore, at concentrations up to 1 nM the nanoflares should not exhibit any important adverse effects.
A very promising application of this technology is the possibility to label any given cell population which is marked by a specific gene. Recently, gene-specific nanoparticles have successfully been used to selectively stain live ventricular myocytes 26, subpopulations of melanoma cells 27, monocytes 28 and cerebellar cell cultures 29. Such fluorescence-marked cell populations can be sorted and purified by flow cytometry as live cells. In the field of oncology it is imaginable to isolate live metastatic tumor cells in animal models based on their expression of CEA, most valuable for the search of potential metastasis promoting genes or target structures. We have recently shown that truly reprogrammed murine iPS colonies can be identified in situ with Nanog-specific nanoparticles based on the detected fluorescence intensity 17. Therefore, the identification of truly reprogrammed iPS colonies could be performed on high throughput platforms, which use robotic fluorescence detection and picking devices to identify the most promising colonies. This technology appears to be suitable in many research areas to select specific cellular subpopulations and it seems possible to apply the nanoparticles in high throughput platforms. In conclusion, this protocol represents a novel approach using fluorescence labeled nanoparticles which allows the detection of intracellular gene expression directly in live cells. This technology may be a valuable tool to purify, expand and further characterize rare cellular subpopulations in many research areas.

In 2006, a novel approach was described to reprogram somatic cells into a pluripotent state. After prescreening of an array of potential reprogramming factors murine fibroblasts could be successfully converted to so-called induced pluripotent stem cells (iPS) by retroviral transduction and overexpression of four transcription factors (Oct4, Sox2, Klf4, c-Myc) 1. Subsequently this technique has effectively been reproduced using somatic cells of different mammalian species including humans 2, monkeys 3, rats 4, dogs 5, sheep 6 and pigs 7. In addition, modified protocols omitting or replacing the potentially oncogenic transcription factor c-MYC have been published 8, 9.
Regardless of the initial reprogramming approach the true pluripotency of growing colonies has to be confirmed, a laborious and time-consuming task. A major drawback of the majority of these analyses is that they cannot be performed on live cells. Established pluripotency factors such as OCT4, SOX2, NANOG or REX1 represent transcription factors located in the nucleus and their detection requires fixation of the cells prior to immunohistochemistry or cell lysis for RT-PCR analysis 10 - 12. Thereafter, these cells are lost for future experiments requiring replicate cultures of each colony.
Thus, a technology to analyze pluripotency on live cells is highly desirable. Several approaches can indeed be performed directly on live cells using antibodies directed against membrane-based antigens (e.g. TRA-1-60, SSEA4) 12, fluorescent reported dyes to detect alkaline phosphatase 13 or small molecules which specifically label embryonic stem (ES) and iPS cells 14. However, the antibodies might nevertheless adversely affect the cells and each method is restricted to a single pluripotency marker. Finally, fluorescent molecular beacons, dual-antisense oligonucleotides with hairpin structures, which allow live staining of cells, have been described 15. Although this approach may be applied to many genes, the technique requires nucleofection of a single cell suspension, which is not applicable to growing iPS colonies.
A couple of years ago gene-specific nanoparticles have been described to analyze survivin expression in human SKBR3 breast cancer cells 16. This manuscript describes a novel innovative approach to detecting pluripotency markers in live ES and iPS cells through the use of gold nanoparticle conjugates. These nanoparticles consist of a central gold particle with a coupled capture strand carrying the complementary RNA sequence and a Cy3-labeled reporter strand. The fluorescent signal of the reporter strand is quenched by the central gold particle. In addition, appropriate nanoparticles serve as negative and positive controls, respectively (Figure 1). For an application the nanoparticles are simply added to the culture medium (Figure 2) and enter the cells via endocytosis (Figure 3). If the target RNA is expressed it displaces the reporter strand which then emits a fluorescent signal. This method does not require manipulation of the target cell and the expression of the target gene can be monitored optically under a fluorescence microscope directly in live cells. Furthermore, the technology can be applied to any desired target gene across species in the case of iPS cells and thus may be employed in many different research areas. Finally, flow cytometric analyses allow the identification and enrichment of Cy3 positive cells.

<table><tbody><tr><td>DMEM/Ham&#039;s F12 medium with stable glutamine</td><td>Biochrome GmbH</td><td>FG4815</td><td></td></tr><tr><td>Fetal calf serum</td><td>Life Technologies</td><td>SV30160.03</td><td></td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>Millipore</td><td>A2213</td><td></td></tr><tr><td>Sodium pyruvate (100x)</td><td>Life Technologies</td><td>11360-039</td><td></td></tr><tr><td>DMEM high glucose with stable glutamine</td><td>Biochrome GmbH</td><td>FG0435</td><td></td></tr><tr><td>Mitomycin C</td><td>Roche</td><td>10 107 409 001</td><td>prevents cell division of MEFs</td></tr><tr><td>MEM non-essential amino acids (100x)</td><td>Life Technologies</td><td>11140-050</td><td></td></tr><tr><td>b-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>Leukemia inhibitory factor (10&lt;sup&gt;7&lt;/sup&gt; U/ml)</td><td>Millipore</td><td>ESG1107</td><td>growth factor for murine ES and iPS cells, prevents differentiation</td></tr><tr><td>0.25% Trypsin/EDTA (1x)</td><td>Life Technologies</td><td>25200-056</td><td>detaches adherent cell cultures</td></tr><tr><td>Knockout serum</td><td>Invitrogen</td><td>10828-028</td><td>supplement for human and porcine iPS cell culture</td></tr><tr><td>bFGF</td><td>Peprotech</td><td>100-18B</td><td>growth factor for human and porcine iPS cells</td></tr><tr><td>Collagenase IV</td><td>Worthington</td><td>LS004188</td><td>dissociation of pig iPS cells</td></tr><tr><td>Matrigel</td><td>Corning</td><td>354277</td><td>effective surface for feeder-free culture of human iPS cells, referred to as basement preparation membrane in the manuscript</td></tr><tr><td>E8 medium</td><td>Stem Cell Technologies</td><td>05940</td><td>defined medium for feeder-free culture of human iPS cells on Matrigel</td></tr><tr><td>EDTA 0.5 M</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>SmartFlare Probes</td><td>EMD Millipore Corporation</td><td>customized item</td><td>detection of intracellular gene expression in live cells, referred to as nanoparticles in the manuscript</td></tr><tr><td>Fluorescence microscope</td><td>Zeiss</td><td>Axiovert 200M</td><td></td></tr><tr><td>HeraCell 150i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo Scientific</td><td>51026282</td><td></td></tr><tr><td>Gelatine (from porcine skin)</td><td>Sigma</td><td>G6144</td><td>enhances attachment of MEFs to surface</td></tr><tr><td>Propidium iodide (1 mg/ml)</td><td>Sigma</td><td>P4864</td><td>labels dead cells in flow cytometric analyses</td></tr><tr><td>BD FACS Aria Illu</td><td>Becton Dickinson</td><td>A4544</td><td>referred to as device 1 in Figure 4</td></tr><tr><td>Navios</td><td>Beckman Coulter</td><td>775213</td><td>referred to as device 2 in Figure 4</td></tr><tr><td>FloJo vX 10.0.7r2</td><td>Treestar</td><td></td><td>software for analysis of flow cytometry data</td></tr></tbody></table>

detection of intracellular gene expression in live cells of murine, human and porcine origin using fluorescence-labeled nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

In a first attempt to verify and establish the intracellular detection of specific gene expression in live cells using nanoparticles we decided to conduct pilot experiments using a constitutively expressed house-keeping gene (GAPDH) in human HEK 293 cells. The concentration of nanoparticles used in these experiments was chosen according to the recommendation of the manufacturer. The addition of a GAPDH-specific probe to the culture medium at a 100 pM final concentration induced a clear-cut Cy3-fluorescence in these cells which was visible under a fluorescence microscope after O/N incubation (Figure 5A). Two controls were included in these experiments to substantiate the result. One control comprises of a so-called &#8220;scramble&#8221; nanoparticle where the reporter strand does not have a matching sequence in mammalian or eukaryotic cells. The &#8220;scramble&#8221; nanoparticle determines the unspecific background fluorescence due to possible probe degradation or incomplete quenching of the fluorescence. The addition of the scramble probe produced an only marginal fluorescent signal, which is negligible (Figure 5B). A permanently fluorescing &#8220;uptake&#8221; control helps to determine the ability of cells to incorporate the nanoparticles by endocytosis which may vary between different cell types. These positive control particles produced a bright fluorescent signal in HEK 293 cells (Figure 5C). Thus, the feasibility of the technology to record a specific fluorescent signal of an intracellular gene with a negligible background signal was confirmed. It must be mentioned that the difference between the background signal and the specific fluorescence decreases over time. The background increases due to probe degradation or incomplete quenching while the specific fluorescence fades gradually (Figure 5D). The same GAPDH-specific nanoparticles have been tested in primary fibroblast cultures of murine, porcine and ovine origin 17. These cells produce a much higher background signal than HEK 293 cells while the target-specific fluorescence is rather weak, possibly due to the relatively low proliferative capacity of these cultures.
These pilot experiments prompted us to use this technology for the detection of pluripotency gene expression in iPS derived from different species. For this purpose NANOG and GDF3 were analyzed, both of which are known to be expressed in pluripotent cells 18. At first iPS cells derived from tail-tip fibroblasts of an Nkx2.5 cardiac enhancer transgenic mouse line 19 were investigated. For both genes a strong fluorescent signal was obtained (Figure 6A) and a negligible Cy3-fluorescence of the scramble control (data not shown). Similar results were obtained on human and porcine iPS cells (Figure 6B and C). Importantly, the fluorescence signal was restricted to the iPS colonies and did not label the fibroblasts used as a feeder layer for mouse and porcine iPS (see arrows in Figure 6A and C). The analysis of NANOG gene expression also revealed that not every sequence predicted in silico to be &#8220;good&#8221; finally is functional. One of three designs of a NANOG-specific nanoparticles induced almost no fluorescent signal comparable to that of the scramble control despite a 100% sequence match in murine iPS cells (Figure 6D). A similar result was obtained on iPS cells of human and porcine origin with this probe (data not shown). Thus, nanoparticles can be used to detect pluripotency gene expression across species borders while the functionality of every single designed probe needs to be evaluated beforehand.
The promising results of the in situ labeling of live cells in cell culture dishes prompted us to analyze gene expression quantitatively by flow cytometry. To this end GAPDH-specific nanoparticles were added at graded concentrations to HEK 293 monolayers in order to compare the labeling efficiency within the target cell population. As assessed by fluorescent microscopy an enhanced Cy3-induced fluorescence was seen with the lowest concentration compared to cells which did not receive the nanoparticles. Increasing concentrations of the nanoparticles induced a clearly concentration dependent fluorescence of HEK 293 cells in situ (Figure 7A). Subjecting these cells to a flow cytometric analysis confirmed the concentration dependent labeling efficiency. The frequency of Cy3 positive cells increased significantly (more than sevenfold) when the highest concentration of nanoparticles was applied (Figure 7B). In further experiments ES cells from the Nkx2.5 cardiac enhancer transgenic mouse line 19 were used to analyze the expression of pluripotency genes by flow cytometry. Pilot experiments with 400 pM of the positive uptake control yielded clearly Cy3 positive live ES colonies under the microscope and flow cytometry detected almost 40% of Cy3 positive cells (Figure 7C). In contrast, comparable to untreated cells approximately 0.1% of cells treated with the scramble stained positive (data not shown). Similarly the addition of nanoparticles specific for Gapdh, Nanog and Gdf3 induced a distinct fluorescence signal in individual colonies in the cell culture dish. The frequency of Cy3 positive cells for Gapdh as determined by flow cytometry was comparable to that seen for both pluripotency genes (Figure 7C). This result is expectable as Nanog and Gdf3 are also thought to be expressed constitutively in pluripotent ES cells. Thus, these data clearly indicate that cells expressing a particular gene can be identified by flow cytometric analyses, qualitatively and by their relative amount.
<img alt="Figure 1" src="/files/ftp_upload/53268/53268fig1.jpg" /> 
Figure 1: Schematic Representation of the Structure of the Nanoparticles. (A) Structure of a gene-specific nanoparticle. (B) Scramble nanoparticle (negative control). (C) Uptake nanoparticle (positive control). Adapted from Lahm et al. 17, reprinted with permission from Wiley.
<img alt="Figure 2" src="/files/ftp_upload/53268/53268fig2.jpg" /> 
Figure 2: Application of Nanoparticles to Cell Cultures. A properly diluted solution of nanoparticles is pipetted simply into the culture medium. After O/N incubation gene-specific fluorescent cells are visible under a fluorescent microscope.
<img alt="Figure 3" src="/files/ftp_upload/53268/53268fig3.jpg" /> 
Figure 3: Active Uptake of Nanoparticles by Cells via Endocytosis. (A) Cells actively engulf the nanoparticles by endocytosis. (B) Target mRNA binds to the capture strand and displaces the fluorescing reporter.
<img alt="Figure 4" src="/files/ftp_upload/53268/53268fig4.jpg" /> 
Figure 4: Gating Strategy for Flow Cytometry. (A) Strategy for HEK 293 cells. (B) Strategy for murine ES cells. Data were acquired using device 1 for HEK 293 cells or device 2 for murine ES cells and were subsequently analyzed by a software tool (see Table of Materials).
<img alt="Figure 5" src="/files/ftp_upload/53268/53268fig5.jpg" /> 
Figure 5: Analysis of GAPDH-expression in HEK 293 Cells. A GAPDH-specific nanoparticle was added to the cells at 100 pM (final concentration) and fluorescence was recorded after O/N incubation. (A)GAPDH-specific nanoparticle. (B) Scramble (negative control). (C) Uptake (positive control). (D) Kinetics of scramble and uptake control. Fluorescence was determined at the indicated time-points after addition of GAPDH-specific nanoparticles. Scale bars represent 50 &#181;M.
<img alt="Figure 6" src="/files/ftp_upload/53268/53268fig6.jpg" /> 
Figure 6: Analysis of Pluripotency Gene Expression in iPS Cells of Different Species. Nanoparticles specific for NANOG or GDF3 were added to the cells at 400 pM (final concentration) and fluorescence was recorded after O/N incubation. (A) Murine iPS cells. (B) Human iPS cells. (C) Porcine iPS cells. Arrows in (A) and (C) designate unlabeled fibroblast feeder-layer cells. (D) Fluorescence in murine iPS cells after addition of the NANOG-design SF3 nanoparticle. Scale bars represent 50 &#181;M.
<img alt="Figure 7" src="/files/ftp_upload/53268/53268fig7.jpg" /> 
Figure 7: Nanoparticle Labeled Live Cells can be Identified by Flow Cytometry. (A) Microscopic view of Cy3 positive HEK 293 cells after addition of different concentrations of nanoparticles. (B) Frequency of Cy3 positive cells determined by flow cytometry. (C) Live staining of murine ES cell colonies and determination of the frequency of Cy3 positive cells. Nanoparticles were added at a final concentration of 400 pM. Scale bars represent 50 &#181;M.

Watch this Scientific Journal Video about Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles at JoVE.com

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

detection-intracellular-gene-expression-live-cells-murine-human

Research

JoVE Journal

Biology

11.0K Views.  Technische Universität München. This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species. 

Video: Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Cancer Research

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Immunology and Infection

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

Bioengineering

Live Imaging and Quantification of Viral Infection in K18 hACE2 Transgenic Mice Using Reporter-Expressing Recombinant SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

This protocol describes the dynamics of viral infections using luciferase- and fluorescence-expressing recombinant (r)SARS-CoV-2 and an in vivo imaging systems (IVIS) in K18 hACE2 transgenic mice to overcome the need of secondary approaches required to study SARS-CoV-2 infections in vivo.

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded &#180;indefinitely&#180;. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

Since the discovery of the green fluorescent protein gene, fluorescent proteins have impacted molecular cell biology. This protocol describes how expression of distinct fluorescent proteins through genetic engineering is used for barcoding individual cells. The procedure enables tracking distinct populations in a cell mixture, which is ideal for multiplexed applications.

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

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