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
Summary
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.
Abstract
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.
Introduction
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
Protocol
Representative Results
Discussion
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 …
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Histopaque-1077 | Sigma Aldrich SRL | 10771-500 ML | density gradient separation method |
| QIAamp DNA Blood Mini Kit (250) | QIAGEN | 51106 | DNA extraction |
| Unmodified DNA Oligonucleotides HPSF 0.01 mmol | Eurofins MWG Operon/Carlo Erba Reagents S.r.l | Resuspend the lyophilized product to 100 picmol/µl | |
| AmpliTaq DNA Polymerase: including 10x Buffer II and 25mM MgCl2 | Applied Biosystems/Life-Technologies | N8080156 | |
| GeneAmp dNTP Blend (100 mM) | Applied Biosystems/Life-Technologies | N8080261 | |
| TOPO TA Cloning Kit for Subcloning | Invitrogen/Life-Technologies | K4500-01 | |
| XL1-Blue Subcloning Grade Competent Cells | Stratagene | 200130 | |
| PureYield Plasmid Miniprep System | Promega | A1223 | |
| SpeI 500U | New England Biolabs | R0133S | |
| HindIII-HF 10,000 U | New England Biolabs | R3104S | |
| PureYield Plasmid Midiprep System | Promega | A2492 | |
| XhoI 5,000 U | New England Biolabs | R0146S | |
| TRIS Utrapure | Sigma Aldrich SRL | T1503 | |
| EDTA | Sigma Aldrich SRL | E5134 | |
| TE buffer (1 mM TRIS and 0.1 mM EDTA) | |||
| TaqMan Universal PCR Master Mix | Applied Biosystems/Life-Technologies | 4364338 | |
| Dual labeled probes HPLC 0.01 mmol | Eurofins MWG Operon/Carlo Erba Reagents S.r.l | Resuspend the lyophilized product to 100 picmol/µl | |
| NanoDrop 2000c spectrophotometer | ThermoFisher | ||
| Applied Biosystems 2720 Thermal Cycler | Applied Biosystems/Life-Technologies | 4359659 | |
| Fast 7500 Real-Time PCR system | Applied Biosystems/Life-Technologies | ||
| SDS Sequence Detection Software 1.4 | Applied Biosystems/Life-Technologies |
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