The authors have no acknowledgements.

<ol>
	<li>Douek, D. C., 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=Changes+in+thymic+function+with+age+and+during+the+treatment+of+HIV+infection.">Changes in thymic function with age and during the treatment of HIV infection.</a> Nature. 396 (6712), 690-695 (1998).</li><li>Zelm, M. C., Szczepanski, T., van der Burg, M., van Dongen, J. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+history+of+B+lymphocytes+reveals+homeostatic+proliferation+and+extensive+antigen-induced+B+cell+expansion.">Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion.</a> J. Exp. 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Neuroimmunol. 240-241, 147-150 (2011).</li><li>Zanotti, C., 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=Peripheral+accumulation+of+newly+produced+T+and+B+lymphocytes+in+natalizumab-treated+multiple+sclerosis+patients.">Peripheral accumulation of newly produced T and B lymphocytes in natalizumab-treated multiple sclerosis patients.</a> Clin. Immunol. 145 (1), 19-26 (2012).</li><li>Sottini, A., 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=Pre-existing+T-+and+B-cell+defects+in+one+progressive+multifocal+leukoencephalopathy+patient.">Pre-existing T- and B-cell defects in one progressive multifocal leukoencephalopathy patient.</a> PLoS One. 7, e34493 (2012).</li><li>Quiros-Roldan, E., 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=Effects+of+combined+antiretroviral+therapy+on+B-+and+T-cell+release+from+production+sites+in+long-term+treated+HIV-1++patients.">Effects of combined antiretroviral therapy on B- and T-cell release from production sites in long-term treated HIV-1+ patients.</a> J. Transl. Med. 10, 94 (2012).</li><li>Mensen, A., 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=Utilization+of+TREC+and+KREC+quantification+for+the+monitoring+of+early+T-+and+B-cell+neogenesis+in+adult+patients+after+allogeneic+hematopoietic+stem+cell+transplantation.">Utilization of TREC and KREC quantification for the monitoring of early T- and B-cell neogenesis in adult patients after allogeneic hematopoietic stem cell transplantation.</a> J. Transl. Med. 11, 188 (2013).</li><li>Serana, F., 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=The+different+extent+of+B+and+T+cell+immune+reconstitution+after+hematopoietic+stem+cell+transplantation+and+enzyme+replacement+therapies+in+SCID+patients+with+adenosine+deaminase+deficiency.">The different extent of B and T cell immune reconstitution after hematopoietic stem cell transplantation and enzyme replacement therapies in SCID patients with adenosine deaminase deficiency.</a> J. Immunol. 185 (12), 7713-7722 (2010).</li><li>Borte, S., 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=Neonatal+screening+for+severe+primary+immunodeficiency+diseases+using+high-throughput+triplex+real-time+PCR.">Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR.</a> Blood. 119 (11), 2552-2555 (2012).</li><li>Baker, M. W., 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=Implementing+routine+testing+for+severe+combined+immunodeficiency+within+Wisconsin's+newborn+screening+program.">Implementing routine testing for severe combined immunodeficiency within Wisconsin's newborn screening program.</a> Public Health Rep. 125 (2), 88-95 (2010).</li><li>Lorenzi, A. 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=Determination+of+thymic+function+directly+from+peripheral+blood:+a+validated+modification+to+an+established+method.">Determination of thymic function directly from peripheral blood: a validated modification to an established method.</a> J. Immunol. Meth. 339 (2), 185-194 (2008).</li><li>De Boer, R. J., Perelson, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+T+lymphocyte+turnover.">Quantifying T lymphocyte turnover.</a> J. Theor. Biol. 327, 45-87 (2013).</li></ol>

The authors have nothing to disclose.

TREC and KREC quantification can be considered a good estimate of recent thymic and bone marrow output provided that some caveats are taken into account. Even though an absolute quantification method employing standard curve requires more reagents and more space on the real-time PCR reaction plate, it ensures highly accurate quantitative results because unknown sample quantities are interpolated from standard curves built upon known amounts of starting material. Moreover this method is better fitted to detect low amount ...

T-Zell-Rezeptor-Exzision Kreise (TRECs) und K-Löschen Rekombination Exzision Kreise (KRECs) sind kleine zirkularisiertes DNA-Elemente, die in einem Anteil von T- und B-Zellen jeweils während einer genomischen DNA-Rekombination herausgeschnitten werden, was zu dem Bildung einer stark diversifizierten Repertoire von T- und B-Zell-Rezeptoren. Sie haben keine Funktion, sondern weil sie stabil sind und nicht repliziert werden, werden sie nach jeder Zellteilung verdünnt, also nur in einer der beiden Tochter persistierenden Zellen. Daher kann ihre Ebenen im peripheren Blut als eine Schätzung des Thymus und des Knochenmarks Ausgangs angenommen werden. Während die TREC-Assay wurde weitgehend in den letzten 15 Jahren um das Ausmaß der Thymusfunktion auszuwerten, um 1 der KREC Assay, die ursprünglich entwickelt wurde B-Zell-Proliferation und ihren Beitrag zur B-Zell-Homöostase in Gesundheit und Krankheit, 2 messen wurde erst kürzlich als Marker der Knochen m vorgeschlagenenarrow Ausgang. 3,4 Hier beschreiben wir die Methode, die wir für die simultane Quantifizierung von beiden TRECs und KRECs entwickelt. 4 Mit diesem kombinierten Verfahren wird die Variabilität DNA Quantifizierung durch Echtzeit-PCR verbunden sind, durch die Verwendung eines einzigartigen Standardkurve durch Verdünnung einer Triple-Insert enthaltenden Plasmid Fragmente TRECs, KRECs und T-Zell-Rezeptor alpha konstant erhalten (TCRAC) beseitigt Gens in einer 1: 1: 1-Verhältnis. Dies ermöglicht eine genauere Bewertung der TREC und KREC Kopienzahl. Ferner ist die gleichzeitige Quantifizierung der zwei Targets in der gleichen Reaktion ermöglicht Reagenz Kostenreduzierung. Die vorgeschlagene TREC / KREC Assay kann nützlich sein, um das Ausmaß der T- und B-Zell-neo-Produktion bei Kindern oder Erwachsenen mit schwerer kombinierter Immundefizienz (SCID), 4 Common Variable Immunodeficiency, 5 Autoimmunerkrankungen, 6-8 und HIV-Infektionen zu messen Ferner. 9 zeigt, kann es verwendet werdenÜberwachung der Immun Erholung nach Transplantation hämatopoetischer Stammzellen, 10 Enzymersatz, 11 und antivirale 9 oder immunmodulierende Therapien. 6-8 Schließlich weil SCID-Patienten werden mit TREC-Test trotz der zugrunde liegenden genetischen Defekte erkannt und Agammaglobulinämie Patienten können mit KREC Quantifizierung identifiziert werden Die TREC / KREC Assay kann auch verwendet werden, um Immun in Neugeborenen-Screening-Programme erkennen. 12 In diesem Fall muss der Test an aus kleinen Flecken von Blut abgetupft extrahierten DNA durchgeführt, und auf Filterpapier getrocknet werden müssen hochempfindlich und spezifisch für sein die Zielkrankheiten, sowie in hohem Maße reproduzierbar und kostengünstig. Die Einführung KREC Quantifizierung im Test sollte Aufführungen von Neugeborenen-Screening für Immundefekte, die routinemäßig in den einigen Teilen der USA (WI, MA, CA) seit 2008 durchgeführt wurde, zu verbessern, wenn Wisconsin war der erste, um introduce die Analyse TRECs in seiner postnatale Screening-Programm. 13

<table><tbody><tr><td>Histopaque-1077</td><td>Sigma Aldrich SRL</td><td>10771-500 ML</td><td>density gradient separation method</td></tr><tr><td>QIAamp DNA Blood Mini Kit (250)</td><td>QIAGEN</td><td>51106</td><td>DNA extraction</td></tr><tr><td>Unmodified DNA Oligonucleotides HPSF 0.01 mmol</td><td>Eurofins MWG Operon/Carlo Erba Reagents S.r.l&amp;nbsp;</td><td></td><td>Resuspend the lyophilized product to 100 pmol/&amp;micro;l</td></tr><tr><td>AmpliTaq DNA Polymerase: including 10x Buffer II and 25 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Applied Biosystems/Life-Technologies</td><td>N8080156</td><td></td></tr><tr><td>GeneAmp dNTP Blend (100 mM)</td><td>Applied Biosystems/Life-Technologies</td><td>N8080261</td><td></td></tr><tr><td>TOPO TA Cloning Kit for Subcloning</td><td>Invitrogen/Life-Technologies</td><td>K4500-01</td><td></td></tr><tr><td>XL1-Blue Subcloning Grade Competent Cells</td><td>Stratagene</td><td>200130</td><td></td></tr><tr><td>PureYield Plasmid Miniprep System</td><td>Promega</td><td>A1223</td><td></td></tr><tr><td>SpeI 500U</td><td>New England Biolabs</td><td>R0133S</td><td></td></tr><tr><td>HindIII-HF 10,000 U</td><td>New England Biolabs</td><td>R3104S</td><td></td></tr><tr><td>PureYield Plasmid Midiprep System</td><td>Promega</td><td>A2492</td><td></td></tr><tr><td>XhoI 5,000 U</td><td>New England Biolabs</td><td>R0146S</td><td></td></tr><tr><td>TRIS Utrapure</td><td>Sigma Aldrich SRL</td><td>T1503</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich SRL</td><td>E5134</td><td></td></tr><tr><td>TE buffer (1 mM TRIS and 0.1 mM EDTA)</td><td></td><td></td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Applied Biosystems/Life-Technologies</td><td>4364338</td><td></td></tr><tr><td>Dual labeled probes HPLC 0.01 mmol</td><td>Eurofins MWG Operon/Carlo Erba Reagents S.r.l&amp;nbsp;</td><td></td><td>Resuspend the lyophilized product to 100 pmol/&amp;micro;l</td></tr><tr><td>NanoDrop 2000c spectrophotometer</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Applied Biosystems 2720 Thermal Cycler</td><td>Applied Biosystems/Life-Technologies</td><td>4359659</td><td></td></tr><tr><td>Fast 7500 Real-Time PCR system</td><td>Applied Biosystems/Life-Technologies</td><td></td><td></td></tr><tr><td>SDS Sequence Detection Software 1.4</td><td>Applied Biosystems/Life-Technologies</td><td></td><td></td></tr></tbody></table>

simultaneous quantification of t-cell receptor excision circles (trecs) and k-deleting recombination excision circles (krecs) by real-time pcr

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Der Test wurde in einer repräsentativen Stichprobe von 87 gesunden Kontrollpersonen durchgeführt: 42 Kinder im Alter von 0 bis 17 (männlich / weiblich: 25/17) und 45 Erwachsene im Alter von 24 bis 60 (Männer / Frauen: 29/16). Die Ergebnisse wurden als TRECs und KRECs pro 10 6 PBMC und dann den TRECs und KRECs pro ml Blut wurden berechnet erhalten. Die Anzahl der TRECs nimmt mit dem Alter aufgrund Thymusinvolution, 4 insbesondere in einem sehr scharfen Mode 0-3 - 4 J...

Watch this Scientific Journal Video about Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR at JoVE.com

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

Simultane Quantifizierung von T-Zell-Rezeptor-Excision Kreis (TRECs) und K-Löschen Rekombination Excision Kreis (KRECs) durch Echtzeit-PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

simultaneous-quantification-t-cell-receptor-excision-circles-trecs-k

Research

JoVE Journal

Immunology and Infection

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

16.4K Aufrufe.  Spedali Civili di Brescia. Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

Serie: Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

Bidirektionale Retrovirale Integrationsstelle PCR Methodik und quantitative Datenanalyse Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

Forschung

Genetik

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

qKAT: Quantitative Flugbuchungssystem Typisierung von Killer-Zelle Immunoglobulin-Like-Rezeptor-Gene

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Generation von Krebs Zellklonen Telomeric Repeat-haltigen RNA TERRA von einem einzigen Telomere in lebenden Zellen ausgedrückt zu visualisieren

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

<meta charset="utf8"></head><body>Detektion von Off-Target-CRISPR-Cas9-Spaltungsereignissen mit CIRCLE-Seq

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

<meta charset="utf8"></head><body>CIRCLE-Seq zur Abfrage von Off-Target-Gen-Editing

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

CIRCLE-Seq zur Abfrage von Off-Target-Gen-Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

Optimierte Einzelzelle TCR Isolation und Generierung von retroviralen Vektoren für In-Vitro und In Vivo Ausdruck menschlichen TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Immunologie und Infektion

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs.
Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between na&#239;ve and effector cells.

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

Gleichzeitige Quantifizierung von Anti-Vektor- und Anti-Transgen-spezifische CD8+ T Zellen über MHC Tetramer Färbung nach der Impfung mit viralen Vektoren

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

Quantitative, Echtzeit-Analyse der Base Excision Repair Aktivität in Zelllysaten Verwendung läsionsspezifischen Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Biologie

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR

Immune cell subtype population frequencies can have a large effect on the efficacy of T cell therapies. Current methods, like flow cytometry, have specific sample requirements, high sample input, are low throughput, and are difficult to standardize, all of which are detrimental to characterization of cell therapy products during their development and manufacturing.
The assays described herein accurately identify and quantify immune cell types in a heterogeneous mixture of cells using isolated genomic DNA (gDNA). DNA methylation patterns are revealed through bisulfite conversion, a process in which unmethylated cytosines are converted to uracils. Unmethylated DNA regions are detected through qPCR amplification using primers targeting converted areas. One unique locus per assay is measured and serves as an accurate identifier for a specific cell type. The assays are robust and identify CD8+, regulatory, and Th17 T cells in a high throughput manner. These optimized assays can potentially be used for in-process and product release testing for cell therapy process.

Here we describe a robust method of determining immune cell identity and purity through epigenetic signatures detected using quantitative PCR (qPCR). DNA demethylation at a specific locus serves as a unique identifier for a particular cell type and allows for identification of CD8+, regulatory, or Th17 T cells.

Simultane Quantifizierung von T-Zell-Rezeptor-Excision Kreis (TRECs) und K-Löschen Rekombination Excision Kreis (KRECs) durch Echtzeit-PCR

Zusammenfassung

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