The authors have no acknowledgements.

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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 세포 수용체 절제 원 (TRECs) 및 K-삭제 재조합 절제 원 (KRECs)는 게놈 DNA 재조합 과정 T- 각각 B-세포의 비율로 절제되어 작은 원형으로 DNA 요​​소이다 선도 매우 다양한 레퍼토리의 T- 및 B- 세포 수용체의 형성. 이들은 어떤 기능이 없지만, 그들이 안정되고 복제 될 수 없기 때문에, 따라서 그들은 단지 두 개의 딸 세포 중 하나로 지속 각 세포 분열 후에 희석된다. 따라서, 말초 혈액에서의 수준은 흉선 및 골수의 출력 추정값으로서 가정 될 수있다. TREC 분석은 주로 흉선 출력의 정도를 평가하기 위해 지난 15 년 동안 사용되었지만, 초기에 개발 된 1 KREC 분석은 B 세포의 증식 및 건강과 질환에서의 B 세포의 항상성에 기여 2 측정 최근 골 m의 마커로서 제안되었다출력 화살표. 3,4 여기, 우리는 우리가 TRECs과 KRECs 모두의 동시 정량을 위해 개발 된 방법을 설명합니다. (4) 이 조합 된 방법으로, 실시간 PCR에 의해 DNA 정량 연관된 가변성 TRECs, KRECs 및 T 세포 수용체 알파 상수의 단편을 함유하는 트리플 삽입 플라스미드를 희석 한 고유의 표준 곡선을 사용 (TCRAC)에 의해 제거된다 1 : 1 : 1 비율의 유전자. 이것은 TREC와 KREC 카피 수의 더 정확한 평가를 할 수있다. 또한, 동일한 반응에서 두 타겟의 동시 정량 시약 비용 감소를 허용한다. 제안 된 TREC / KREC 분석은 중증 복합 면역 결핍 (SCID), 4 공통 변수 면역 결핍, 5자가 면역 질환, 6-8과 HIV 감염 어린이 또는 성인의 T-의 범위와 B 세포 신 생산을 측정하기 위해 유용 할 수 있습니다 . (9) 또한,이 사용 가능환자 KREC 정량화를 사용하여 식별 될 수 agammaglobulinemia SCID 환자 근본적인 유전 적 결함에도 불구 TREC 분석법을 인식하고 있기 때문에, 최종적. 6-8, 조혈 줄기 세포 이식, 10 효소 대체 (11)와 바이러스 9 또는 면역 요법 후에 면역 회수를 모니터링하고, , TREC / KREC 분석은 또한 신생아 스크리닝 프로그램에서 immunodeficiencies를 검출하는데 사용될 수있다. (12)이 경우, 시험 혈액 도말의 작은 반점에서 추출한 DNA에 대해 수행하고 여과지상에서 건조되어야 고도로 민감하고 특이이어야 표적 질환뿐만 아니라 재현성이 높은 비용 효율적인. 위스콘신부터 소개하는 첫번째되었을 때 시험에 KREC 정량의 도입은 일상적으로 2008 미국 (WI, MA, CA)의 일부 지역에서 수행 된 immunodeficiencies에 대한 신생아 선별 검사의 성능을 개선한다그 산후 검진 프로그램에 TRECs의 전자 분석. (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.

분석은 87 건강한 대조군의 대표 샘플에서 수행 하였다 : (42) 어린이는 0 세 - 17 (남성 / 여성 : 17분의 25)와 24 세 (45) 성인 - 60 (남성 / 여성 : 16분의 29)을. 결과를 계산 하였다 TRECs 10 6 PBMC 당 KRECs 후 혈액 1 ㎖ 당과 TRECs KRECs로서 수득 하였다. 4 년 - TRECs의 수는 인해 0-3 매우 날카로운 방식으로 특히 흉선 퇴화, 4 세 감소한다. 그것은 여성보다 남성에게 더 빠르게 감소하기 때문 성인에서, TREC 번호도 성별에 의존한다. (5)이, 연령 및 가능 성별에 기초하여 차별화 매우 정밀한 경우되어야 적절한 참고 범위를 설정하는 문제를 생성 할 수있다 정상의 추정이 필요하다. <img alt="그림 3" src="/files/ftp_upload/52184/52184fig3display.jpg" /> 건강한 DO 그림 3. TREC 정량<html> nors. TRECs의 양은 TRECs / 건강한 소아 10 (A)와 동일한 피험자 (C, D)에서 TRECs / ㎖로 계산 한 다음 성인 (B) 및 6 개의 세포로 하였다. 클리어 원 : 여성; 채워진 원 : 남성. 선은 로그 변환 데이터를 사용하여 선형 회귀에 의해 수득 하였다. 어린이, 두 회귀 라인은 더 나이 TREC 감소의 가변 속도를 표현하기 위해 설치되었다. 성인의 경우, 점선은 남성 (연속 선)에서보다 느린 여성에서 볼 TRECs의 나이가 감소 추세를 ​​나타냅니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52184/52184fig3highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 성인의 골수 출력 수명 동안 비교적 안정하고, 성별에 의존하지 않는 반면 KRECs의 수는 단지 아이들 TRECs 유사한 패턴 나이 감소한다. 항상 &quot;&gt; :&quot;유지 - together.within 페이지를 = FO &quot;_content <img alt="그림 4" src="/files/ftp_upload/52184/52184fig4display.jpg" /> 도 4 :. 건강한 기증자 KREC 정량화 KRECs의 양은 건강한 소아 (A)와 동일한 피험자 (C, D)에서 KRECs / ㎖로 계산 한 다음 성인 (B)과의 KRECs / 106 개 세포로 하였다. 클리어 원 : 여성; 채워진 원 : 남성. 선은 로그 변환 데이터를 사용하여 선형 회귀에 의해 수득 하였다. 어린이, 두 회귀 라인이 더 나이 KREC 감소의 가변 속도를 표현하기 위해 설치되었다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52184/52184fig2highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 대표적인 목적을 위해, 간단한 로그 - 선형 회귀 모델은 연령 TREC / KREC 감소의 패턴을 묘사에 장착했지만,보다 정제 비선형 모델 I가 끼워 질 수있다N 시도가 더 나이가 겉으로는 지수 KREC / TREC 감소를 예측합니다.

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.

T 세포 수용체 절제술 원의 동시 정량 (TRECs)와 K-삭제 재조합 절제술 원 (KRECs) 실시간 PCR에 의한

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

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Universidad San Sebastian

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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 조회수.  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.

비디오: 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.

양방향 레트로 바이러스 통합 사이트 PCR 방법론 및 정량적 데이터 분석 워크 플로

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

연구

유전학

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: 킬러 세포 같은 면역 글로불린 수용 체 유전자의 양적 반자동 입력

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.

Telomeric 반복 포함 된 RNA 테라에서 살아있는 세포에서 단일 Telomere 표현 시각화 하 암 세포 클론의 세대

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-Seq를 사용한 off-target CRISPR-Cas9 분할 이벤트 검출

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>off-target 유전자 편집에 대한 조사를 위한 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.

off-target 유전자 편집에 대한 조사를 위한 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.

단일 셀 TCR 고립과 Retroviral 벡터 생체 외에서 의 생성과 Vivo에서 표현의 인간의 TCRs 간소화

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 ...

면역학 및 감염병학

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).

반대로 벡터 및 안티 transgene 특정 CD8의 동시 정량화+ T 세포가 MHC를 통해 내가 Tetramer 바이러스 성 벡터와 예방 접종 후 얼룩

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.

병변 특정 분자 비콘을 활용 세포 Lysates의 기본 절단 복구 활동의 양적, 실시간 분석

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

생물학

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.

T 세포 수용체 절제술 원의 동시 정량 (TRECs)와 K-삭제 재조합 절제술 원 (KRECs) 실시간 PCR에 의한

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양방향 레트로 바이러스 통합 사이트 PCR 방법론 및 정량적 데이터 분석 워크 플로

qKAT: 킬러 세포 같은 면역 글로불린 수용 체 유전자의 양적 반자동 입력

Telomeric 반복 포함 된 RNA 테라에서 살아있는 세포에서 단일 Telomere 표현 시각화 하 암 세포 클론의 세대

off-target 유전자 편집에 대한 조사를 위한 CIRCLE-Seq

off-target 유전자 편집에 대한 조사를 위한 CIRCLE-Seq

off-target 유전자 편집에 대한 조사를 위한 CIRCLE-Seq

단일 셀 TCR 고립과 Retroviral 벡터 생체 외에서 의 생성과 Vivo에서 표현의 인간의 TCRs 간소화

반대로 벡터 및 안티 transgene 특정 CD8의 동시 정량화⁺ T 세포가 MHC를 통해 내가 Tetramer 바이러스 성 벡터와 예방 접종 후 얼룩

병변 특정 분자 비콘을 활용 세포 Lysates의 기본 절단 복구 활동의 양적, 실시간 분석

후성유전학 기반 정량적 PCR을 이용한 면역 세포 정체성 및 순도 측정