Name: pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica
Uploaded: 2011-08-31T14:00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Work in the lab is supported by NSF (MCB0919933) and NIH (GM088790).

1. 	Trunk fragment regeneration assay
<ol>
<li>Stop feeding a cohort of worms for at least 5 days before the regenerative assay to ensure animals are free of waste and ingested food (&tilde;30 intact worms, each worm &tilde;8-10 mm long post-starvation).</li>
<li>On the day of the assay, rinse a pre-frozen leveled ice dish with water and cover the flat iced surface with plastic wrap. Using forceps, place one filter paper on the plastic wrap.</li>
<li>Moisten the filter paper with a few drops of spring water. Transfer planarians onto filter (&lt;20 worms per filter) using a transfer pipette. Worms can be repositioned on filter, if necessary, by repipetting with more spring water. Remove excess fluid.</li>
<li>Using a scalpel, amputate the head by a single cut made approximately halfway between the anterior apex of the animal and the anterior end of the pharynx (Figure 1A).</li> 
<li>Using a scalpel, amputate the tail region by a single cut made approximately halfway between the tail end of the animal and posterior end of the pharynx (Figure 1A). Remove excess mucus on the scalpel using a paper towel moistened with 70% ethanol after cutting. Wipe excess ethanol from scalpel.</li>
<li>Transfer filter via forceps into a Petri dish (100 x 25mm depth) containing spring water. Wait &tilde;3 minutes (for wound closure, observable by a pinching and darkening of the wound).</li>
<li>Rinse trunk fragments from filter paper into Petri dish containing desired medium for regenerative assay and transfer to thermostatted incubator (24 &deg;C).</li>
<li>Score regenerative phenotype after &tilde;1 week, when regenerated structures are identifiable (Figure 1A).</li>
</ol>
2.	Pharmacological manipulation of regeneration: bipolarity induced by praziquantel
<ol>
<li>Prepare praziquantel (PZQ) stock by solubilizing drug in DMSO (62.4mg PZQ in 1mL for 200mM stock). Use on same day.</li>
<li>Make 50mL of drug-containing solution (90 &mu;M PZQ) in buffered Montjuch salts. Vortex for &tilde;1 minute to ensure dispersion.</li>
<li>Perform trunk fragment regenerative assay as detailed above [protocol #1], exposing a cohort of trunk fragments to PZQ at the final step [1.7].</li>
<li>After 24-48 hours, exchange PZQ-containing media for spring water, washing worms several (&gt;3) times.</li>
<li>Return worms to incubator. Score bipolarity (two-heads, Figure 1B) one week after cutting.</li> 
</ol>
3. 	Genetic manipulation of regeneration: in vivo RNAi feeding protocol
<ol>
<li>Prior to assay, prepare chicken liver homogenate. Discard fatty, yellow colored sections from foodstock, and pur&eacute;e remaining liver. Filter pur&eacute;e through mesh strainer and aliquot suspension for storage (1.5mL tubes, -20 &deg;C).</li>
<li>Prior to assay, prepare bovine red blood cells (RBCs) by aliquoting supply into 1mL samples, stored at -20&deg;C.</li>
<li>Select worms for RNAi. Three cohorts are used: gene of interest, positive control (gene with known RNAi phenotype; Figure 1C, D &amp; E) and negative control (gene with no RNAi phenotype, also useful for assessment of knockdown levels compared to experimental cohort). For each cohort, use &tilde;250 worms (&tilde;8-10 mm long after starving for at least 5 days). Store in spring water in plastic tub. </li>
<li>For each RNAi cohort, thaw stock of E. coli expressing dsRNA targeting gene of interest [8], along with a tube of chicken liver homogenate [3.1] and a tube of RBCs [3.2].</li>
<li>Pellet bacteria (13,000g, 1 minute). Remove supernatant and resuspend pellet in 2xYT media (&tilde;700&mu;l). Recentrifuge, discard supernatant, place pellet on ice.</li>
<li>Create a large bore P200 pipette tip by cutting off the end of the tip. Thoroughly resuspend the bacterial pellet in a mixture of chicken liver homogenate (150 &mu;L) and RBCs (50 &mu;L) to create RNAi feeding mix. Remove air bubbles by centrifugation.</li>
<li>For feeding, remove the majority of the water from the plastic tub containing worms (leave &tilde;1 inch depth). Swirl tub to concentrate worms at the center of this container, and pipette RNAi feeding mix in a circle corralling worms. Use a transfer pipette to carefully coax escapees back onto the RNAi feeding mix without perturbing other diners!</li>
<li>After feeding (&tilde;1 hour), identify planarians that fed well. These worms show a deep red coloration from ingested RBCs. Discard others.</li>
<li>Carefully replace feeding solution with fresh spring water, minimizing disturbance to prevent egestion of food. To do this, gently localize the worms to one side of the tube using a transfer pipette, pour out turbid water, and carefully replace with fresh water poured down the opposite wall of the tub. Resubmerge planarians quickly as prolonged exposure to air also results in food egestion.</li>
<li>Loosely seal container lid and return to incubator (24&deg;C)</li>
<li>Repeat RNAi feeding protocol over multiple cycles (&tilde;2-3 days apart), interspersed with regenerative cycles [protocol #1] to screen for RNAi phenotypes. A standard protocol for feeding and regeneration effective for many genes is shown in Figure 2, which takes &tilde;1 month in total duration.</li> 
</ol>
Modify this scheme for different genes, as optimal protocol will depend on factors such as mRNA stability, tissue localization, protein perdurance, or development of a phenotype that precludes multiple feeding cycles after regeneration. Assess knockdown levels by simply screening for penetrance of phenotype (if apparent), or by qPCR approaches to compare targeted mRNA levels with negative control cohort.
4. Representative results:
The trunk fragment regeneration assay is robust such that all worms should regenerate with normal anterior-posterior ('head to tail') polarity. This can be scored as soon as five days after cutting, facilitated by the emergence of the anterior eyespots ( asterixed, Figure 1A). However, complete morphological restoration of lost structures occurs after one week. Pharmacological manipulation of trunk fragment regeneration by PZQ to yield two-headed worms (Figure 1B) is also robust (EC50=87 (+-) 11% bipolar fragments, 70&mu;M PZQ for 48 hours20). Bipolarity occurs over a single regenerative cycle. Lower efficacy in these assays likely relates to issues with drug solubility and/or storage, or use of a different flatworm species where PZQ is ineffective. For drugs with lower penetrance, an anteriorization index is often used to score intermediate phenotypes aside from complete bipolarity22. Phenotypes resulting from RNAi of positive control constructs are shown in Figure 1: RNAi of Dugesia japonica six-1 (Dj-six-1) results in an eyeless phenotype23 (Figure 1C), RNAi of Dugesia japonica PC2 (Dj-PC2) results in a loss of the light aversion response24 (Figure 1D) and RNAi of Dugesia japonica &beta;catenin-1 (Dj-&beta;catenin-1) results in a two-headed phenotype16-19, 21 from trunk fragments (Figure 1E). These phenotypes can be achieved using the following simple RNAi protocols: Dj-six-1 (FFxFx), Dj-PC2 (FFFx), Dj-&beta;catenin-1 (FFxFx), where F=feeding cycle and x=regenerative cycle respectively.
<img src="/files/ftp_upload/3058/3058fig1.jpg" alt="Figure 1"/> 
Figure 1: Trunk fragment regeneration assay. A Left, Image of planarian (top) and schematic (middle) to show location of excised trunk fragment. Right, timecourse of trunk fragment regeneration showing appearance of regenerating fragment at indicated times (days). B Image of two-headed planarian produced by PZQ exposure. C 'Eyeless' worm produced by Dj-six-1 RNAi. D Loss of light aversion response in immobile Dj-PC2 RNAi worms (stained red), compared to mobile control worm (stained green). E Bipolar planarian produced by Dj-&beta;catenin-1 RNAi. In B-E, the original anterior end of the RNAi worms is oriented to the left.
<img src="/files/ftp_upload/3058/3058fig2.jpg" alt="Figure 2"/> 
Figure 2: Sequence of feeding and cutting cycles for typical RNAi protocol comprising multiple feedings (F), and regenerative cycles (x) that is effective for knockdown of many genes in D. japonica. Modification of this protocol for individual genes may be needed to yield optimal results, for example using a fewer (parentheses) or a greater number of feeding and regenerative cycles.

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+specifically+disrupts+gene+expression+during+planarian+regeneration.">Double-stranded RNA specifically disrupts gene expression during planarian regeneration.</a> Proc Natl Acad Sci U S A. 96, 5049-5054 (1999).</li><li>Gurley, K. A., Rink, J. 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S., S, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+biological+activity+of+praziquantel+requiring+voltage-operated+Ca2++channel+beta+subunits:+subversion+of+flatworm+regenerative+polarity.">A novel biological activity of praziquantel requiring voltage-operated Ca2+ channel beta subunits: subversion of flatworm regenerative polarity.</a> PLoS Negl Trop Dis. 3, e464-e464 (2009).</li><li>Oviedo, N. 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No conflicts of interest declared.

The protocols described here detail assays for studying and manipulating regeneration of the planarian Dugesia japonica. They are simple and do not require specialized equipment such that they can be easily performed in the laboratory or classroom. Assays can be performed individually, or combined (for chemical genetic screening of drug efficacies in vivo) and can be performed at the candidate gene level, or are adaptable to unbiased, higher throughput screening8. Whether for studying the intriguing biology of planarians in their own right, or for assessing in vivo function of mammalian homologues in a alternative model for studying tissue regeneration, these approaches should catalyze interest from a diverse range of researchers.

<table border="1">
	<tbody>
		<tr>
			<th>Reagent</th>
			<th>Vendor</th>
			<th>Catalogue Number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Spring water</td>
			<td>Kandiyohi. Premium Waters Inc. Minneapolis, MN </td>
			<td>n/a</td>
			<td>Other forms of spring water work well also. Trial first in viability assays.</td>
		</tr>
		<tr>
			<td>1 x buffered Montju&iuml;ch salts: NaCl (1.6mM), CaCl2 (1mM), MgSO4 (1mM), MgCl2 (0.1mM), KCl (0.1mM), NaHCO3 (1.2mM), HEPES (1.5mM). pH 7.4 at 24&deg;C.</td>
			<td>Multiple vendors</td>
			<td>n/a</td>
			<td>Artificial water for drug treatments during regenerative assays to ensure pH buffering. 5/8 Holtfreter's solution is an alternative.</td>
		</tr>
		<tr>
			<td>2xYT Broth</td>
			<td>Fisher Scientific</td>
			<td>BP2467-500</td>
			<td>Media = 31 g/L . Autoclaved. </td>
		</tr>
		<tr>
			<td>Petri Dish (100x25mm)</td>
			<td>Fisher Scientific</td>
			<td>08-757-11</td>
			<td>Housing worms during regeneration cycles</td>
		</tr>
		<tr>
			<td>Square Dish (100x100x15mm)</td>
			<td>Fisher Scientific</td>
			<td>08-757-11A</td>
			<td>Fill with water, freeze for ice tray used as worm cutting surface</td>
		</tr>
		<tr>
			<td>Plastic tub: Ziploc Twist 'n Loc (16oz).</td>
			<td>Various retailers</td>
			<td>n/a</td>
			<td>Convenient water tight containers for RNAi cohorts</td>
		</tr>
		<tr>
			<td>Chicken Liver</td>
			<td>Commercial grocery</td>
			<td>n/a</td>
			<td>Bias towards organic supplies, owing to avoidance of antibiotics.</td>
		</tr>
		<tr>
			<td>Hand Blender</td>
			<td>Any kitchen supplier</td>
			<td>n/a</td>
			<td>Use for making chicken liver puree</td>
		</tr>
		<tr>
			<td>Wire 1mm Mesh strainer</td>
			<td>Any kitchen supplier</td>
			<td>n/a</td>
			<td>Use for straining chicken liver puree</td>
		</tr>
		<tr>
			<td>Bovine red blood cells</td>
			<td>Lampire Biological Laboratories</td>
			<td>7240807</td>
			<td>100% Washed and pooled RBC suspension</td>
		</tr>
		<tr>
			<td>Circular filter papers</td>
			<td>Whatman #3</td>
			<td>1003 055</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transfer Pipette</td>
			<td>Fisher Scientific </td>
			<td>13-711-41</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile, surgical blades</td>
			<td>Multiple Vendors</td>
			<td>n/a</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&plusmn;Praziquantel</td>
			<td>Sigma Aldrich</td>
			<td>P4668</td>
			<td>Store powder aliquots in the dark at 4&deg;C. Desiccate. </td>
		</tr>
		<tr>
			<td>Dj-six-1</td>
			<td>&nbsp;</td>
			<td>GenBank AJ557022.1</td>
			<td>RNAi positive control for &#x201C;eye-less&#x201D; phenotype23</td>
		</tr>
		<tr>
			<td>Dj-&#x03B2;catenin-1</td>
			<td>&nbsp;</td>
			<td>GenBank AB181913.1 </td>
			<td>RNAi positive control for two-headed phenotype17</td>
		</tr>
		<tr>
			<td>Dj-PC2</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>RNAi positive control for loss of light aversion phenotype24</td>
		</tr>
	</tbody>
</table>

pharmacological and functional genetic assays to manipulate regeneration of the planarian dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Watch this Scientific Journal Video about Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica at JoVE.com

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

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Biology

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...