We would like to acknowledge the contributions of all colleagues and collaborators to our original studies, whose results have been referred to in this manuscript (for illustrative purposes). The authors&#39; work is supported by grants from the National Institute of Dental and Craniofacial Research of the National Institutes of Health (1R01DE026043) to AB and from the University of California Tobacco-Related Disease Research Program to AB (TRDRP-26IR-0015) and ST (TRDRP-25IP-0001). The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, writing of the report, or in the decision to submit for publication.

1. Genomic DNA Isolation from Mouse Embryonic Fibroblasts
NOTE: Primary mouse embryonic fibroblasts are isolated from embryos derived from BB transgenic mice with C57BL/6 genetic background, according to the published protocol53. The starting material for this protocol consists of 1 x 106 to 1 x 107 embryonic fibroblast cells treated with a test compound versus control. The harvesting and counting of these cells using standard methods are described in references10,54,55.
<ol>
	<li>Prepare buffer A (0.3 M Sucrose, 60 mM KCl, 15 mM NaCl, 60 mM Tris-HCl, pH 8.0, 0.5 mM spermidine, 0.15 mM spermine, and 2 mM EDTA), buffer B (150 mM NaCl and 5 mM EDTA, pH 7.8), and buffer C (20 mM Tris-HCl, pH 8.0, 20 mM NaCl, 20 mM EDTA, and 1% SDS) in advance, and preserve at room temperature (RT) for up to one year54,55.</li>
	<li>Resuspend cells in 2&#8211;4 mL buffer A in a 15 mL conical centrifuge tube by repeatedly pipetting up and down using a wide bore 1,000 &#181;L pipette tip.</li>
	<li>Add one volume (2&#8211;4 mL) buffer A containing 1% octylphenoxypolyethoxyethanol (e.g., Nonidet P40) using a glass serological pipette.</li>
	<li>Incubate the tube on ice for 5 min.</li>
	<li>Centrifuge the tube at 1,000 x g for 5 min at RT.</li>
	<li>Discard the supernatant and wash the pellet with 10&#8211;15 mL buffer A using a glass serological pipette.</li>
	<li>Resuspend the pellet in 2&#8211;4 mL buffer B.</li>
	<li>Add one volume (2&#8211;4 mL) buffer C containing 600 &#181;g/mL proteinase K.</li>
	<li>Incubate the tube for 3 h at 37 &#176;C.</li>
	<li>Add RNase A to a final concentration of 100 &#181;g/mL.</li>
	<li>Incubate the tube for an additional 1 h at 37 &#176;C.</li>
	<li>Add one volume (2&#8211;4 mL) phenol (saturated in 0.1 M Tris-HCl, pH 8.0):chloroform:isoamyl alcohol (25:24:1 vol/vol). 
	NOTE: Phenol can pose a severe health hazard and must be handled with extreme caution. Phenol is highly corrosive to the skin and readily absorbed through it, and exhibits other effects. When handling phenol, always use double gloving, wear protective eyewear, and work in a chemical fume hood.</li>
	<li>Mix well by inverting the tube for 5 min on a tube rotator at 4 &#176;C.</li>
	<li>Centrifuge the tube at 1,000 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the aqueous phase (top layer) to a new tube using a glass serological pipette.</li>
	<li>Repeat phenol:chloroform:isoamyl alcohol extraction 1 to 3 times until the aqueous phase is clear and the interface is no longer turbid.</li>
	<li>Add 1/10 volume (200&#8211;400 &#181;L) 3M Sodium acetate, pH 5.2.</li>
	<li>Add 2.5 volume (5&#8211;10 mL) 100% ethanol (chilled) and invert the tube gently by hand for 2&#8211;3 min.</li>
	<li>Spool the DNA with a glass hook and transfer it to a new tube containing 1&#8211;5 mL 70% Ethanol and wash it thoroughly. Alternatively, centrifuge the tube at high speed (3,500 x g) at 4 &#176;C to pellet the DNA and wash it thoroughly with 1&#8211;5 mL 70% Ethanol.</li>
	<li>Centrifuge the tube at high speed (3,500 x g) at RT, remove the supernatant, and air-dry the DNA for 10-15 min.</li>
	<li>Dissolve the DNA in 10&#8211;100 &#181;L TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), and store at 4 &#176;C (for short storage of up to one month) or at -20 &#176;C (for longer storage period). The optimal concentration for DNA for the &#955; Select cII assay is 0.5&#8211;1.0 &#181;g/&#181;L (in TE buffer)56.</li>
</ol>
2. In Vitro Packaging Reaction
<ol>
	<li>Per one packaging reaction, add ~5 &#181;g genomic DNA (final volume: 8&#8211;12 &#181;L, genomic DNA was isolated in Section 1 from mouse embryonic fibroblasts treated in vitro with a test compound or control) to a microcentrifuge tube containing the first reaction mix (~10 &#181;L). 
	NOTE: In-house prepared or commercially available &#955; packaging extracts are used in different laboratories. In the commercial packaging extract kit used here56 (see the Table of Materials), red tubes contained the first reaction mix (~10 &#181;L) and blue tubes contained the second reaction mix (~70 &#181;L for at least 5 reactions).</li>
	<li>Incubate the tube for 90 min at 30 &#176;C.</li>
	<li>Add the required volume (~12 &#181;L) of the second reaction mix to the tube.</li>
	<li>Incubate the tube for an additional 90 min at 30 &#176;C.</li>
	<li>Add 1.1 mL of SM buffer to the tube. 
	NOTE: 1 mL of this solution (i.e., packaging reaction mixture) will be used for screening &#955; cII-mutants. The remainder will be used for titering.
	<ol>
		<li>Prepare SM buffer by mixing 5.8 g NaCl, 2.0 g MgSO4.7H2O, 50 mL 1 M Tris-HCl (pH 7.5), and 5 mL 2% (w/v) gelatin. Add dH2O to a final volume of 1 liter, autoclave for 30 min. Store at room temperature for up to 1 year.</li>
	</ol>
	</li>
	<li>Vortex the tube containing the packaged DNA sample for 10 s at RT (vigorous vortexing).</li>
	<li>Pulse spin the tube in a microcentrifuge and store on ice until ready to use. If the sample is not going to be used within the same day of packaging, add 50 &#181;L of chloroform per mL of packaged DNA sample, vortex gently, and store at 4 &#176;C for up to 2 weeks.</li>
</ol>
3. Preparing the E. coli G1250 Bacterial Culture
<ol>
	<li>At least two days before plating, make a few bacterial streak plates from Escherichia coli (E. coli) G1250 on TB1-kanamycin agar plates.
	<ol>
		<li>Prepare TB1-kanamycin agar plates as follows. Mix 5.0 g NaCl, 10.0 g Casein peptone, and 12.0 g agar. Add 800 mL dH2O. Add 1 mL 0.1% Thiamine hydrochloride. Adjust pH to 7.0 with NaOH or HCl. Add dH2O to a final volume of 1 liter.</li>
		<li>Mix well and autoclave for 30 min. Allow to cool to 55 &#176;C. Add 50.0 mg Kanamycin and mix. Pour into sterile 100-mm petri dishes (20&#8211;25 mL per dish). Store plates at 4 &#176;C for up to two weeks.</li>
	</ol>
	</li>
	<li>Incubate the bacterial streak plates in a stationary 30 &#176;C incubator for at least 24 h.</li>
	<li>One day before plating, combine 10 mL of TB1 liquid medium with 100 &#181;L of 20% (w/v) maltose-1 M MgSO4 solution in a sterile 50 mL screw-cap conical tube.
	<ol>
		<li>Prepare TB1 liquid medium as follows. Mix 5.0 g NaCl and 10.0 g Casein peptone, then add 800 mL dH2O, 1 mL 0.1% Thiamine hydrochloride, and adjust the pH to 7.0 with NaOH or HCl. Then add dH2O to a final volume of 1 liter, mix well, and autoclave for 30 min. Store the medium at RT for up to three months.</li>
		<li>Prepare 20% (w/v) Maltose-1 M MgSO4 as follows. Mix 20.0 g Maltose and 24.6 g MgSO4. 7H2O, then add dH2O to a final volume of 100 mL. Filter sterilize and store at 4 &#176;C for up to 6 months.</li>
	</ol>
	</li>
	<li>Inoculate the liquid medium with several colonies from the bacterial streak plate using a sterile inoculating loop56.</li>
	<li>Incubate the liquid culture overnight in a 30 &#176;C shaking incubator with vigorous shaking (250&#8211;300 rpm).</li>
	<li>On the day of plating, centrifuge the conical tube containing the G1250 liquid culture at 1,500 x g for 10 min at RT to pellet the bacterial cells.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 10 mL of 10 mM MgSO4.</li>
	<li>Measure the absorbance of a 1:10 dilution of the cell suspension at wavelength 600 nm using a UV-Vis spectrophotometer (e.g., 100 &#181;L cell suspension + 900 &#181;L 10 mM MgSO4)56.</li>
	<li>Dilute the cell suspension to a final OD600 of 0.5 with 10 mM MgSO4. The prepared suspension of G1250 E. coli with OD600 = 0.5 is referred to as &#39;the G1250 plating culture&#39;.</li>
	<li>Store the G1250 plating culture on ice and use within 1-2 h.</li>
</ol>
4. Plating the Packaged DNA Samples
<ol>
	<li>Per one packaged DNA sample, prepare sixteen sterile 14 x 100 mm2 round-bottom tubes and sixteen TB1 agar plates. Ten from each set will be used for screening and six for titering (three for Titer 20 and three for Titer 100).
	<ol>
		<li>Prepare TB1 agar plates as follows. Mix 5.0 g NaCl, 10.0 g Casein peptone, and 12.0 g Agar, then add 800 mL dH2O, and 1 mL 0.1% Thiamine hydrochloride. Adjust the pH to 7.0 with NaOH or HCl, and add dH2O to a final volume of 1 L.</li>
		<li>Mix well and autoclave for 30 min. Allow the mixture to cool to 55 &#176;C, then pour it into sterile 100 mm Petri dishes (20&#8211;25 mL per dish). Store the plates at 4 &#176;C for up to two weeks. 
		NOTE: TB1 agar plates should be prepared at least 24 h prior to use.</li>
	</ol>
	</li>
	<li>Aliquot 200 &#181;L of the G1250 plating culture into each round-bottom tube.</li>
	<li>For titering, make a 1:100 dilution of the packaged DNA sample and mix well by vortexing (i.e., 10 &#181;L packaged DNA sample + 990 &#181;L SM buffer).</li>
	<li>Add 20 &#181;L of the 1:100 dilution to each of the three Titer 20 tubes.</li>
	<li>Add 100 &#181;L of the 1:100 dilution to each of the three Titer 100 tubes.</li>
	<li>For screening, add 100 &#181;L of the &#39;undiluted&#39; packaged DNA sample to each of the ten screening tubes.</li>
	<li>Process all titering and screening tubes (2 x 3 + 10 = 16 tubes), as follows: mix well by vortexing for approximately 10 s, and then incubate at room temperature for 30 min to allow the host E. coli to adsorb the phages.</li>
	<li>Add 2.5 mL microwaved molten TB1 top agar (cooled to 55 &#176;C) to each titer or screening tube, mix immediately by vortexing (gently), and pouring into the appropriate titer or screening TB1 agar plates.
	<ol>
		<li>Prepare TB1 top agar as follows. Mix 5.0 g NaCl, 10.0 g Casein peptone, and 7.0 g Agar, then add 800 mL dH2O. Add 1 mL 0.1% Thiamine hydrochloride, then adjust the pH to 7.0 with NaOH or HCl. Finally, add dH2O to a final volume of 1 liter.</li>
		<li>Mix well and autoclave for 20&#8211;25 min. Store at room temperature for up to three months.</li>
		<li>Prior to use, melt the prepared TB1 top agar in a microwave, mix well, and allow to cool to 55 &#176;C in a water bath. 
		NOTE: The molten and cooled TB1 top is added to the contents of each titer or screening tube, and after mixing, is poured into the appropriate titer or screening TB1 agar plates.</li>
	</ol>
	</li>
	<li>Let the plates stand for 15&#8211;30 min at room temperature, with the lid ajar to prevent condensation.</li>
	<li>Invert the plates and place the ten screening plates in a stationary 24 &#176;C incubator and incubate for 46&#8211;48 h (i.e., selective conditions) and the six titer plates in a stationary 37 &#176;C incubator and incubate for 24 h/overnight (i.e., non-selective conditions). 
	NOTE: For quality assurance and standardization of results, commercially available control phage solutions containing a mixture of wildtype and mutant cII with known mutant frequency are plated alongside the packaged DNA samples and included in all assay runs56.</li>
</ol>
5. Examining the Titer and Screening Plates to Determine the cII Mutant Frequency
<ol>
	<li>Following the 24 h/overnight incubation at 37 &#176;C, count the number of plaques formed in each of the three Titer 20 plates and Titer 100 plates. To more easily identify the plaques, hold the plate next to a white light box and against a dark background with lid removed (see Figure 2).</li>
	<li>Make an average (mean) of the number of plaques counted in each set of Titer 20 plates and Titer 100 plates (triplicate, each).</li>
	<li>Choose the average number from the set of titer plates that falls closest to the range of 50&#8211;200 plaques per plate set.</li>
	<li>Calculate the total number of plaques screened in the ten screening plates as follows: 
	Total plaques screened = (Mean number of plaques in the chosen set of titer plates &#247; Number of &#181;L of dilution used per chosen titer plate) x Dilution factor x Number of &#181;L of packaged DNA sample per screening plate [100 &#181;L/plate] x Number of screening plates [10]. 
	NOTE: For example, if the mean number of plaques per plate in the set of Titer 20 plates is 128 and the corresponding number in the set of Titer 100 plates is 478, the number 128 will be used for calculation of the total number of plaques screened, as follows: 
	(128 &#247; 20) x 100 [dilution factor] x 100 [&#181;L/plate] x 10 [plates] = 640,000 
	This is equivalent to multiplying the mean number of plaques by 5,000 or 1,000, if the average number is based on counts from Titer 20 plates or Titer 100 plates, respectively.</li>
	<li>Following 46&#8211;48 h incubation at 24 &#176;C, count the total number of plaques formed in the ten screening plates (follow the instructions provided in Section 5.1). 
	NOTE: The cII mutant frequency is calculated by dividing the total number of plaques formed in the screening plates by the total number of plaques screened. Using the above example, if the total number of plaques counted in the ten screening plates is 112, the cII mutant frequency for this DNA sample would be 112 &#247; 640,000 = 17.5 x 10-5.</li>
</ol>
6. Verification of the Putative &#955; cII Mutants, PCR Amplification, and DNA Sequencing
<ol>
	<li>Core the plaque in question with a sterile, wide-bore pipet tip and expel it (by pipetting up and down) into a sterile microcentrifuge tube containing 500 &#181;L of sterile SM buffer.</li>
	<li>Incubate for at least 2 h at room temperature, or at 4 &#176;C overnight, to allow the phage particles to elute from the agar plug.</li>
	<li>In a sterile 14 x 100 mm2 round-bottom tube, mix 200 &#181;L of the G1250 plating culture with 1 &#181;L of the cored phage solution, and incubate for 30 min at RT.</li>
	<li>Plate the sample using 2.5 mL of 55 &#176;C molten TB1 top agar and incubate the plate at 24 &#176;C for 46&#8211;48 h (selective conditions), as described in Sections 4.8&#8211;4.10.</li>
	<li>Once the secondary plaques have formed, use a pipette tip to carefully pick a single well-isolated verified &#955; cII-mutant plaque (avoid touching the lower agar). 
	NOTE: Replating the putative cII mutant plaques serves two purposes: (i) plating artefacts may sometimes be mistaken for small-size plaques; and (ii) an agarose core taken from a screening plate may contain non-mutant phage(s) together with a mutant phage. Secondary plaques from a low-density replating will provide an uncontaminated mutant template for PCR and subsequent DNA sequence analysis.</li>
	<li>Transfer the plaque to a microcentrifuge tube containing 25 &#181;L double-distilled water by pipetting up and down.</li>
	<li>Place the tube in boiling water for 5 min.</li>
	<li>Centrifuge at maximum speed (18,000 x g) for 3 minutes at RT.</li>
	<li>Transfer 10 &#181;L of the supernatant immediately to a new microcentrifuge tube containing 40 &#181;L of a PCR mastermix in which the final concentrations of the reagents are 1 x Taq PCR buffer, 10 pmol each of the forward and reverse primers, 12.5 nmol of each dNTP, and 2.5 U of Taq DNA polymerase. 
	NOTE: The forward and reverse primers are as follows: 5&#39;-CCACACCTATGGTGTATG-3&#39; (positions -68 to -50 relative to the cII start codon) and 5&#39;-CCTCTGCCGAAGTTGAGTAT-3&#39; (positions +345 to +365 relative to the cII start codon), respectively.</li>
	<li>Amplify the template using the following cycling parameters: a 3 min denaturation at 95 &#176;C, followed by 30 cycles of 30 s at 95 &#176;C, 1 min at 60 &#176;C, and 1 min at 72 &#176;C, with a final extension of 10 min at 72&#160;&#176;C.</li>
	<li>Purify the 432 bp PCR product containing the cII gene and flanking regions using commercially available PCR purification kits, according to the manufacturer&#39;s instructions.</li>
	<li>Perform DNA sequencing using an appropriate sequencing platform, and analyze the resulting DNA sequences to detect mutations in the cII transgene (see the note below). 
	NOTE: This is best achieved by alignment with the reference cII sequence using software, such as the web-based T-Coffee sequence alignment server. For instructions on how to use the program, visit: <a href="http://tcoffee.crg.cat/">http://tcoffee.crg.cat/</a></li>
</ol>

All the authors declare no conflict of interest.

The &#955; Select cII assay is used for detection of mutations in the cII transgene recovered from the genomic DNA of cells derived from organs/tissues of BB rodents3. The genome of these transgenic animals contains multiple tandem copies of the chromosomally integrated &#955;LIZ shuttle vector, which carries the cII (294 bp) and lacI (1,080 bp) transgenes, as the mutational reporter genes1,2,...

A wide range of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic Big Blue (referred to hereafter as BB) mice and the &#955; Select cII Mutation Detection System have been employed for mutagenicity experiments by this group and many other research groups worldwide1,2,3,4,5,6,7,8,9. For the past 16 years, we have investigated the mutagenic effects of various chemical and/or physical agents using these transgenic animals or their corresponding embryonic fibroblast cell cultures treated with a test compound, and subsequently analyzed the phenotype and genotype of the cII transgene by the &#955; Select cII assay and DNA sequencing, respectively10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. The genome of these transgenic animals contains a bacteriophage &#955; shuttle vector (&#955;LIZ) integrated on chromosome 4 as a multi-copy head-to-tail concatemer1,2,25. The &#955;LIZ shuttle vector carries two mutational reporter genes, namely the lacI and cII transgenes1,2,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47. The &#955; Select cII assay is based on the recovery of the &#955;LIZ shuttle vectors from the genomic DNA of cells derived from organs/tissues of transgenic animals1,2,25. The recovered &#955;LIZ shuttle vectors are then packaged into &#955; phage heads capable of infecting an indicator host Escherichia coli. Subsequently, the infected bacteria are grown under selective conditions to allow for scoring and analysis of mutations in the cII transgene1,3.
Here, we describe a detailed protocol for the &#955; Select cII assay, which consists of isolation of genomic DNA from cells/organs of transgenic animals treated in vitro/in vivo with a test compound, retrieval of the &#955;LIZ shuttle vectors from the genomic DNA, packaging of the vectors into infectious &#955; phages, infection of the host E. coli with the bacteriophages, identification of the cII-mutants under selective conditions to determine the cII mutant frequency, and DNA sequence analysis to establish the cII mutation spectrum. The protocol can be applied to transgenic mouse/rat cell cultures treated in vitro with a chemical/physical agent of interest, or tissues/organs of the corresponding animals treated in vivo with the test chemical/agent1,2,4,48,49,50,51,52. A schematic presentation of the &#955; Select cII assay is shown in Figure 1.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>MO Bio Laboratories, Inc.</td>
			<td>12112-05</td>
			<td>Bacteriological grade</td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1 Cycle Sequencing Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>4337455</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Casein Peptone</td>
			<td>Alfa Aesar</td>
			<td>H26557</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>J. T. Baker</td>
			<td>2124-01</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP 229-1 / M-13750</td>
			<td>None</td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher Scientific</td>
			<td>BP 9724-500</td>
			<td>None</td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>8104</td>
			<td>50 PCR purification reactions</td>
		</tr>
		<tr>
			<td>Sodium Acetate Trihydrate</td>
			<td>Fisher Scientific</td>
			<td>M-15756</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Taq5000 DNA Polymerase&#160;</td>
			<td>Qiagen</td>
			<td>201207</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Thiamine Hydrochloride</td>
			<td>Macron Fine Chemicals</td>
			<td>2722-57</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Transpack Packaging Extract</td>
			<td>Stratagene Corp., Acquired by BioReliance | Sigma-Aldrich Corp.</td>
			<td>200223</td>
			<td>50 packaging reactions</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Fisher Scientific</td>
			<td>BP 152-1 / EC 201-064-4</td>
			<td>None</td>
		</tr>
		<tr>
			<td>Trypton</td>
			<td>Biosciences</td>
			<td>RC-110</td>
			<td>None</td>
		</tr>
	</tbody>
</table>

the lambda select cii mutation detection system

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (&#955;) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

Depending on data distribution, parametric or non-parametric tests are used to determine the significance of difference in the cII mutant frequency between treatment and control groups (i.e., induced versus spontaneous mutant frequencies). Comparison of the induced cII mutant frequencies across different treatment groups is made by various (pairwise) statistical tests, as applicable. The hypergeometric test of Adams and Skopek is commonly used to compare the ove...

Watch this Scientific Journal Video about The Lambda Select cII Mutation Detection System at JoVE.com

The Lambda Select cII Mutation Detection System

We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

The Lambda Select cII Mutation Detection System

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of ...

the-lambda-select-cii-mutation-detection-system

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JoVE Journal

Immunology and Infection

7.8K Views.  University of Southern California. We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

Video: The Lambda Select cII Mutation Detection System

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...