Name: real-time quantification of the effects of is200/is605 family-associated tnpb on transposon activity
Uploaded: 2023-01-20T13:40:00
Description: Watch this Scientific Journal Video about Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity at JoVE.com

Financial support for this research was provided by startup funds from the University of California.

1. Preparation of bacterial cultures
<ol>
	<li>Grow E. coli strain MG1655 with plasmid transposon constructs (previously described in Kim et al.8) overnight in LB with the appropriate antibiotics (25 &#181;g/mL of kanamycin, see Table of Materials) at 37 &#176;C. 
	NOTE: The sequences of the constructs used and the related sequences are available as GenBank20 accession numbers OP581959, OP581957, OP581958, OP717084, and OP71...

<ol>
	<li>Cowley, M., Oakey, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposable+elements+re-wire+and+fine-tune+the+transcriptome.">Transposable elements re-wire and fine-tune the transcriptome.</a> PLoS Genetics. 9 (1), 1003234 (2013).</li><li>Schneider, D., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+insertion+sequence+elements+during+experimental+evolution+of+bacteria.">Dynamics of insertion sequence elements during experimental evolution of bacteria.</a> Research in Microbiology. 155 (5), 319-327 (2004).</li><li>Chao, L., Vargas, C., Spear, B. B., Cox, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposable+elements+as+mutator+genes+in+evolution.">Transposable elements as mutator genes in evolution.</a> Nature. 303 (5918), 633-635 (1983).</li><li>Coufal, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1+retrotransposition+in+human+neural+progenitor+cells.">L1 retrotransposition in human neural progenitor cells.</a> Nature. 460 (7259), 1127-1131 (2009).</li><li>Kano, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L1+retrotransposition+occurs+mainly+in+embryogenesis+and+creates+somatic+mosaicism.">L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism.</a> Genes &amp; Development. 23 (11), 1303-1312 (2009).</li><li>Belancio, V. P., Deininger, P. L., Roy-Engel, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LINE+dancing+in+the+human+genome:+transposable+elements+and+disease.">LINE dancing in the human genome: transposable elements and disease.</a> Genome Medicine. 1 (10), 97 (2009).</li><li>Goodier, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrotransposition+in+tumors+and+brains.">Retrotransposition in tumors and brains.</a> Mobile DNA. 5, 11 (2014).</li><li>Kim, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+transposable+element+activity+in+individual+live+cells.">Real-time transposable element activity in individual live cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (26), 7278-7283 (2016).</li><li>Lutz, R., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+and+tight+regulation+of+transcriptional+units+in+Escherichia+coli+via+the+LacR/O,+the+TetR/O+and+AraC/I1-I2+regulatory+elements.">Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements.</a> Nucleic Acids Research. 25 (6), 1203-1210 (1997).</li><li>Calos, M. P., Miller, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+sequence+change+resulting+from+the+IQ1+mutation,+which+greatly+increases+promoter+strength.">The DNA sequence change resulting from the IQ1 mutation, which greatly increases promoter strength.</a> Molecular and General Genetics MGG. 183 (3), 559-560 (1981).</li><li>Markwardt, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+cerulean+fluorescent+protein+with+enhanced+brightness+and+reduced+reversible+photoswitching.">An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching.</a> PLoS One. 6 (3), 17896 (2011).</li><li>Nagai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nature Biotechnology. 20 (1), 87-90 (2002).</li><li>Kersulyte, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposable+element+ISHp608+of+Helicobacter+pylori:+nonrandom+geographic+distribution,+functional+organization,+and+insertion+specificity.">Transposable element ISHp608 of Helicobacter pylori: nonrandom geographic distribution, functional organization, and insertion specificity.</a> Journal of Bacteriology. 184 (4), 992-1002 (2002).</li><li>Shmakov, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+evolution+of+class+2+CRISPR-Cas+systems.">Diversity and evolution of class 2 CRISPR-Cas systems.</a> Nature Reviews Microbiology. 15 (3), 169-182 (2017).</li><li>Bao, W., Jurka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologues+of+bacterial+TnpB_IS605+are+widespread+in+diverse+eukaryotic+transposable+elements.">Homologues of bacterial TnpB_IS605 are widespread in diverse eukaryotic transposable elements.</a> Mobile DNA. 4 (1), 12 (2013).</li><li>Siguier, P., Gourbeyre, E., Chandler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+insertion+sequences:+their+genomic+impact+and+diversity.">Bacterial insertion sequences: their genomic impact and diversity.</a> FEMS Microbiology Reviews. 38 (5), 865-891 (2014).</li><li>Altae-Tran, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+widespread+IS200/IS605+transposon+family+encodes+diverse+programmable+RNA-guided+endonucleases.">The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases.</a> Science. 374 (6563), 57-65 (2021).</li><li>Karvelis, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-associated+TnpB+is+a+programmable+RNA-guided+DNA+endonuclease.">Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease.</a> Nature. 599 (7886), 692-696 (2021).</li><li>Kaur, D., Kuhlman, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IS200/IS605+family-associated+TnpB+increases+transposon+activity+and+retention.">IS200/IS605 family-associated TnpB increases transposon activity and retention.</a> bioRxiv. , (2022).</li><li>Benson, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GenBank.">GenBank.</a> Nucleic Acids Research. 41, 36-42 (2013).</li><li>Shaner, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nature Biotechnology. 22 (12), 1567-1572 (2004).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Ton-Hoang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Stranded+DNA+transposition+is+coupled+to+host+replication.">Single-Stranded DNA transposition is coupled to host replication.</a> Cell. 142 (3), 398-408 (2010).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+growth+of+Escherichia+coli.">Robust growth of Escherichia coli.</a> Current Biology. 20 (12), 1099-1103 (2010).</li></ol>

The unique method presented here for real-time imaging of transposable element activity in live cells is a sensitive assay that can directly detect transposition in live cells and in real-time and correlate this activity with the expression of accessory proteins. While the throughput is lower than can be accomplished by bulk methods, this method achieves detailed measurements of TE activity and protein expression in individual living cells.
A variety of tools and techniques can be employed to ...

Transposable elements (TEs) are genetic elements that mobilize within their host genomes by excision or catalyze copying followed by genomic reintegration. TEs exist in all domains of life, and transposition restructures the host genome, mutating coding and control regions1. This generates mutations and diversity that play an important role in evolution2,3, development4,5, and several human diseases6, including cancer7.
Using novel genetic constructs that couple aspects of transpositional activity to fluorescent reporters, our previous work described the development of an experimental system based on the bacterial TE IS608, a representative of the widespread IS200/IS605 family of TEs, that allows for the real-time visualization of transposition in individual live cells8 (Figure 1). The TE system is displayed in Figure 1A. The TE comprises the transposase coding sequence, tnpA, flanked by Left End (LE) and Right End (RE) imperfect palindromic repeats (IPs), which are the recognition and excision sites for TnpA. tnpA is expressed using the promoter PLTetO1, which is repressed by the tet repressor and is inducible with anhydrotetracycline (aTc)9. The TE splits the -10 and -35 sequences of a constitutive PlacIQ1 promoter10 for the blue reporter mCerulean311. As shown in Figure 1C, when the production of tnpA is induced, the TE can be excised, leading to promoter reconstitution. The produced cell expresses mCerulean3 and fluoresces blue. The N-terminus of TnpA is fused to the yellow reporter Venus12, allowing measurement of the TnpA levels by yellow fluorescence.
IS608 and other members of the IS200/IS605 family of transposons also typically encode a second gene of the thus far unknown function, tnpB13. The TnpB proteins are a tremendously abundant but imperfectly characterized family of nucleases encoded by several bacterial and archaeal TEs14,15, which often consist of only tnpB16. Furthermore, recent studies have renewed interest in TnpB by finding that TnpB functions as a CRISPR/Cas-like programmable RNA-guided endonuclease that will yield either dsDNA or ssDNA breaks under diverse conditions17,18. However, it remains unclear what role TnpB may play in regulating transposition. To perform real-time visualization of the effects of TnpB on IS608 transposition, a version of the transposon, including the coding region of TnpB with an N-terminal fusion to the red fluorescent protein mCherry, was created.
Complementing more detailed bulk-level studies performed by the Kuhlman lab19, it is shown here how real-time imaging of transposon activity can quantitatively reveal the impact of TnpB or any other accessory proteins on transpositional dynamics. By fusing TnpB to mCherry, the individual transpositional events are identified by blue fluorescence and correlated with expression levels of TnpA (yellow fluorescence) and TnpB (red fluorescence).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 Ton Clear Epoxy</td>
			<td>Devcon</td>
			<td>31345</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>5066</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>AX1385-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrotetracycline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>37919</td>
			<td></td>
		</tr>
		<tr>
			<td>Argon Laser</td>
			<td>Melles Griot</td>
			<td>35-IMA-840-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue Filter Cube</td>
			<td>Chroma</td>
			<td>Ex: Z457/10X, Em: ET485/30M</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Eclipse Ti-E Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Discontinued</td>
		</tr>
		<tr>
			<td>Eppendorf epTIPS Boxes and Refill Trays, Volume: 0.1 to 10 &#181;L, Length: 3.4 cm, 1.33 in., PP (Polypropylene)</td>
			<td>Eppendorf North America Biotools</td>
			<td>22491504</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf epTIPS Boxes and Refill Trays, Volume: 50 to 1000 &#181;L, Length: 7.1 cm, 2.79 in., PP (Polypropylene)</td>
			<td>Eppendorf North America Biotools</td>
			<td>22491555</td>
			<td></td>
		</tr>
		<tr>
			<td>Ferrous Sulfate Acs 500 g</td>
			<td>Fisher Scientific</td>
			<td>706834</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td></td>
			<td>Fiji (imagej.net)</td>
		</tr>
		<tr>
			<td>Fisher BioReagents&#160;LB Broth, Miller (Granulated)</td>
			<td>Fisher Scientific</td>
			<td>BP9723-2&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Cover Slide</td>
			<td>Fisher Scientific</td>
			<td>12-542B&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>1355006</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate Cert Ac</td>
			<td>Fisher Scientific</td>
			<td>XXM63SP3KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Heater</td>
			<td>World Precision Instruments</td>
			<td>96810-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>17001H</td>
			<td></td>
		</tr>
		<tr>
			<td>ProScan III Stage</td>
			<td>Prior</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Red Filter Cube</td>
			<td>Chroma</td>
			<td>Ex: ET560/40X, Em: ET645/75M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire 561 LP Laser</td>
			<td>Coherent</td>
			<td>1170412</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide, Microscope</td>
			<td>Fisher Scientific</td>
			<td>125535B</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiamine Hydrochloride</td>
			<td>Sigma-Aldrich (SIAL)</td>
			<td>T1270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-LU4 Laser Launch</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yellow Filter Cube</td>
			<td>Chroma</td>
			<td>Ex: Z514/10X, Em: ET535/30M</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time quantification of the effects of is200/is605 family-associated tnpb on transposon activity

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent reporters coupled to transposition. In particular, it demonstrates how real-time imaging can be used to assess the effects of the accessory protein TnpB on the activity of the transposable element IS608, a member of the IS200/IS605 family of transposable elements. The IS200/IS605 family of transposable elements are abundant mobile elements connected with one of the most innumerable genes found in nature, tnpB. Sequence homologies propose that the TnpB protein may be an evolutionary precursor to CRISPR/Cas9 systems. Additionally, TnpB has received renewed interest, having been shown to act as a Cas-like RNA-guided DNA endonuclease. The effects of TnpB on the transposition rates of IS608 are quantified, and it is demonstrated that the expression of TnpB of IS608 results in ~5x increased transposon activity compared to cells lacking TnpB expression.

This method of visualizing transposon activity in live cells by fluorescence microscopy, while having lower throughput than bulk fluorescence measurements, allows direct visualization of transposon activity in individual live cells. Transposon excision events result in the reconstitution of the promoter for mCerulean3 (Figure 1), allowing identification of cells undergoing transposon activity by bright blue fluorescence (Figure 2, TnpB+: Supplementary Mo...

Watch this Scientific Journal Video about Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity at JoVE.com

Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity

A protocol is outlined to perform live real-time imaging to quantify how the accessory protein TnpB affects the dynamics of transposition in individual live Escherichia coli cells.

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent ...

This protocol allows us to quantify excision statistics on a single-cell basis, as opposed to looking at bulk behavior. At this level, we can track the behavior of individual cells over time, including their TnpA and TnpB levels for cells that excise versus cells that don't, which can allow us to, for example, measure the effects of identified mutational events on growth rate. This method is a simple, low-cost way to measure live cell mutational dynamics.The general approach is applicable to any system that requires live cell fluorescent imaging. Prepare a slide by boiling M63 with 0.5%glucose and 1.5%agarose in the microwave to melt the agarose. Ensure that the medium is completely melted and well-mixed.Allow the mixture to cool to 55 degrees Celsius before adding antibiotics and inducers. Place a microscope slide on the workbench. Stack one slide perpendicular to the first.Ensure that there is a gap equal to one slide thickness between the bottom and top slide. Pipette one milliliter of the M63 agarose mixture into the gap between the slide slowly to create a small gel square. Once the gel has solidified, slide the top slide to remove it.Trim the agarose pad with a razor blade or knife. Then, pipette 2.5 microliters of the bacterial culture and put the cover slip on top. Seal the space between the slide and the cover slip with epoxy.Allow the epoxy to dry and the cells to settle onto the agarose pad for one hour at 37 degrees Celsius. Place the prepared sample on a fluorescence microscope in an environment heated and maintained at 37 degrees Celsius. For each wavelength, find the field-of-view containing minimal fluorescence.Set up a protocol to acquire images in a grid at different wavelengths and at regular time intervals. Transposon excision events result in the reconstitution of the promoter for mCerulean3, allowing the identification of cells undergoing transposon activity by bright blue fluorescence. TnpB enhances transposon excision rate.Cells expressing accessory protein TnpB experience four to five times higher levels of transposon activity compared to TnpB-negative cells. In TnpB-positive cells, cells undergoing transposon excision events have higher expression levels of mCherry TnpB than the general population. Moreover, for cells undergoing excision events, TnpB-positive cells express only marginally higher levels of venous TnpA-transposase than TnpB-negative cells, which is higher than the yellow fluorescence of the general population.The most likely place to struggle is in the preparation of the cell culture for the experiment. You should make sure cells are grown to exponential phase and that the slide is inoculated with an appropriate number of cells. Remember to watch out for bubbles when sealing the slide so the agarose gel does not dry out.

real-time-quantification-effects-is200is605-family-associated-tnpb-on

Research

JoVE Journal

Biology

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...