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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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. 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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 of targets. From the methodological point of view, a critical point is that the frozen plasmid dilutions corresponding to the standard curve points may degrade sooner than expected. This requires that new dilutions from the originally stored plasmid need to be prepared before each experimental procedure, in particular the most dilute ones. In the final calculations, a non-perfect equivalence between lymphocyte plus monocyte count as an estimate of the PBMC quantity may add some bias in the conversion between values expressed per 106 and values expressed per ml of blood. An improvement can be made to determine the amount of two targets (TRECs and KRECs) from a known amount of DNA extracted directly from whole blood, thus eliminating the need of the monocyte plus lymphocyte counts.14 In this specific case, a reference gene is not strictly necessary to perform the quantification, but may be used as an additional DNA quality and quantity check. 
The result interpretation can be affected by some issues. First of all, it should be taken into account that reference ranges change with gender and age, determining marked differences between children and adults. Furthermore, in case of certain immune deficiencies or autoimmune syndromes, a homeostatic peripheral replication of existing memory cells can be present, which dilutes TREC/KREC content and lowers the result of the assay when expressed only per 106 lymphocytes. This may lead to the erroneous conclusion that the thymic/bone marrow output is lower than it actually is. Therefore, it is advisable to always estimate lymphocyte neoproduction by also calculating TRECs/KRECs per ml of blood, which makes results independent of the proliferation rate of memory cells. Third, whatever is the unit of measure, the test result may lead to biased overestimates, due to persistence of some long-lived na&#239;ve lymphocytes that still bear TRECs or KRECs. Therefore, those who are interested in the best possible estimate of &#8220;true&#8221; lymphocyte neoproduction can take advantage of some more complicated mathematical models that also takes into account the lymphocyte apoptosis rate.15
An improvement of assay sensitivity to enhance its use is to perform TRECs and KRECs quantification starting from a limited amount of capillary whole blood spotted on Guthrie card filter paper. This modified version of the assay can be applied with success to the newborn screening of congenital immune deficiencies affecting lymphocyte production,12 and was proved beneficial in terms of cost-efficacy. This simplified version of assay also paves the way to new potential applications, especially considering the high number of potent immune modulating drugs that have been developed in the last few years. However, the &#8220;classical&#8221; TREC/KREC assay herein described has been already applied with success as a routine test in the follow up of children with severe congenital immune deficiencies following hematopoietic stem cell transplantation,4 or for the monitoring of the therapy outcome of autoimmune diseases and acquired immune deficiencies.6-9,11

T細胞受容体切除サークル（TRECs）とK-削除組換え切除サークル（KRECs）を導く、ゲノムDNAの再結合プロセスの間に、それぞれ、T細胞およびB細胞の割合で切除される小さな環状DNAエレメントであるT-およびB細胞受容体の高度に多様なレパートリーの形成。これらは、機能を有さないが、それらは安定であり、複製できないので、それらは、それぞれの細胞分裂後に希釈され、したがって、2つの娘細胞のいずれかでのみ持続する。したがって、末梢血におけるそのレベルは、胸腺および骨髄の出力の推定値として想定することができる。 TRECアッセイは、主として胸腺出力の程度を評価するために、過去15年間にわたって使用されてきたが、最初に開発された1 KRECアッセイは、B細胞増殖および健康および疾患におけるB細胞の恒常性への寄与、2を測定するごく最近骨mのマーカーとして提案されている出力を矢印。3,4ここで、我々はTRECsとKRECs両方の同時定量化のために開発された方法について説明します。4 この組み合わせ方法では、リアルタイムPCRによってDNAの定量に関連する変動はTRECs、KRECsおよびT細胞受容体α定数の断片を含むトリプル挿入プラスミドを希釈したユニークな標準曲線の使用（TCRAC）により除去される1：1比1遺伝子。これはTRECとKRECコピー数をより正確に評価することが可能となる。また、同じ反応における2つの標的の同時定量試薬コストを低減することができる。 提案TREC / KRECアッセイは、重症複合免疫不全（SCID）、4共通変数免疫不全、5自己免疫疾患、6-8とHIV感染の小児や成人におけるT-の程度およびB細胞ネオ産生を測定するために役立ちます図9また、ために使用することができる造血幹細胞移植、10酵素補充、11および抗ウイルス9または免疫調節療法後の免疫の回復を監視する。6-8 SCID患者は、基礎となる遺伝的欠陥にもかかわらず、患者無ガンマグロブリン血症TRECアッセイを用いて認識されるため、最終的に、KREC定量を用いて同定することができる、TREC / KRECアッセイはまた、新生児スクリーニングプログラムにおいて免疫不全を検出するために用いることができる。この場合には12、テストが濾紙上にブロットし、乾燥させ、血液の小さなスポットから抽出されたDNAに対して実施する必要があり、高感度かつ特異的でなければならない対象疾患、ならびに高度に再現可能で費用対効果。 テストでKREC定量の導入は、日常的にウィスコンシン州はintroducする最初になった2008年以来、米国（WI、MA、CA）の一部で行われている免疫不全のために新生児スクリーニングの性能を向上させる必要がありますその出生後のスクリーニングプログラムにおけるTRECsの電子分析。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.

24 45歳の成人 - 60（男性/メス：16分の29）： - 17（17分の25オス/メス）42 0歳の子供：アッセイは、87健常対照の代表的なサンプルで実施した。結果は、10 6 PBMCあたりTRECsとKRECsとして得られたし、その後の血液1mlあたりTRECsとKRECsを算出した。 4年- TRECsの数は、0から3の非常にシャープなファッションに特に胸腺退縮、4に年齢とともに減少します。それは、女性よりも男性においてより急速に減少するので、成人では、TREC番号は、性別に依存する。5これは非常に正確な場合には、年齢、おそらく性別に基づいて区別されるべき適切な基準範囲を設定することで問題を作成すること正規性の推定が必要である。 <img alt="図3" src="/files/ftp_upload/52184/52184fig3display.jpg" /> 健康DO図3. TREC定量<html>否定論理和をとる。TRECsの量は、同じ対象（C、D）にTRECs個/ mlとして計算次にTRECs健康な小児における/ 10 6細胞（A）および成人（B）として決定した。クリアサークル：メス。黒丸：男性。線は対数変換データを用いた線形回帰によって得られた。小児では、2回帰ラインがより良い年齢とともに減少するTRECの可変速度を表現するために適合させた。成人では、破線は、男性（実線）よりも遅い女性で見られるTRECs、年齢とともに減少の傾向を表す。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52184/52184fig3highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 成人の骨髄出力は生涯を通じてかなり安定しており、性別に依存しないのに対し、KRECsの数は、子供たちだけでTRECsと同様のパターンで、年齢とともに減少する。 常に &quot;&gt;：&quot; =キープtogether.withinページFO」_content <img alt="図4" src="/files/ftp_upload/52184/52184fig4display.jpg" /> 図4：健康なドナーにおけるKREC定量は KRECsの量は健康な小児（A）と同一の被験者（C、D）でKRECs個/ mlとして計算し、その後成人（B）およびKRECs / 10 6細胞として測定した。クリアサークル：メス。黒丸：男性。線は対数変換データを用いた線形回帰によって得られた。小児では、2回帰ラインはより良い年齢とともにKREC低下の変動金利を表現するために適合させた。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52184/52184fig2highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 代表的な目的のために、単純な対数線形回帰モデルは、年齢とともにTREC / KREC低下のパターンを示すように適合させた、より洗練された非線形モデルは、iを取り付けることができるより良い年齢とともに一見、指数KREC / TRECの減少を予測するnの試み。

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.

リアルタイムPCRによるT細胞受容体切除サークル（TRECs）とK-削除換え切除サークル（KRECs）の同時定量

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

Universidad San Sebastian

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 ビュー.  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.

ビデオ: 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.

双方向レトロウイルスの統合サイトPCRの方法論と定量的データ解析ワークフロー

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

遺伝学

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: キラー細胞免疫グロブリン様受容体遺伝子の定量的半自動化された入力

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.

生きている細胞の単一テロメアから表現されるテロメア繰り返し含む RNA テラを視覚化するがん細胞クローンの生成

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>CIRCLE-SeqによるオフターゲットCRISPR-Cas9切断イベントの検出

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>オフターゲット遺伝子編集の検討のためのCIRCLE-Seq

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.

オフターゲット遺伝子編集の検討のためのCIRCLE-Seq

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.

合理化単細胞 TCR 分離し体外の Retroviral ベクトルの生成、人間 Tcr の生体内で発現

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 ...

免疫・感染学

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).

反ベクトルと反遺伝子特定 CD8 の同時定量+ T はマイク ウイルスベクターを用いたワクチン後染色テトラマー MHC を介して細胞

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.

病変の特異的分子ビーコンを活用した細胞ライセート中の塩基除去修復活性の定量、リアルタイム分析

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

生物学

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.

リアルタイムPCRによるT細胞受容体切除サークル（TRECs）とK-削除換え切除サークル（KRECs）の同時定量

要約

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この動画の章

双方向レトロウイルスの統合サイトPCRの方法論と定量的データ解析ワークフロー

qKAT: キラー細胞免疫グロブリン様受容体遺伝子の定量的半自動化された入力

生きている細胞の単一テロメアから表現されるテロメア繰り返し含む RNA テラを視覚化するがん細胞クローンの生成

オフターゲット遺伝子編集の検討のためのCIRCLE-Seq

オフターゲット遺伝子編集の検討のためのCIRCLE-Seq

オフターゲット遺伝子編集の検討のためのCIRCLE-Seq

合理化単細胞 TCR 分離し体外の Retroviral ベクトルの生成、人間 Tcr の生体内で発現

反ベクトルと反遺伝子特定 CD8 の同時定量⁺ T はマイクウイルスベクターを用いたワクチン後染色テトラマー MHC を介して細胞

病変の特異的分子ビーコンを活用した細胞ライセート中の塩基除去修復活性の定量、リアルタイム分析

エピジェネティックベースの定量PCRを用いた免疫細胞の同一性と純度の測定

リアルタイムPCRによるT細胞受容体切除サークル（TRECs）とK-削除換え切除サークル（KRECs）の同時定量

要約

さらに動画を探す

この動画の章

双方向レトロウイルスの統合サイトPCRの方法論と定量的データ解析ワークフロー

qKAT: キラー細胞免疫グロブリン様受容体遺伝子の定量的半自動化された入力

生きている細胞の単一テロメアから表現されるテロメア繰り返し含む RNA テラを視覚化するがん細胞クローンの生成

オフターゲット遺伝子編集の検討のためのCIRCLE-Seq

オフターゲット遺伝子編集の検討のためのCIRCLE-Seq

オフターゲット遺伝子編集の検討のためのCIRCLE-Seq

合理化単細胞 TCR 分離し体外の Retroviral ベクトルの生成、人間 Tcr の生体内で発現

反ベクトルと反遺伝子特定 CD8 の同時定量+ T はマイク ウイルスベクターを用いたワクチン後染色テトラマー MHC を介して細胞

病変の特異的分子ビーコンを活用した細胞ライセート中の塩基除去修復活性の定量、リアルタイム分析

エピジェネティックベースの定量PCRを用いた免疫細胞の同一性と純度の測定

反ベクトルと反遺伝子特定 CD8 の同時定量⁺ T はマイクウイルスベクターを用いたワクチン後染色テトラマー MHC を介して細胞