The HCC4017 and HBEC30KT cell lines were kindly provided by Dr. John Minna&#8217;s lab. This work was supported by the Cecil H. and Ida Green funds to Dr. Li Zhang.

CAUTION: While working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Dispose off all waste following local radiation safety guidelines.
1. Preparation of Cells
<ol>
	<li>Seed cells in 3.5 cm plates such that the confluency reaches 80%-90% on the day of assay.
	<ol>
		<li>Note that seeding confluency for cells depends upon the cell type and their growth rate. When treating cells with a reagent for a certain number of days, seed cells so that the confluency is 80%-90% on the day of heme measurement. If the number of cells obtained from 3.5 cm plates is insufficient, use 6 cm plates with same amount of radioactive ALA as in the next step. 
		Note: Use of individual plates is recommended because it is easier to handle individual plates.</li>
	</ol>
	</li>
	<li>A day before heme measurement, add 0.1-0.3 &#181;Ci [14C] 5-ALA and cold ALA to reach a final concentration of 20 &#181;M of total ALA to each culture dish in a work area fit for working with radioactive materials and put the plates back into the incubator. Ideally, incubate with radioactive ALA for 16 hr. 
	Note: Radiolabeled 5-ALA required may vary between cell lines. A radioactive count per minute (cpm) of at least 100 is required to minimize the error associated with counting. It is advisable to determine a minimum amount of [14C] 5-ALA required for the cell lines under study. For most experiments 0.1-0.3 &#181;Ci is sufficient to get a good measurement. Addition of cold ALA is optional.</li>
</ol>
2. Heme Extraction
<ol>
	<li>Place the cell culture dishes on ice and take them to the area fit for radioactivity work.
	<ol>
		<li>Perform the whole extraction and evaporation process in an explosion-proof hood. Because the stored ether can explode, use the ether in small bottles and evaporate any remaining liquids, do not store ether in an open bottle for more than one month. Do not use acetone near open flames and heaters as it is highly flammable. Store acetone containing reagents in glass bottles as it dissolves tissue culture plastics.</li>
	</ol>
	</li>
	<li>Aspirate off the medium using a pipette. Wash cells one time with 1 ml of 1x cold PBS. Aspirate off PBS.</li>
	<li>Add 1 ml of 1x cold PBS to the plates and collect the cells at the bottom of the plate using a rubber policeman. Transfer the collected cells from each plate to a labeled pre-chilled 2 ml tube. Collect all cells in 2 ml tubes. 
	Note: Use a fresh rubber policeman for each plate for scraping cells from each plate. For each plate you would require four 2 ml tubes, a 1.5 ml tube and a scintillation vial.</li>
	<li>Spin at 15,000 x g for 1 min at 4 &#176;C.</li>
	<li>Take out PBS using a pipette, give a short spin and remove the residual PBS. Resuspend cells in 100 &#956;l of 1x PBS. Vortex to resuspend cells and keep on ice.</li>
	<li>Take 10 &#956;l from step 5 and transfer to a 1.5 ml microcentrifuge tube and store on ice to measure protein concentration. 
	Note: Cell lysis buffer should be added to the cells and kept on ice to measure protein concentration later.</li>
	<li>Spin the remaining 90 &#181;l from step 5 at 5,000 x g for 30 sec at 4 &#176;C; remove PBS completely using a 200 &#181;l pipette tip.</li>
	<li>Add 300 &#181;l of heme extraction buffer (see Materials List) to each tube. Then vortex for 15 sec to mix and put on ice for 1 min, repeat this 3 times.
	<ol>
		<li>Add heme extraction buffer (HEB) and vortex the tube quickly to resuspend the pellet, do only one or two tubes at a time because the pellet should be resuspended quickly without being left in the HEB for too long. Once HEB is added to all the tubes and pellet is resuspended then follow the 15 sec vortex and 1 min on ice cycle as in step 8. Note that pellet may not go completely into the HEB if the pellet is left in the HEB buffer for a longer time and not resuspended quickly.</li>
	</ol>
	</li>
	<li>Add 1.2 ml of diethyl ether to each tube and vortex for 30 sec. Spin at 15,000 x g for 5 min at 4 &#176;C. 
	Note: Diethyl ether runs out of pipette tips quickly. To control this, pipette diethyl ether up and down once or twice, doing so helps hold the diethyl ether in the pipette. Repeat this every time a fresh pipette tip is used.</li>
	<li>Transfer top (ether) phase with 1 ml pipette to a new microcentrifuge tube. Add 0.5 ml of 2 N HCl, vortex to mix and spin as in step 9. 
	Note: It is difficult to see the separation between ether and HCl phases. Therefore, add small amounts of Trypan blue in 2 N HCl solution to be used during the experiment, enough to give it a pale blue color. This will help distinguish the two phases. The lower phase will be blue and the upper ether phase will be colorless. Transfer the upper colorless ether phase to a new tube and discard the lower HCl (blue) phase.</li>
	<li>Repeat step 10 two more times to wash the ether phase with 2 N HCl. Transfer only the top ether phase to a new tube during each repetition and discard the lower HCl phase.</li>
	<li>After the third wash with HCl, transfer the top phase with 1 ml pipette to a scintillation vial. Dry the samples by evaporating the diethyl ether by keeping the samples in the chemical hood O/N.</li>
</ol>
3. Measurement of Radioactivity
<ol>
	<li>Next day, add 5 ml of scintillation fluid to the dried samples from step 2.12.</li>
	<li>Shake well to mix. Measure the radioactivity by using a scintillation counter.</li>
</ol>
4. Calculations
<ol>
	<li>Lyse the cells from step 2.6 using equal amounts of CelLytic M buffer (see Materials List) and spin at 14,000 x g for 5 min at 4 &#176;C.</li>
	<li>Take an aliquot of the supernatant and measure the protein concentration using BCA protein assay kit. 
	Total amount of protein in mg (TP) = (90 &#181;l * conc. of protein in &#181;g/&#181;l) / 1,000 
	Heme synthesis level (radioactivity/TP) = pmol/mg of protein.</li>
</ol>

We have nothing to disclose.

Heme plays a key role in the generation of cellular energy via respiration26. Altered heme metabolism is known to be associated with various diseases including cancer28,41. Inhibition of heme synthesis is known to cause cell cycle arrest and apoptosis in Hela cells26,41. It has been shown that high heme synthesis level is associated with the progression of lung cancer cells28. Therefore, it would be of great importance to measure the levels of heme biosynthesis in cells under d...

Heme, a complex of ferrous iron and protoporphyrin IX is a central molecule for transporting and utilizing oxygen in virtually all living organisms1-3. The unique structure of heme enables it to function as a carrier of diatomic gases and electrons, as well as to perform various other functions1-5. For example, heme binds to oxygen in hemoglobin and myoglobin for the transfer and storage of oxygen6,7. It also works as an electron carrier in the cytochromes during respiration and acts as an electron donor for redox reactions catalyzed by cytochrome P450 enzymes8,9. One of the most significant features of heme is that, it can play regulatory roles in cellular and molecular processes such as gene transcription, protein synthesis and micro-RNA biogenesis4. For example, it affects the transcription of many genes by controlling the activity of mammalian transcriptional repressor Bach1 and the mammalian nuclear receptor Rev-erb&#945;10-15. Heme regulates the activation of heme activator protein (Hap) 1 which plays an important role in the activation of genes involved in respiration and controlling oxidative damage, in response to heme or oxygen16. Heme also regulates gene transcription in neuronal cells via nerve growth factor (NGF) signaling3,17-20. It also regulates protein synthesis in mammalian erythroid cells by modulating the activity of heme-regulated eIF2&#945; kinase (HRI)21-24. Furthermore, heme affects the activity of key signaling proteins such as tyrosine kinase Jak2 and Src, which are essential for proper cell functioning and cell growth4,20,25. It was found that in HeLa cells heme inhibition causes cell cycle arrest and activation of markers associated with senescence and apoptosis26. Both Heme deficiency or increased levels of heme are associated with severe health effects in humans27. Recent molecular and epidemiological studies have shown a positive association of high heme intake and increased risk of diseases, such as type-2 diabetes, coronary heart disease and several cancers including lung cancer, colorectal cancer and pancreatic cancer27,28. Using a matched pair of normal and cancer lung cells authors&#39; lab have found that cancer cells have increased levels of oxygen consumption, heme synthesis and proteins involved in heme uptake and oxygen utilization28. Interestingly, inhibition of heme synthesis decreased oxygen consumption, proliferation, migration and colony formation of cancer cells28. Thus, the fluctuation in the levels of endogenous heme plays an important role in the regulation of molecular and cellular processes3,4,28,29.
In mammals, the biosynthesis of heme occurs in eight steps, involving enzymes located in the mitochondria and the cytosol4 (Figure 1). Heme biosynthesis begins in the matrix of mitochondria with the condensation of glycine and succinyl-coA to form 5-aminolevulinic acid (5-ALA), catalyzed by ALA synthase (ALAS)4,31. This is the rate limiting step in heme biosynthesis in nonerythroid cells. 5-ALA is then exported out to the cytosol where the next four steps occur to form coproporphyrinogen III (CPgenIII), which is then imported back to the mitochondria, where it is converted into protoporphyrin IX (PPIX). Finally, one molecule of iron is incorporated to the protoporphyrin IX (PPIX) to produce heme, a reaction catalyzed by ferrochelatase (FECH)2,4.
The level of heme biosynthesis depends primarily on the level of ALAS enzyme which is tightly controlled by intracellular iron and heme4. The biosynthesis of heme can be affected by genetic defects, availability of certain minerals and vitamins (e.g., riboflavin, zinc), exposure to toxins (e.g., aluminum, lead), anoxia, fever, and levels of certain steroids (e.g., estrogen)32-35. The level of heme synthesis is altered in various diseased conditions. Decreased heme biosynthesis can cause anemia as well as neurological diseases3,36. Alternatively, increased heme biosynthesis plays an important role in the progression of certain cancers28,37. Heme has been shown to be critical for the growth, differentiation and survival of mammalian adipose, erythroid and neuronal cells4,38-41. For example, heme deficiency leads to neurite damage in primary mouse cortical neurons via the inhibition of glutamate NMDA (N-methyl-D-aspartate) receptor17. Additionally, inhibition of heme synthesis causes programmed cell death in the human epithelial cervix carcinoma HeLa cells26,41. Therefore, measuring the heme biosynthesis levels in various cells under different conditions is important for studying etiology and progression of many diseases.
Here we describe a fast and sensitive method to measure the level of intracellular heme synthesis by using [4-14C] 5-aminolevulic acid. This is an alternative method to other methods using 55Fe or 59Fe. We prefer using 14C because its radiation is very weak. In contrast, strong protection is required for working with Fe isotopes. Furthermore, this method is intended to measure and compare heme synthesis in different cells in parallel in a quick manner. In order to measure absolute heme levels, one may use the previously established method involving the use of HPLC42,43.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>Sigma</td>
			<td>650501</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethy ether</td>
			<td>Sigma</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl (Hydrochloric acid)</td>
			<td>Fisher</td>
			<td>A481-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Scintillation cocktail&#160;</td>
			<td>MP Biomedicals</td>
			<td>882470</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco</td>
			<td>15250</td>
			<td></td>
		</tr>
		<tr>
			<td>Radiolabeled 4-14C aminolevulinic acid</td>
			<td>Perkin-Elmer life sciences</td>
			<td></td>
			<td>Store @&#160; -80 &#176;C</td>
		</tr>
		<tr>
			<td>CelLytic M</td>
			<td>Sigma</td>
			<td>C2978</td>
			<td>Mammalian cell lysis reagent</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Specific reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Component</td>
			<td>Dispense</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Heme extraction buffer- Acetone: HCl:Water (25:1.3:5)</td>
			<td>Acetone</td>
			<td>25ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated HCl</td>
			<td>1.3ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>5ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This method was used to compare the levels of heme synthesis in normal (HBEC30KT) vs. cancer (HCC4017) lung cells. Figure 2 shows a higher level of heme synthesis in cancer cells (HCC4017) than normal lung cells (HBEC30KT). The level of heme synthesis was also measured in normal and cancer cells in the presence of mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cells were treated with 10 &#956;M CCCP for 24 hr before the measurement of heme synthesis levels. As expected, the leve...

Watch this Scientific Journal Video about Measurement of Heme Synthesis Levels in Mammalian Cells at JoVE.com

Measurement of Heme Synthesis Levels in Mammalian Cells

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 ...

measurement-of-heme-synthesis-levels-in-mammalian-cells

Research

JoVE Journal

Biology

12.0K Views.  University of Texas at Dallas. 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.

Video: Measurement of Heme Synthesis Levels in Mammalian Cells

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

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.

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.

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 ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

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 ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

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 ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.