Name: substrate generation for endonucleases of crispr/cas systems
Uploaded: 2012-09-08T17:00:00
Duration: 11 min 53 s
Description: Watch this Scientific Journal Video about Substrate Generation for Endonucleases of CRISPR/Cas Systems at JoVE.com

The authors would like to thank Jeanette Schermuly for the preparation of recombinant T7 RNA polymerase and Norman Ebelt for assistance in preparing Figure 1. This work was funded by funds from the Deutsche Forschungsgemeinschaft (DFG FOR1680) and the Max Planck Society.

1. Generation of Long Pre-crRNA Substrates via PCR
<ol>
	<li>Design PCR primers targeting the spacer regions of a CRISPR cluster. Add the T7 RNA polymerase (T7RNAP) promoter sequence (5' -taatacgactcactata-3') to the forward primer and restriction sites for cloning the PCR product into a vector to both primers (e.g. BamHI and Hind III for pUC19, Fig. 2A). 
	Note: The T7RNAP requires a guanidine residue for proper initiation of transcription.</li>
	<li>Amplify your pre-crRNA sequence of interest from genomic DNA by PCR.</li>
	<li>Separate the PCR products by agarose gel electrophoresis and gel extract the desired band. Digest the PCR product with the restriction enzymes to create sticky ends (e.g. BamHI and HindIII, Fig. 2A). Purify your PCR product with a PCR purification kit to eliminate cleavage by-products.</li>
	<li>Set up a ligation reaction that contains T4 DNA ligase, T4 DNA ligase buffer and a 3:1 molar ratio of the cleaved PCR product and the dephosphorylated linear pUC vector with corresponding sticky ends. Incubate the mixture at 16 &deg;C overnight. Transform the ligation mixture into competent Escherichia coli DH5&alpha; cells by standard protocols and use blue white screening to identify successful ligation.</li>
	<li>Isolate plasmids from white colonies using a plasmid preparation kit. Identify positive clones by plasmid sequencing. Alternatively, colony PCR might be utilized for screening.</li>
</ol>
2. Generation of Intermediate Pre-crRNA Substrates via Annealing of DNA Oligonucleotides
<ol>
	<li>Design forward and reverse oligonucleotides with the desired CRISPR repeat/spacer sequence. The oligonucleotides contain the sequence of a T7 RNAP promoter as well as terminal restriction sites (e.g. BamHI and Hind III for pUC19). Terminate the oligonucleotides to ensure that sticky ends form after annealing (see Fig. 2B).</li>
	<li>5'-phosphorylate 1 nmol of each oligonucleotide in a separate 20&mu;l reaction containing 5 &mu;l of T4 polynucleotide kinase (PNK), 2 &mu;l of T4 PNK 10x buffer, 2 &mu;l ATP (10 mM). Incubate each sample for 1 hour at 37 &deg;C.</li>
	<li>Hybridize the two phosphorylated oligonucleotides. Combine 1&mu;l of the phosphorylated forward oligo mixture (from 2.2.), 1 &mu;l of the phosphorylated reverse oligo mixture (from 2.2.), 1 &mu;l of T4 DNA ligase 10x buffer in a 10 &mu;l reaction. Incubate the samples for 5 min at 95 &deg;C on a heating block or in boiling water, turn off the heat source and let the mixture cool down to room temperature (~2-3 hours). 
	Note: In this critical step, the slow cooling process favors the annealing of the two oligonucleotides compared to the formation of structures within each single oligonucleotide.</li>
	<li>Ligate 4 &mu;l of the hybridization mix, 1&mu;l of digested and dephosphorylated pUC vector (0.1 &mu;g/&mu;l) with T4 DNA ligase, T4 DNA ligase 10x buffer and 10 mM ATP in a 20 &mu;l ligation mixture. Incubate the sample at 16 &deg;C overnight.</li>
	<li>Transform the ligated plasmids into competent Escherichia coli DH5&alpha; cells by standard protocols and utilize blue white screening. Isolate plasmids and identify positive clones by digestion (to screen for inserts of the desired size) and subsequent plasmid sequencing.</li>
</ol>
3. Generation of Short Cas RNA Substrates via Custom RNA Oligonucleotide Synthesis
Design short Cas RNA substrates (e.g. single repeat sequences, Fig. 2C) and utilize custom RNA oligonucleotide synthesis facilities.
Note: The inclusion of a deoxyribonucleotide at a specified position of an RNA oligonucleotide (Fig. 2C) can be used to pinpoint the site of RNA cleavage.
4. In vitro T7 RNA Polymerase Transcription
<ol>
	<li>Isolate plasmids with your designed construct (from 1.9. or 2.7.) using a maxiprep plasmid purification kit.</li>
	<li>Linearize the plasmid with the restriction enzyme that cleaves downstream of the cloned fragment (e.g. HindIII). Ensure complete digestion. 
	Note: If a divergent defined 3' terminus of the RNA transcript is desired, the designed construct should contain an additional specific restriction site for "run-off" transcription upstream of the HindIII sequence.</li>
	<li>Purify the linearized plasmid by phenol:chloroform (1:1) extraction and ethanol precipitation. Recover the nucleic acids by resuspending the pellet in DEPC treated sterile water.</li>
	<li>Set up an in vitro T7 RNAP run off transcription mixture that contains 40 mM Hepes/KOH (pH 8), 22 mM MgCl2, 5 mM dithiothreitol, 1 mM spermidine, 4 mM of each nucleoside triphosphate (ATP, CTP, GTP, UTP), 40-100 &mu;g/ml of digested plasmid and 0.1 mg/ml T7 RNAP in DEPC treated water. Incubate for 3 hours at 37 &deg;C.</li>
	<li>Analyze the obtained RNA transcripts on a denaturing 8 M urea 12% polyacrylamide gel (Fig. 3A). The RNA transcripts can be purified via Mono Q anion exchange chromatography13 and recovered by ethanol precipitation of the RNA fractions and resuspension of the pellet in DEPC treated sterile water. For future use, store the RNA at -80 &deg;C.</li>
</ol>
5. Cas6 Endonuclease Assay
<ol>
	<li>Set up a 20 &mu;l in vitro T7 RNAP run off transcription mixture (see 4.4) that contains a reduced amount of 2 mM ATP and is complemented with 2.5 &mu;l &alpha;-[32P]-ATP (10 mCi/ml, 5000 Ci/mmol). Purify reaction products via gel extraction from a denaturing 8 M urea 12% polyacrylamide gel. Visualize bands by autoradiography.</li>
	<li>Produce and purify the desired recombinant Cas proteins. In this example, Cas6 from Clostridium thermocellum was purified via heat precipitation and Ni-NTA chromatography.</li>
	<li>Set up an endonuclease assay reaction (e.g. for Clostridium thermocellum Cas6, the reaction mixture contains 20 mM Hepes (KOH pH8), 250 mM KCl, 2 mM MgCl2, 1 mM DTT, 12,000 cpm RNA substrate and 1 &mu;M enzyme and was incubated at 37 &deg;C for 30 min).</li>
	<li>Load 5 &mu;l of the reaction mixture (+ 10 &mu;l RNA loading buffer containing 95% formamide) on a 8 M urea 12% polyacrylamide gel. Visualize the cleavage products after electrophoresis by autoradiography.</li>
</ol>
6. Representative Results
An example of RNA substrates for the analysis of Cas endonuclease activity is shown in Figure 3A. An aliquot of 5 &mu;l of an analytical 100 &mu;l in vitro transcription reaction were loaded. Please note that the efficiency of RNA production varies between different constructs. Some factors that were observed to influence the amount of obtained RNA are (i) the initial sequence following the +1G required for transcription initiation, (ii) the possibility of RNA structure formation during transcription and (iii) the choice of the restriction site for the generation of the run-off cleavage position.
The investigation of RNA endonuclease activity requires both highly purified recombinant Cas proteins (Fig. 3B) and proper negative controls. Ideally, this negative control sample differs as little as possible from the investigated Cas endonuclease reaction. This can be achieved by incubation of the RNA with reaction buffer and cell-lysate without Cas expression (and following the identical purification procedure). An ideal negative control is the addition of a deoxyribonucleotide at the proposed cleavage site. In Figure 3C, the cleavage of a 5' terminal labeled repeat sequence is shown for Clostridium thermocellum Cas6. Under identical conditions, this repeat is not a substrate anymore when a deoxyribonucleotide is introduced at position -9. This method also provides information about the cleavage site. Finally, a long internally labeled pre-crRNA is cleaved by Cas6 and two cleavage fragments are observed.
<img alt="Figure 1" src="/files/ftp_upload/4277/4277fig1.jpg" /> 
 Figure 1. Schematic overview of CRISPR/Cas activity. The overview follows the insertion of a viral DNA sequence (protospacer) into the CRISPR cluster (adaptation), the transcription and processing of the CRISPR array into small crRNAs by a Cas6 endonuclease, the uptake of crRNAs into the Cascade complex and the interference of a repeated viral attack based on complementarity between crRNA and protospacer. Protospacer adjacent motifs (PAM) mark viral protospacer sequences.
<img alt="Figure 2" src="/files/ftp_upload/4277/4277fig2.jpg" /> 
 Figure 2. Generation of RNA substrates for Cas proteins. The scheme shows the workflow for generating (A) long pre-crRNA substrates, (B) intermediate pre-crRNA substrates and (C) short Cas RNA substrates for pre-crRNA production. Example sequences are presented for the CRISPR array of Clostridium thermocellum. <a href="https://www.jove.com/files/ftp_upload/4277/4277fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4277/4277fig3.jpg" /> 
 Figure 3. Cas6 endonuclease assay. A.) Toluidine blue stained polyacrylamide gel of a custom-designed RNA oligonucleotide (250 pmol) and two in vitro RNA transcripts (5 &mu;l of a typical 100 &mu;l reaction). B.) SDS-PAGE gel of a Cas6 preparation (80 pmol) from Clostridium thermocellum after heat precipitation at 50&deg;C for 1 hour and Ni-NTA chromatography. C.) Detection of endonucleolytic Cas6 activity for 5' terminal labeled repeat sequences and pre-crRNA in vitro transcripts. The introduction of a dNTP at position -9 abolishes Cas6 cleavage for a short Cas RNA substrate (S, Fig. 2C). A 5' terminal 8 nt tag is also generated for crRNA maturation from long pre-crRNA substrates (L, Fig. 2A). The bands were separated on a denaturing 8 M urea 12% polyacrylamide gel and visualized by autoradiography.

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No conflicts of interest declared.

The presented methods enable the generation of Cas endonuclease substrates of different size ranges and with varying freedom in sequence design. The most straight-forward approach for the generation of synthetic RNA oligonucleotide substrates is limited to short RNA designs due to increasing costs and technical limitations in creating longer RNA oligomers. While successful RNA synthesis has been reported for unmodified RNA oligomers of over 100 nucleotides length, the practical and economical maximum for custom RNA synthesis lies below 40 nucleotides. However, any given sequence can be synthesized and the targeted introduction of modified nucleotides (e.g. deoxyribonucleotides) can be utilized to analyze cleavage sites in detail. Longer pre-cRNAs should be generated via in vitro transcription.
The annealing of oligonucleotides that contain a T7 RNA polymerase promoter allows for the production of intermediate length pre-crRNAs. The maximum length of routinely and economically synthesized DNA oligonucleotides is just above 150 nucleotides and represents the maximum of synthetic pre-crRNAs via this method. The assembly of several annealed DNA oligonucleotide pairs that form sticky ends with each other can extend this maximal length but necessitates increasing challenges in the cloning of the construct. The main advantage of this method is the capability to generate pre-crRNA constructs (and therefore Cas endonuclease substrates) with any desired sequence. This allows the testing of synthetic crRNA designs.
Finally, larger pre-crRNAs can be obtained from PCR amplificates of entire genomic CRISPR elements or fractions thereof. Alterations to the in vitro transcription template plasmid can be introduced via site-directed mutagenesis for the generation of pre-crRNA variants. These constructs can be used to analyze the global endonucleolytic cleavage pattern within an entire pre-crRNA.
The pioneer work for the production of unmodified RNA via T7 RNA polymerase in vitro transcription was based on transfer RNAs 14 and brome mosaic virus RNA 15. The consensus T7 RNA polymerase promoter consists of a recognition domain (-17 through -5) and an initiation domain (-4 through +6) with transcription initiation at an essential guanosine +1 16. Positions downstream of +1 can be varied which allows for the transcription of almost any desired RNA sequence. The presented methods for generating in vitro run-off transcription templates allow for in vitro synthesis of complete pre-crRNAs that match genomic CRISPR regions or synthetic pre-crRNA variants. The transcripts are generated with a 5'-terminal triphosphate present unless the transcription reaction is primed with GMP to obtain 5' monophosphate termini 14. Such termini are required if the transcripts are to be labeled with T4 polynucleotide kinase and &gamma;-[32P]-ATP. Cleavage activity of Cas6 and Cas6-like enzymes generates crRNA that contain 5'-hydroxyl and a 2',3'-cyclic phosphate termini. These RNAs can then be analyzed for recognition by the Cascade complex.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Antarctic Phosphatase</td>
 <td>NEB</td>
 <td>M0289L </td>
 <td></td>
 </tr>
 <tr>
 <td>in vitro Transcription Grade Ultrapure Nucleotide Triphosphates</td>
 <td>Jena Bioscience</td>
 <td>NU-1010 - NU-1013</td>
 <td></td>
 </tr>
 <tr>
 <td>Liquid chromatography, ktapurifier 100</td>
 <td>GE Healthcare</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>DNA oligonucleotides</td>
 <td>MWG Operon</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>RNA oligonucleotides</td>
 <td>MWG Operon</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Gel Extraction Kit</td>
 <td>QIAGEN</td>
 <td>28704 </td>
 <td></td>
 </tr>
 <tr>
 <td>PCR purification Kit</td>
 <td>QIAGEN</td>
 <td>28104</td>
 <td></td>
 </tr>
 <tr>
 <td>Spin Miniprep Kit</td>
 <td>QIAGEN</td>
 <td>27106</td>
 <td></td>
 </tr>
 <tr>
 <td>Plasmid Maxi Kit</td>
 <td>QIAGEN</td>
 <td>12163</td>
 <td></td>
 </tr>
 <tr>
 <td>BamHI</td>
 <td>NEB</td>
 <td>R0136L </td>
 <td></td>
 </tr>
 <tr>
 <td>HindIII</td>
 <td>NEB</td>
 <td>R0104L </td>
 <td></td>
 </tr>
 <tr>
 <td>T4 DNA Ligase</td>
 <td>NEB</td>
 <td>M0202S </td>
 <td></td>
 </tr>
 <tr>
 <td>T4 Polynucleotide Kinase</td>
 <td>Ambion</td>
 <td>AM2310</td>
 <td></td>
 </tr>
 <tr>
 <td>T7 RNA Polymerase</td>
 <td>own preparation</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Deoxynucleotide Solution Mix</td>
 <td>NEB</td>
 <td>N0447L </td>
 <td></td>
 </tr>
 <tr>
 <td>Mini-PROTEAN Tetra Electrophoresis System</td>
 <td>Bio-Rad</td>
 <td>165-8001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Storm 840 Scanner</td>
 <td>GE Healthcare</td>
 <td>163723 </td>
 <td></td>
 </tr>
 <tr>
 <td>Storage Phosphorscreen</td>
 <td>Molecular Dynamics</td>
 <td>63-0034-81 </td>
 <td></td>
 </tr>
 <tr>
 <td>Mono Q 5/50 GL</td>
 <td>GE Healthcare</td>
 <td>17-5166-01 </td>
 <td></td>
 </tr>
 <tr>
 <td>Phusion High-Fidelity DNA Polymerase</td>
 <td>NEB</td>
 <td>M0530L </td>
 <td>use the GC buffer</td>
 </tr>
 <tr>
 <td>Toluidine Blue</td>
 <td>Sigma</td>
 <td>T3260</td>
 <td></td>
 </tr>
 <tr>
 <td>pUC19</td>
 <td>NEB</td>
 <td>N3041S </td>
 <td></td>
 </tr>
 <tr>
 <td>&gamma;-[32P]-ATP, &alpha;-[32P]-ATP </td>
 <td>Hartmann Analytic</td>
 <td>SRP-401, SRP-307</td>
 <td></td>
 </tr>
 </tbody>
</table>

substrate generation for endonucleases of crispr/cas systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

Watch this Scientific Journal Video about Substrate Generation for Endonucleases of CRISPR/Cas Systems at JoVE.com

Substrate Generation for Endonucleases of CRISPR/Cas Systems

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

substrate-generation-for-endonucleases-of-crisprcas-systems

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The dissection of the stomatogastric nervous system (STNS) from the Jonah crab (Cancer borealis) is done in two parts; the gross and fine dissection. In the gross dissection the entire stomach is dissected from the crab. During the fine dissection the STNS is extracted from the stomach using a dissection microscope and micro-dissection tools (see figure 1). The STNS includes the STG, the oesophageal ganglion (OG), and the commissural ganglia (CoG) as well as the nerves that innervate the stomach muscles. Here, we show how to perform a complete dissection of the STNS in preparation for an electrophysiology experiment where the cells in the STG would be recorded from intracellularly and the peripheral nerves would be used for extracellular recordings. The proper technique for finding the desired nerves is shown as well as our technique of desheathing the ganglion to reveal the somata and neuropil.

Cancer Borealis Stomatogastric Nervous System Dissection

The stomatogastric nervous system (STNS) of the Jonah crab (C. borealis) can be used for electrophysiology, immunohistochemistry, and cell culture studies. The STNS extraction is done in two parts: the gross and fine dissection.

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...