The original GFP-Pem1 was a gift from Dr. Lei Li. This work was supported by grants from the NIH and the Ellison Medical Foundation to V.G. and A.S.

In this protocol we describe the method for analysis of DNA DSB repair with chromosomally integrated reporter constructs1,2 where DSBs are induced in vivo by transient expression a rare cutting endonuclease I-SceI3. The integrated assay provides the advantage of analyzing DSB repair within the chromosomal context. However, this protocol requires prolonged cell passaging, which may be problematic when working with primary cells.
An alternative approach is an extrachromosomal assay where reporter constructs are digested in vitro with I-SceI or HindIII endonucleases and then transfected into cells as linear DNA. The exctrachromosomal assay protocol4 is similar to the integrated assay described below with the following modifications. For the extrachromosomal assay omit the integration of reporter constructs and instead co-transfect the cells with 0.5 &mu;g of NHEJ reporter plasmid linearized in vitro with I-SceI or HindIII and 0.1 &mu;g of pDsRed2-N1 as transfection control. For the HR assay co-transfect 2 &mu;g HR reporter plasmid linearized with I-SceI or HindIII and 0.1 &mu;g pDsRed2-N1. Analyze the cells by FACS three days after transfection. The extrachromosomal assay allows for the analysis of DSB repair in primary isolates, avoiding extensive cell passaging, and facilitating the analysis of multiple cell lines.
1. Reporter Constructs for NHEJ and HR
The NHEJ reporter cassette5 contains a GFP gene with an engineered 3 kb intron from the Pem1 gene (GFP-Pem1). The Pem1 intron contains an adenoviral exon flanked by recognition sequences for HindIII and I-SceI endonucleases (Figure 1a) for induction of DSBs. The I-SceI sites are in inverted orientation. I-SceI has a nonpalindromic recognition sequence, hence two inverted sites generate incompatible DNA ends (Figure 1c). Incompatible DNA ends best mimic the naturally occurring DSBs. The intact NHEJ cassette is GFP negative as the adenoviral exon disrupts the GFP ORF. Upon induction of DSBs by I-SceI, the adenoviral exon is removed and NHEJ restores function of the GFP gene (Figure 2). The unique feature of this NHEJ reporter is that it can detect a wide spectrum of NHEJ events since the intron can tolerate deletions and insertions.
The HR reporter cassette1 (Figure 1b) is also based on the GFP-Pem1. In the HR cassette, the first exon of the GFP-Pem1 contains a 22 bp deletion combined with the insertion of three restriction sites, I-SceI/HindIII/I-SceI. The deletion ensures that GFP cannot be reconstituted by an NHEJ event. The two I-SceI sites are in inverted orientation, so that I-SceI digestion leaves incompatible ends (Figure 1c). The first copy of GFP-Pem1 is followed by a promoter-less/ATG-less first exon and intron of GFP-Pem1. The intact construct is GFP-negative. Upon induction of a DSB by I-SceI digestion the functional GFP gene is reconstituted by intramolecular or intermolecular gene conversion between the two mutated copies of the first GFP-Pem1 exon. Since the second copy of the GFP gene is lacking the first ATG codon and the second exon, crossing over or single-strand annealing will not restore the GFP activity. This design allows for the exclusive detection of gene conversion, which is the predominant HR pathway in mammalian cells (Figure 3).
2. Integration of the Reporter Constructs into the Genome
<ol>
 <li>Make a high quality preparation of NHEJ or HR reporter plasmid. For best results use Qiagen Endo Free Kit. Measure plasmid concentration and purity by absorbance at 260/280 nm.</li>
 <li>Linearize 10 &mu;g of the reporter plasmid by digesting with 50 U of NheI in 50 &mu;l (total volume of the reaction) for 6 hours in 37&deg;C incubator (do not use the dry block, to prevent evaporation). Purify DNA from the digestion solution with Qiagen QiaexII Kit, elute DNA into 20 &mu;l of 10 mM Tris-HCl pH 8.0. Measure DNA concentration and purity by absorbance at 260/280 nm. Usually, you will lose 20-30% of DNA during purification. Confirm the yield and digestion by running a small aliquot (2 &mu;l) on a gel, along with an undigested reporter construct. The purified linearized reporter construct can be stored at 20&deg;C.</li>
 <li>In preparation for transfection, bring the cells to the best growing condition. If starting from a frozen vial, split the cells twice before transfection. If starting from a plate of confluent cells, split them once.</li>
 <li>Grow the cells to 70-80% confluence (usually two days, if plated 5x105 cells/100 mm plate, for normal human fibroblasts). It is critical for the best transfection efficiency to bring the cells into logarithmic growth. Actively growing culture contains cells in M stage (seen as rounded cells attached to the surface). </li>
 <li>Transfect the cells using Amaxa Nucleofector following manufacturer's recommendations. For fibroblasts use NHDF solution and program U20. For transfection, harvest the cells from two 100 mm plates (~2x106 cells). It is critical not to over trypsinize the cells. Stop trypsinization as soon as 80% of cells detached from the plate. Resuspend the cells in NHDF solution. Cells are sensitive to NHDF solution, therefore, minimize the incubation time and mix the cells gently. Transfect the cells with 0.5 &mu;g of linearized reporter construct. These transfection conditions, when used with normal human fibroblasts result in the integration of a single copy of a reporter construct for the majority of integrants. </li>
 <li>Select the cells with chromosomally integrated reporter constructs by adding 1 mg/mL geneticin (G418) 24h after transfection. Continue selection for 7-10 days.</li>
 <li>Pick the G418 resistant colonies (you can pick individual colonies or pool the resistant clones). Expand the culture to approximately five 100 mm plates. Freeze three plates for future use and expand remaining two plates to the desired cell number (usually ten 100 mm plates are sufficient for five transfections). </li>
 <li> When the cells reach 70-80% confluence (two days after plating 5x105 cells per 100 mm plate for fibroblasts), the cells are ready to be transfected with I-SceI expressing plasmid.</li>
</ol>
3. Induction of DSB by Transient Expression of I-SceI Endonuclease
<ol>
 <li>Transfect ~2x106 cells (two plates) of logarithmically growing culture with a mixture of 5 &mu;g I-SceI expressing plasmid and 0.1 &mu;g pDsRed2-N1 (Clontech) as a transfection efficiency control using Amaxa Nucleofector (use NHDF solution and U20 program for fibroblasts). </li>
 <li>At the same time prepare three calibration controls for FACS by trasfecting ~2x106 cells with (i) 5 &mu;g EGFP-expressing plasmid; (ii) 5 &mu;g pDsRed2-N1; (iii) 5 &mu;g of a control plasmid that does not express a fluorescent protein (we are using a plasmid encoding HPRT minigene).</li>
 <li>Grow the cells for four days to provide sufficient time for expression of GFP and DsRed proteins.</li>
 <li> (Optional) Three days after transfection verify the efficiency of transfection and expression of GFP and DsRed proteins by fluorescent inverted microscope with filters for detection of GFP and DsRed.</li>
</ol>
4. Analysis of the GFP+ and DsRed+ Cells by Flow Cytometry
<ol>
 <li>Four days after transfection harvest I-SceI transfected cells and the control cells. Resuspend the cells in 500 &mu;L of PBS and transfer cells to the FACS tubes. At this stage it is critical to harvest all the cells and have cells of round shape. To achieve this, it is recommended to use a longer trypsinization time or a stronger trypsin. Confirm that 99% of cells are detached from the plate and have round shape by observing the cells under microscope. Keep the cells on ice protected from light (cover ice bucket with foil). It is possible to store the cells on ice for a few hours. For the best results analyze the cells immediately after harvesting.</li>
 <li>Calibrate FACS with GFP, DsRed, and the negative controls. Adjust voltage and color compensation in order to include all the fluorescent cells in the analysis (Figure 4). Keep in mind that the fluorescent intensity of the experimental samples will be lower than that of the controls.</li>
 <li>Analyze I-SceI transfected cells by FACS. We count at least 20,000 cells for each treatment. To prevent potential interference between GFP and DsRed fluorescence it is recommended to keep the percent of GFP+ or DsRed+ cells below 25%. If the percentage of DsRed+ or GFP+ cells is higher reduce the amount of pDsRed2-N1 or I-SceI plasmid. In our experiments we only needed to adjust the amount of pDsRed2-N1 plasmid, but not of the I-SceI plasmid.</li>
 <li>The percent of GFP+ cells corresponds to the efficiency of DNA DSB repair and the percent of DsRed+ cells indicates the efficiency of transfection. Calculate the relative efficiency of DNA DSB repair as the ratio of GFP+ cells to DsRed+ cells.</li>
 <li> The repaired end junctions may be sequenced to test the accuracy of repair. The reporter constructs contain E.coli origin of replication and kanamycin resistance genes (Figure 1). This enables rescuing the constructs by digesting genomic DNA with EcoRI enzyme, recircularization, and transformation into competent E.coli cells. The rescued plasmids are then analyzed by restriction digestion and sequencing.</li>
</ol>
5. Representative Results
Typical FACS results of the control transfections and of NHEJ and HR experiments are shown in Figures 4 and 5. Fluorescent intensity and the number of fluorescent cells will be lower in the I-SceI transfected cells containing integrated NHEJ and HR constructs (Figure 5) compared to the controls (Figure 4) due to the difference in the copy number of GFP and DsRed constructs in these cells. Each cell in the experiment contains only one copy of the integrated reporter construct, thus successful repair events will reconstitute the GFP gene in a fraction of the cells. Transfection with 0.1 &mu;g pDsRed2-N1 in human fibroblasts delivers approximately 1 plasmid copy per cell.
The efficiency of NHEJ and HR is calculated as a ratio of GFP+/DsRed+ cells. NHEJ is a more efficient process than HR in human cells2. In human fibroblasts the NHEJ efficiency is typically 0.6-1.3, and HR efficiency is 0.05-0.3. Since DSB efficiency is measured as a ratio GFP+/DsRed+ cells variations in the mixture of I-SceI and DsRed2-N1 plasmids may affect the result. The plasmid quality may also affect the results by changing transfection efficiency. Therefore, to obtain consistent measurements, it is important to use the same plasmid mix within one experiment. Furthermore, the DSB repair efficiency is affected by cell type, cell cycle phase, and chromosomal location of the integrated constructs.
 <img src="/files/ftp_upload/2002/2002fig1.jpg" alt="Figure 1" /> 
 Figure 1. Reporter constructs for the analysis of DNA DSB repair by NHEJ (A) and HR (B). (C) Incompatible DNA ends generated by digestion of two inverted I-SceI sites. SD, splice donor; SA, splice acceptor; shaded squares, polyadenylation sites; Neo/Kana, single ORF controlled by two promoters: SV40 conferring neomycin resistance in mammalian cells; and &beta;-lactamase conferring kanamicyn resistance in E. coli; Ori, E. Coli pUC origin of replication.
<img src="/files/ftp_upload/2002/2002fig2.jpg" alt="Figure 2" /> Figure 2. Reporter construct for the analysis of NHEJ. In this construct the GFP gene is inactive; however upon induction of a DSB and successful NHEJ the construct becomes GFP+. Shown below is a repair product of this reporter by NHEJ.
<img src="/files/ftp_upload/2002/2002fig3.jpg" alt="Figure 3" /> Figure 3. Reporter construct for the analysis of HR by gene conversion. Upon induction of the DSBs by I-SceI, HR by gene conversion (GC) reconstitutes an active GFP gene. Shown below are repair products of this reporter by the major DNA repair pathways: GC, crossing-over (X-
over), single strand annealing (SSA), and NHEJ. X-over repair product is shown for intramolecular recombination, exramolecular recombination will give the same product but located on one chromosome. Genes expressing active GFP protein (GFP+) are indicated by green color.
<img src="/files/ftp_upload/2002/2002fig4.jpg" alt="Figure 4" /> Figure 4. Calibration of the parameters for FACS analysis. (A) Fibroblasts transfected with the pHPRT plasmid, used as a negative control for excluding auto fluorescent cells. (B) Fibroblasts transfected with pEGFP plasmid, used for setting the gating for GFP+ cells (green area). (C) Fibroblasts transfected with pDsRed2-N1 plasmid, used for setting the gating for DsRed+ cells (red area).
<img src="/files/ftp_upload/2002/2002fig5.jpg" alt="Figure 5" /> Figure 5. Typical results of the analysis of NHEJ (A) and HR (B) in normal human fibroblasts with chromosomally integrated reporter constructs.

<ol>
	<li>Mao, Z., Seluanov, A., Jiang, Y., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRF2+is+required+for+repair+of+nontelomeric+DNA+double-strand+breaks+by+homologous+recombination.">TRF2 is required for repair of nontelomeric DNA double-strand breaks by homologous recombination.</a> Proc Natl Acad Sci U S A. 104, 13068-13073 (2007).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+nonhomologous+end+joining+and+homologous+recombination+in+human+cells.">Comparison of nonhomologous end joining and homologous recombination in human cells.</a> DNA Repair (Amst). 7, 1765-1771 (2008).</li><li>Rouet, P., Smih, F., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+site-specific+endonuclease+stimulates+homologous+recombination+in+mammalian+cells.">Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells.</a> Proc Natl Acad Sci U S A. 91, 6064-6068 (1994).</li><li>Mao, Z., Jiang, Y., Xiang, L., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+homologous+recombination,+but+not+by+nonhomologous+end+joining,+is+elevated+in+breast+cancer+cells.">DNA repair by homologous recombination, but not by nonhomologous end joining, is elevated in breast cancer cells.</a> Neoplasia. 11, 683-691 (2009).</li><li>Seluanov, A., Mittelman, D., Pereira-Smith, O. M., Wilson, J. H., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+end+joining+becomes+less+efficient+and+more+error-prone+during+cellular+senescence.">DNA end joining becomes less efficient and more error-prone during cellular senescence.</a> Proc Natl Acad Sci U S A. 101, 7624-7629 (2004).</li><li>Mao, Z., Bozzella, M., Seluanov, A., Gorbunova, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+by+nonhomologous+end+joining+and+homologous+recombination+during+cell+cycle+in+human+cells.">DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells.</a> Cell Cycle. 7, 2902-296 (2008).</li></ol>

No conflicts of interest declared.

The fluorescent NHEJ and HR reporter assays provide a quantitative way for separately measuring each DSB repair pathway in vivo. The assays are very sensitive, as FACS can reliably detect 10 GFP+ cells in 20,000 cells. The assays may be adapted for measuring repair events in "real time" by detecting the appearance of GFP+ cells within minutes or hours after induction of DSBs2. Furthermore, the analysis of GFP+ cells does not rely on additional cell proliferation, allowing DSB repair to be analyzed a...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>EndoFree Plasmid Maxi kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12362</td>
<td>&nbsp;</td>
<tr>
<td>Qiaex II Gel Extraction Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>20021</td>
<td>&nbsp;</td>
<tr>
<td>Amaxa Nucleofector</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>AAD-1001</td>
<td>&nbsp;</td>
<tr>
<td>Geneticin (G418)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11811-031</td>
<td>&nbsp;</td>
<tr>
<td>pDsRed2-N1</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>632406</td>
<td>&nbsp;</td>
<tr>
<td>Round bottom tubes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352058</td>
<td>FACS tubes</td>
</tr>		</tbody>
		</table>
		</div>

analysis of dna double-strand break (dsb) repair in mammalian cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Watch this Scientific Journal Video about Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells at JoVE.com

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

analysis-of-dna-double-strand-break-dsb-repair-in-mammalian-cells

Research

JoVE Journal

Biology

31.5K 조회수.  University of Rochester. 이 문서는 GFP 기반의 형광을 설명합니다 생체내에 별도로 상동 재조합을 계량하고 nonhomologous 포유 동물 세포에 입사 최종 assays.

비디오: Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

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 ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.