我々は、この原稿でグラフィックデザインや写真撮影のためにデヴィッド·R·ロドリゲス、マサチューセッツ州に感謝したいと思います。この作品は、UTHSCSAフローサイトメトリー中核施設とUTHSCSAアドバンスト核酸基盤施設にGCCRI、NIH / NIA（5R21AG033339）とがんセンターサポート助成金（P30CA054174）からの資金によってサポートされていました。

著者らは、競合する経済的利益を宣言していません。

本明細書に記載のin vivo変異導入アッセイはもともとKohler らによって生成されたLacIトランスジェニックマウスモデルに基づいている。18このモデルは、lacIリレポーター遺伝子を有するλファージベクターを利用する。ベクトルは、感染性ファージ粒子に比較的単純な回復とその後のパッケージングを可能に隣接する2つのCOSサイトは、Eを?...

ほとんどの組織では、分化した細胞は、限られた寿命を持っている。機能的完全性を維持するために、長寿命の、組織特異的幹細胞は、連続的に順番に、その特定の組織の機能に必要な完全に分化した細胞を生じる前駆細胞を産生する。幹細胞は、自己複製と呼ばれるプロセスを通じて、自分の区画を補充する。このように、幹細胞は、彼らがそのためインチ常駐組織の機能的完全性を維持する責任があり、それは彼らが損傷を受けたDNAを検出し、潜在的に修復するために、堅牢なメカニズムが装備されていることが不可欠です。そうでなければ、彼らは彼らの子孫によって継承できる複数のゲノム（潜在的に有害な）摂動を、取得することができる。幹細胞は、生物の寿命の間に彼らのゲノムをセーフガードする方法を理解することは重要な問題であり、ゲノムの不安定性は、癌および（1,2に概説されている）他のいくつかの加齢に伴う疾患にリンクされている理由を私たちが理解するのに役立つことがあります。 3-6の直接DNA修復の特定のタイプを測定することが可能であることを実証した。なお、後者は低い忠実度および製造従ってリスク増加の修復プロセスであり、造血系において、例えば、二本鎖DNAの切断が相同組換え（HR）又は非相同末端結合（NHEJ）によって修復することができることが見出されたエラーが。両方が、造血幹細胞（HSC）4,5に利用されているが、マウスにおいて、早期前駆細胞がHR 4を利用し 、一方、それは造血幹細胞で主にNHEJであると思われる。同様の観察は、皮膚6中の幹細胞のために作られました。興味深いことに、ヒトHSC人事ではないNHEJにおいて、二本鎖切断3のための選択の修復機構であると思われる。 2種間のこの機能的な違いは本物であるか、単に技術的または実験の差を表しているかどうかは、見守らなければならない。 損傷を受けたDNAを修復する幹細胞のレパートリーは、例えば、塩基除去修復（BER）、ヌクレオチド除去修復（NER）およびミスマッチ修復（MMR）のような他のDNA修復機構を含む可能性がある。 MMR修正塩基塩基ミスマッチ、挿入/欠失ループながらBERとNERは、一本鎖DNAに、単一または複数の塩基対の病変を修復するための責任であり、DNA損傷のこれらのタイプは、NHEJまたはHRによって修復することができない。この概念をサポートすることは、HSCコンパートメント7-9に、これらの経路の1の変化と異常の間のリンクを示す造血系だけでなく、骨髄異形成症候群10月16日の発症リスクの増加に起因する疾患からのいくつかの研究であるHSCは、その疾患が進行するにつれて17ゲノム不安定性を増大と関連している。まだ今のように、造血幹細胞を直接、BER、NER、およびMMRの測定が報告されていない。 機構的レベルでの組織の完全性を制御する様々なプロセスを解明することに加えて、これらのプロセスのいずれかの収差の影響が遺伝子対正常に、例えば 、試験することができるように、突然変異したDNAの程度を測定することができることが不可欠である幹細胞を操作または若いに対する古い中。しかし、関連アッセイの開発が原因組織特異的幹細胞の不足および「幹細胞性」を維持し、培養条件の欠如のために困難である。さらに、そのようなアッセイは、環境や遺伝子操作に修正可能である必要があります。これらの制限および要件に可能な解決策は、具体的にはDNAの突然変異を検出するために設計されているマウスモデルの使用である。 MUL変異検出のためのTIPLEトランスジェニックマウスモデルが開発されている。例えば、トランスジェニックマウスのLacI 18 のLacI遺伝子はLacオペレーターのサプレッサーをコードする変異レポーターとして機能するLacIリレポーター系をコードする回復可能なλファージベクターを運ぶ。 LacIリ遺伝子の変異の際に、Lacオペレーターが活性化され、β-ガラクトシダーゼが生成される。それが青色に変わっβ-ガラクトシダーゼの発色性基質を切断するのX-gal（5 - ブロモ-4 - クロロ - 電子 - インドリル-β-D-ガラクトピラノシド）。 のLacIベクトルに隣接するCOSサイトはラムダファージタンパク質およびEのその後の感染により容易に回収することができ大腸菌 。感染したEをインキュベートした後大腸菌は、X-gal基質を含有するアガロースに、プラークをスコア付けすることができる。明確なプラークは非変異体を保有しながら、青斑は、推定される変異型ラック-I有するファージを含んでいる。目の中で青いプラークの周波数（電子明確なもの）は、DNAから抽出された元の細胞集団における突然変異頻度を示す。また、LacIリターゲットをホストするλファージは、容易に、比較的高いスループット分析のためのPCR技術を用いて配列決定することができる。複数の変異型のLacI遺伝子の配列を決定することは今度は特定のDNA修復経路で可能な欠陥または特定の遺伝毒性事象を指すことがあり、突然変異スペクトルに関する重要な情報を明らかにします。 のLacIトランスジェニックシステムは、複数の研究室19全体で標準化されており、試薬は、市販されている。 LacIリシステムの一つの主要な欠点は、大規模な欠失または再配列を検出する能力が限られているので、分裂中期スプレッド上の他の方法、 例えば 、多色FISHは、この欠陥を補完するために使用される必要がある。 LacIリマウスモデルのλファージベクターの中では、はるかに小さい遺伝子、CIIがあります、変異解析のために利用できる。そのサイズおよび変異体を選択することができるという事実は、このlacI遺伝子解析未満の労働集約的かつ安価なアッセイを20にする。しかしながら、lacI遺伝子は、より広範囲に突然変異誘発21に研究されており、発色基質22-25に関する表現型応答を生成するアミノ酸残基の明確な理解が存在するように突然変異遺伝子の感度はよく特徴付けられている。 変異検出のための他のマウスモデルがΦX174またはLacZを導入遺伝子の使用を含む。 T→G：オリジナルAとΦX174トランスジェニックマウスモデル、C復帰突然変異試験26又は塩基対置換のスペクトルの検出を可能に前進突然変異試験27は 、LacIリモデルより安価なシステムを表す。しかし、フォワードアッセイにおける突然変異画面は些細な突然変異の仕様ではありませんΦX174導入遺伝子のスペクトラムは、次のようにのLacIのものと十分に特徴づけされていません。 LacZを導入遺伝子を有するマウスモデルでは、LacZの突然変異レポーターを大腸菌を用いた回収され、ガラクトースおよびガラクトース28を含有する培地に対して感受性である大腸菌宿主細胞。このシステムの欠点は、LacZの対象の回復はまた、E.のライゲーションおよびエレクトロポレーションに続いて、制限エンドヌクレアーゼ消化を含むことであることが困難少数の細胞のためのシステムを適合すること大腸菌ホスト。多数の細胞が必要な場合には（ 例えば百万個以上）、（1、常により多くのマウスで始めることができます）幹細胞/前駆細胞集団を操作するための絶対条件ではありませんが、それはすぐに非現実的で費用対法外になります。また、LacZの比較的大きなサイズの、感受性突然変異レポーターを提供しつつ、DNA配列分析およびdetermために面倒でコストがかかる突然変異スペクトルの追うもの。このモデルの主な利点は、しかしながら、その大きな欠失および挿入を検出する能力、ならびに染色体再編成である。 LacIリ、ΦX174およびLacZトランスジェニックマウスモデル内のすべてのセルがレポーター系を運ぶので、これらのマウスモデルのいずれかは、それらが確実に採取することができるように、幹細胞および前駆細胞を含む、関心対象の任意の細胞型において突然変異誘発を測定するために使用することができる十分な数の。我々はのLacIマウスモデルとのLacI突然変異試験で豊富な経験を持っていたので、我々は造血幹細胞および前駆細胞集団内変異導入解析のための更なるこのシステムを追求することにしました。 造血組織は、リンの極めてまれな集団として同定可能であり、長期再増殖幹細胞を含むその個々の成分の細胞表面表現型の点でよく特徴付けられている- IL7R <suP&gt; - SCA-1 +するcKit + +（LSK）/ Flk2は- CD150 + CD48 -細胞29。 それは、DNA修復を研究になると、細胞はまだHSCのために良いの代表であり、最も原始的な骨髄球前駆細胞（CMP）の人口とは大きく異なる- Mohrin ら 4は LSK/Flk2のわずかに大きい人口があることを明らかにした。また、ときHSCに富むLSK（FLK-2 +およびFLK-2 - ） - IL7R -細胞が林と比較したSCA-1-するcKit + +（LS-K前駆細胞）は、有意差が残っていた純度の低い間NHEJ能力5、細胞に富むLSKの人口および前駆細胞を幹。この細胞数は極めて困難であり、我々は、少なくとも2×10 5個の細胞をこの変異誘発アッセイにおいて、一貫した信頼性のある結果を得るために必要であることを見つけたので、細胞-我々の研究ではHSCに富むLSK（のFlk-2 +およびFlk-2）を使用CD150 + CD48集団またはさえLSK/Flk2 - - （マウス、コストと実用性の点で）人口1はLSK/Flk2をソートする際に取得する。もともとはKohler らによって開発された一方に基づいて、このプロトコルは、 図18は、自発的なDNA突然変異頻度はLSK細胞および分化した骨髄細胞ならびに未分離の骨髄および脾臓細胞の規定された集団において決定することができる方法を詳細に記載している。

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">LacI transgenic mice&#160;</td>
			<td style="width:245px;">BioReliance Corporation</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">RecoverEase DNA Isolation Kit, including the RNace-It ribonuclease cocktail</td>
			<td style="width:245px;">Agilent Technologies (Stratagene)</td>
			<td style="width:195px;">720202</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:252px;">Transpack Packaging Extract, including the orange and blue transpack tubes and the SCS-8 E. coli</td>
			<td style="width:245px;">Agilent Technologies (Stratagene)</td>
			<td style="width:195px;">200221</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA size standards &#8211; lambda ladder</td>
			<td>Bio Rad</td>
			<td>170-3635</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.025 mM pore size membranes&#160;</td>
			<td>Fisher (Millipore)</td>
			<td>VSWP 025 00</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">245 mm2 bioassay dishes (trays)&#160;</td>
			<td>Fisher (Corning)</td>
			<td>07-200-600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NZY broth (powder)</td>
			<td>Fisher (Teknova)</td>
			<td>N1144</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Fisher</td>
			<td>E-3120-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N, N-dimethylformamide</td>
			<td>Fisher</td>
			<td>AC34843-5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal</td>
			<td>Research Products Intl Corp (RPI)</td>
			<td>B71800-10.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3-115-852</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Extender Taq Polimerase kit</td>
			<td>5 PRIME</td>
			<td>2200500</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agencourt AMPure XP cleanup kit</td>
			<td>Beckman/coulter</td>
			<td>A63880</td>
		</tr>
	</tbody>
</table>

identifying dna mutations in purified hematopoietic stem/progenitor cells

近年では、ゲノム不安定性がしっかり多くの発達障害、癌、および加齢に関連していることが明らかになった。幹細胞は生涯を通じて組織の恒常性および修復を確実にする責任があることを考えれば、幹細胞集団は、組織のゲノムの完全性を維持するために重要であると仮定することは妥当である。そのため、重要な関心は、幹細胞ベースの疾患の病因を理解することを目指し、幹細胞およびその子孫ゲノムの完全性に関する内因性および環境要因の影響を評価する際に生じている。 LacIリトランスジェニックマウスは、LacIリ遺伝子が変異レポーターとして機能するLacIリレポーター系をコードする回復可能なλファージベクターを運ぶ。変異したlacI遺伝子の結果は青に回し、切断する発色基質のβ-ガラクトシダーゼの製造である。 LacIリレポーター系は、iを搭載してnの全ての幹/前駆細胞を含む細胞、および容易に回収し、続いてE.を感染させるために使用することができる大腸菌 。感染したEをインキュベートした後正しい基質を含むアガロース上で大腸菌は 、プラークをスコアすることができ、明確なプラークが野生型を保有しながら、青いプラークは、変異型lacI遺伝子を示している。プラーク（クリア間の）青色の周波数は、DNAがより抽出された元の細胞集団における突然変異頻度を示す。変異型lacI遺伝子の配列を決定することは遺伝子の変異の位置および変異の種類が表示されます。 LacIリトランスジェニックマウスモデルは、in vivo変異導入アッセイとして十分に確立されている。また、アッセイのためのマウスおよび試薬は、市販されている。ここでは、このモデルを自発的に幹細胞富化林におけるDNA突然変異体を天然の周波数を測定するように構成することができる方法を詳細に説明する- IL7R -のSca-1 +するcKit + +（LSK）細胞および造血系の他の部分集団。

<html> in vivoでの変異誘発アッセイは、多くのイベント（すべてのPFU）の中で稀な事象（変異のPFU）を測定する。少数の細胞でアッセイを行うことにより、結果が大幅に正偽及び偽陰性の結果に影響されることが可能である。この問題に対処するために、我々は、3つの異なる動物から採取未分画骨髄細胞との連続希釈実験を行った。我々は、1.4×10 6、7.0×10 5を使用して、これら?...

Watch this Scientific Journal Video about Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells at JoVE.com

精製された造血幹/前駆細胞における DNA 変異の同定 |免疫学と感染症 |ビデオ

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

ここでは、LacIリトランスジェニックマウスモデルを用いて精製した造血細胞の数が少ないため、生体内突然変異誘発アッセイを説明します。 lacI遺伝子は、DNA突然変異の頻度、場所、タイプ、自然に生じまたは遺伝毒性物質への曝露後を決定するために単離することができる。

精製造血幹細胞/前駆細胞のDNAの変異を特定する

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

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12.9K ビュー.  UT Health Science Center at San Antonio. ここでは、LacIリトランスジェニックマウスモデルを用いて精製した造血細胞の数が少ないため、生体内突然変異誘発アッセイを説明します。 lacI遺伝子は、DNA突然変異の頻度、場所、タイプ、自然に生じまたは遺伝毒性物質への曝露後を決定するために単離することができる。 

ビデオ: Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

カポジ肉腫関連ヘルペスウイルス（KSHV）によって誘発される調節不全遺伝子を特定

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

免疫・感染学

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

マウスB細胞を精製における誘導とクラススイッチ組み換えの評価

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

ヒト造血細胞のためのコロニー形成細胞（CFC）アッセイ

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

細胞星状に7つのステップ

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

同種造血幹細胞移植後のウイルス感染を予防/治療するMultivirus -特異的T細胞の生成

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

慢性EAEを有するマウスにおける神経幹/前駆細胞の全身注射

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

光活性化単一分子追跡を使用してライブ細菌細胞におけるタンパク質-DNA相互作用を可視化する

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

造血前駆細胞の養子移入を使用して、パイエル板におけるマウス形質細胞様樹状細胞の開発を評価

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...