Research in the Keestra-Gounder lab is supported by grants from the NIAID of the NIH under Award Number R21AI122092 and from the American Diabetes Association under Award Number 1-18-JDF-035.

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+cross-linking+method+for+identification+of+NF-kappaB+gene+network+by+chromatin+immunoprecipitation.">Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.</a> Biotechniques. 39 (5), 715-725 (2005).</li><li>Ernst, O., Vayttaden, S. J., Fraser, I. D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+NF-kappaB+Activation+in+TLR-Activated+Macrophages.">Measurement of NF-kappaB Activation in TLR-Activated Macrophages.</a> Methods in Molecular Biology. 1714, 67-78 (2018).</li><li>Rodriguez, M. S., Thompson, J., Hay, R. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+small+Rho+GTPases+is+a+pathogen-induced+process+detected+by+NOD1.">Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1.</a> Nature. 496 (7444), 233-237 (2013).</li><li>Wong, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+characterization+of+NF-kappaB+binding+uncovers+non-canonical+motifs+and+advances+the+interpretation+of+genetic+functional+traits.">Extensive characterization of NF-kappaB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.</a> Genome Biology. 12 (7), R70 (2011).</li><li>Gupta, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+luciferase+mutants+as+sensors+of+proteome+stress.">Firefly luciferase mutants as sensors of proteome stress.</a> Nature Methods. 8 (10), 879-884 (2011).</li><li>Cogswell, J. 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The authors have nothing to disclose.

The major contribution of the protocol described is that it provides a fast and easy method to detect NF-&#954;B activation in cells, which allows for high throughput analysis of multiple stimulatory conditions or drugs affecting NF-&#954;B activation. Here, we describe a protocol for NF-&#954;B activation in Salmonella-infected HeLa cells. These cells can be used for infection with other pathogens as well to study the impact of bacterial infection on NF-&#954;B activation. In addition, NF-&#954;B-dependent luci...

The nuclear factor-&#954;B (NF-&#954;B) family of proteins are important transcription activators that regulate gene expression in various biological pathways. Activation of NF-&#954;B induces transcription of target genes, many of which are important for immune and inflammatory responses, cell proliferation, stress responses and cancer progression1,2. NF-&#954;B plays an integral role in mediating early inflammatory outcomes for pathogen clearance. Given the many biological processes mediated by NF-&#954;B activation, disruptions in its signaling can have serious consequences for health and disease. Loss of function mutations in NF-&#954;B signaling are associated in several immune deficiency phenotypes, while gain of function mutations are associated with several types of cancers, including B-cell lymphomas and breast cancers3. Additionally, many pathogens have been shown to directly modulate the activation state of NF-&#954;B through expression of virulence factors4,5,6,7.
Activation of NF-&#954;B is known to be a consequence of many variable stimuli including bacterial products such as lipopolysaccharides (LPS), flagellin and peptidoglycans known as pathogen-associated molecular patterns (PAMPs). These PAMPs are detected by pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) and Nod-like receptors (NLRs) leading to the activation of NF-&#954;B and the subsequent expression of an array of NF-&#954;B-dependent inflammatory genes8. In addition to PRR activation by PAMPs, other bacterial products, such as bacterial effector proteins, can induce the activation of NF-&#954;B. Interestingly, bacteria also express effector proteins that actively attenuate the NF-&#954;B pathway and enhance their pathogenicity, underscoring the importance of NF-&#954;B as an essential mediator of immunity9.
There are five different subunits that form the NF-&#954;B dimers; p50, p52, RelA (p65), RelB and cRel. The two main NF-&#954;B heterodimers are the p50:RelA and the p52:RelB dimers. The activated NF-&#954;B dimers bind to DNA sites, known as &#954;B sites, in the promoter and enhancer regions of various target genes. Under normal homeostatic conditions, NF-&#954;B interacts with a family of inhibitor proteins known as I&#954;B proteins to remain inactive. Upon stimulation, I&#954;B is phosphorylated by I&#954;B Kinase (IKK), which allows it to be targeted for ubiquitination, and subsequently degradation. Degradation of I&#954;B activates NF-&#954;B by revealing a nuclear localization signal. NF-&#954;B then translocates to the nucleus, where it binds &#954;B sites in the promoter region of target genes and promote transcription10. Thus, activation of NF-&#954;B upregulates mRNA expression of NF-&#954;B target genes, and this change can be measured through RNA quantification assays such as RT-qPCR11.
Several methods exist and are commonly used for the measurement of NF-&#954;B activation, including electrophoretic mobility shift assays (EMSA), nuclear translocation, and gene reporter assays. EMSA is used to detect protein complexes with nucleic acids. Stimulated cells are fractionated to isolate nuclear proteins, including the translocated NF-&#954;B, which is then incubated with radiolabeled nucleotides containing the NF-&#954;B binding domain. The samples are run on a gel and imaged by autoradiography of 32P-labeled nucleic acid. If NF-&#954;B is present in the protein fraction, it will bind the nucleotides, which will migrate slower through the gel and present as discrete bands. Nuclear fractions of cells lacking activated NF-&#954;B (e.g., unstimulated control cells) will produce no bands as the nucleotides migrate faster to the end of the gel. A major drawback of this method is that it is largely quantitative in the binary sense (i.e., on or off) and does not adequately capture meaningful differences in NF-&#954;B binding capacity. Additionally, this method does not consider chromatin structures that are functionally important for NF-&#954;B target genes12,13.
Similar to the previous method, there is a &#34;non-shift&#34; assay in which multi-well plates are coated with nucleotides containing the NF-&#954;B binding sequence. Following treatment of cells with nuclear fractions of protein, NF-&#954;B will bind to the nucleotides bound to the well. Anti-NF-&#954;B antibodies are then added, which will interact with the bound NF-&#954;B and produce a colorimetric signal proportional to the amount of NF-&#954;B, indicating the degree of NF-&#954;B activation. This method is advantageous over the EMSA in that it does not require radiolabeled nucleic acids and is quantitative, in comparison. However, a caveat of this method is that it again does not differentiate between chromatin states of NF-&#954;B target genes14.
Another method by which NF-&#954;B activation may be detected is by chromatin immunoprecipitation (ChIP), whereby DNA and interacting proteins are cross-linked with formaldehyde and immunoprecipitated with specific anti-NF-&#954;B antibodies. The specific nucleotide fragments are then purified and identified through PCR amplification or direct high throughput sequencing. Results generated from this method provide semi-quantitative results of NF-&#954;B binding activity with target genes. However, the results are highly dependent on the fixation conditions and purification processes at each step15.
In nuclear translocation assays, cells are stimulated to induce NF-&#954;B activation and then fixed. Anti-p65 antibodies are added to fixed cells. Alternatively, the p65 subunit itself can be tagged with a fluorescent peptide such as green fluorescent green (GFP). In either case, immunofluorescence will allow imaging of localization of p65 to determine cellular distribution. By measuring the proportion of cytosolic and nuclear localized protein, investigators can determine the relative activation state of NF-&#954;B. A drawback of this method is that the immunofluorescence is comparatively time consuming, requires expensive antibodies, and needs relatively greater technical expertise16.
Reporter genes are commonly used tools to study the regulatory and expression patterns of a gene of interest. Typically, reporter genes are constructed from the promoter sequence of a gene of interest fused to a gene coding for an easily detectable protein. Proteins with enzymatic activities, fluorescence, or luminescence properties are commonly chosen for their ability to be assayed and quantified. Thus, the read-out (e.g., luminescence, fluorescence) serves as a signal for detection of the gene expression. These reporter constructs can then be introduced into different cell types, such as epithelial cells or macrophages.
Described in the protocol is the use of a cloned HeLa cell line (HeLa 57A) that is stably transfected with a luciferase reporter containing three copies of the &#954;B consensus of the immunoglobulin &#954;-chain promoter region17. Expression of luciferase is dependent on the activation of NF-&#954;B, which occurs following cell stimulation. Stimulated cells are easily lysed using cell lysis buffer provided in the luciferase assay kit. A portion of the cell lysate is then mixed with luciferase assay buffer that contains luciferin. Luciferin is the substrate of luciferase and is required for the generation of light in the presence of luciferase. After combining the assay buffer with the lysate, the solution will emit light in a process known as luminescence. The amount of light produced, given in lumens, is proportional to the amount of luciferase present in the lysate and serves as a measure of NF-&#954;B activation. The lumen readings are interpreted in comparison to an unstimulated standard to account for baseline NF-&#954;B activity and the signal itself is stable for several minutes to allow for reliable measurement. In addition, the HeLa 57A cell line is stably transfected with a NF-&#954;B-independent &#946;-galactosidase reporter. The &#946;-galactosidase reporter is constitutively expressed, and &#946;-galactosidase activity can be measured to control for cell viability or variation in cell numbers17. The luciferase values can then be adjusted to the &#946;-galactosidase values and reported as fold increase over the unstimulated control cells.
Since NF-&#954;B is a transcription factor responsible for the increased expression of NF-&#954;B-dependent target genes, a follow up experiment to control for NF-&#954;B-dependent increased gene expression is quantitative reverse transcription polymerase chain reaction (RT-qPCR). RT-qPCR is a highly sensitive method by which changes in the gene expression can be quantified over several orders of magnitude. Stimulated and control cells are harvested for RNA via phenol-chloroform extraction. Following phase separation, RNA is extracted as the major component of the aqueous layer. RNA is then precipitated and washed to produce a pure pellet. This pellet is then reconstituted and further cleaned of contaminant DNA via DNase treatment. The pure RNA is then reverse transcribed to create complementary DNA (cDNA). This cDNA can then be analyzed through quantitative PCR techniques, where the abundance of a specific mRNA sequence is quantified to determine gene expression. This technique does not elucidate translational control, post translational modification, protein abundance, or protein activity. However, many genes, particularly those involved in pro-inflammatory processes, are regulated via NF-&#954;B and their mRNA abundance is indicative of their expression.
The method proposed here utilizes a fast and simple way by which NF-&#954;B activation can be detected via luminescence assays of cellular lysate. RT-qPCR of NF-&#954;B target gene expression can be used to quantify expression of particular genes, as well as validate functional activity of NF-&#954;B activation. The major advantages of such a system are its simplicity and speed, which allows for high throughput screening of a range of conditions that modulate NF-&#954;B activation. This protocol is suitable for other cell lines expressing an NF-&#954;B::luciferase reporter, and has been demonstrated in stably transfected RAW264.7 cells18. The amount of time required to handle samples, starting from cell lysis to generating a luminescence signal, is minimal and takes the span of about an hour. Measurement of NF-&#954;B requires only basic laboratory equipment such as opaque plates, a plate reader capable of measuring luminescence, and simple data analysis software such as a spreadsheet program.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesive film</td>
			<td>VWR International</td>
			<td>60941-070</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Fisher Bioreagents</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher</td>
			<td>11665092</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse treatment kit</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Promega</td>
			<td>U1511</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Fisher Bioreagents</td>
			<td>BP2818100</td>
			<td>molecular grade</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F0926</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa 57A cells</td>
			<td></td>
			<td></td>
			<td>Ref # 15</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>isopropanol</td>
			<td>Fisher Bioreagents</td>
			<td>BP26181</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Bioreagents</td>
			<td>BP906-5</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Fisher Bioreagents</td>
			<td>BP1425-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny broth</td>
			<td>Fisher Bioreagents</td>
			<td>BP1426-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher Chemical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>spectrophotometer</td>
		</tr>
		<tr>
			<td>promega luciferase assay system</td>
			<td>Promega</td>
			<td>E1501</td>
			<td>Cell lysis buffer &#38; luciferin substrate</td>
		</tr>
		<tr>
			<td>Random Hexamers</td>
			<td>Thermo Scientific</td>
			<td>SO142</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time GAPDH forward primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-CCAGGAAATGAGCTTGAC 
			AAAGT-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time GAPDH reverse primer</td>
			<td></td>
			<td></td>
			<td>5-&#39;CCCACTCCTCCACCT 
			TTGAC-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time IL-23 forward primer</td>
			<td></td>
			<td></td>
			<td>5-&#39;GAGCCTTCTCTGCTCCC 
			TGAT-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time IL-23 reverse primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-AGTTGGCTGAGGCCCAGTAG-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time IL-6 forward primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-GTAGCCGCCCCACACAGA-3&#39;</td>
		</tr>
		<tr>
			<td>Real-time IL-6 reverse primer</td>
			<td></td>
			<td></td>
			<td>5&#39;-CATGTCTCCTTTCTCAGG 
			GCTG-3&#39;</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase</td>
			<td>Applied Biosystems</td>
			<td>4308228</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse inhibitor</td>
			<td>Thermo Scientific</td>
			<td>EO0381</td>
			<td></td>
		</tr>
		<tr>
			<td>RT buffer</td>
			<td>Promega</td>
			<td>A3561</td>
			<td></td>
		</tr>
		<tr>
			<td>SL1344</td>
			<td></td>
			<td></td>
			<td>Ref # 17</td>
		</tr>
		<tr>
			<td>SL1344 &#916;sipA sopB::MudJ sopE2::pSB1039</td>
			<td></td>
			<td></td>
			<td>Ref # 18</td>
		</tr>
		<tr>
			<td>SL1344 &#916;sopE &#916;sipA sopB::MudJ sopE2::pSB1039</td>
			<td></td>
			<td></td>
			<td>Ref # 18</td>
		</tr>
		<tr>
			<td>SYBR green</td>
			<td>Applied Biosystems</td>
			<td>4309155</td>
			<td>2x mastermix</td>
		</tr>
		<tr>
			<td>Tri-reagent</td>
			<td>Molecular Research Center</td>
			<td>TR 118</td>
			<td>guanidine thiocyanate</td>
		</tr>
		<tr>
			<td>Trypsin -EDTA</td>
			<td>Thermo Fisher</td>
			<td>25300054</td>
			<td>0.05% Trypsin-EDTA</td>
		</tr>
		<tr>
			<td>ultrapure water</td>
			<td>Fisher Bioreagents</td>
			<td>BP248450</td>
			<td></td>
		</tr>
		<tr>
			<td>Well plate for PCR</td>
			<td>VWR International</td>
			<td>89218-294</td>
			<td>384-well plate</td>
		</tr>
	</tbody>
</table>

nf-&#954;b-dependent luciferase activation and quantification of gene expression in salmonella infected tissue culture cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

The assay described here focuses on activation of the transcription factor NF-&#954;B using a NF-&#954;B-dependent luciferase reporter that is stably transfected into a line of HeLa cells. Activated NF-&#954;B translocates to the nucleus where it binds &#954;B binding sites of target genes, including the pro-inflammatory cytokines IL6 and IL23. A general overview of NF-&#954;B activation is depicted in Figure 1. The gram-negative bacterium <...

Watch this Scientific Journal Video about NF-ÃŽÂºB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells at JoVE.com

NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...

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10.4K Views.  University of Colorado Anschutz Medical Campus. Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in cell lines expressing NF-κB::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

Video: NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in ...

In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5&#39;-UTR and 3&#39;-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...