この作品は、ワシントン大学、プログラム、プロジェクトグラントNS032636（BJS）にNIH神経青写真コアグラント（P30 NS057105、BJS）によってサポートされており、神経疾患のためのホープセンターでされました。

1。レンチウイルスベクターのパッケージングレンチウイルスベクターは、レンチウイルストランスファーベクターとリン酸カルシウムトランスフェクション法により293T細胞にパッケージングするために必要な他のプラスミドのトランスフェクションによって生成されます。我々は、このプロトコルでは10〜100 mmの組織培養皿を使用しています。これは、スケールアップまたはダウンしてアプリケーションに応じてすることができます。 293T細胞株は、10％ウシ胎児血清（FBS）、100単位/ mlペニシリン、37°C​​で100μg/ mlのストレプトマイシンを添加し、高グルコース（4500 mg / L）をしてダ​​ルベッコ改変イーグル培地（DMEM）で維持され5％CO 2インキュベーター。 <ol><li>培地中の10〜100 mmの組織培養皿（3×10 6細胞/シャーレ）に30〜40％コンフルエントでシード293T細胞。インキュベーターにセルを返します。 </li><li> 20から24時間の培養後、細胞密度を確認してください。細胞はトランスフェクション時の約80％コンフルエントでなければなりません。 </li><li> 50 mlチューブを準備します。 4.4ミリリットルを追加TE79/10（1 mMのトリス、0.1mMのEDTA、pH7.9）を引いた、次のプラスミドDNAの合計量。 100μgのレンチウイルストランスファープラスミド（ 図1）、58μgのpMDLg / pRRE、31μgののpCMV-G、25μgのPRSV-REV、600μlの2M CaCl 2を追加します。穏やかに混合する。 </li><li>別の50 mlチューブを準備します。 5ミリリットル2倍HBS（0.05 MのHEPES、0.28 MのNaCl、1.5mMののNa 2 HPO 4、pHは7.12）を追加します。 </li><li> 10 mlのピペットでDNA-CaCl 2の混合物を取って、チューブをボルテックスしながら滴下し、2×HBSを含むチューブに追加します。 </li><li> 30分間室温（RT）で沈​​殿反応を保持します。 </li><li>インキュベーターから培養皿を取り外します。ボルテックスでよく沈殿反応を混在させること。細胞を含む各100 mmディッシュに懸濁液1 mlを追加します。懸濁液を静かに皿の中の培地を旋回しながらゆっくりと滴下しなければなりません。インキュベーターにこれらの料理を返し、5時間放置する。 </li><li>培養液から培地を除去します。 6mMのBUナトリウムを含む6 mlの新鮮な培地を追加します。各ディッシュにtyrate。インキュベーターに文化を返します。構文内の蛍光レポーターがある場合は、一晩培養後、蛍光顕微鏡下でレポーター遺伝子の発現を確認してください。それがユビキタスプロモーター（例えば、CMVプロモーター）により駆動されている場合は、通常細胞の80％以上は、レポーター遺伝子を発現する。 </li><li>トランスフェクションの2日後（40から44 h）は、2〜50 mlのチューブ（約30ml各チューブ）に10皿から上清を収集します。 -80℃の冷凍庫で上清を凍結したり、次のステップに進みます。 </li></ol> 2。ベクターの濃度および精製<ol><li>新たに10分間上清中に任意の細胞破片を除去するために収集または900グラム（2000回転程度）で上清を融解を遠心分離します。 </li><li> 0.2μmのSFCAシリンジフィルターを60 mlの注射器を取り付けます。シリンジに50 mlチューブから上清を移します。ポリアロマーの遠心チューブに上清をフィルタリングします。 </li><li> 5 mlのピ​​ペットで5ミリリットルの20％スクロース（PBSで調製）を取る。挿入上清を含む遠心管の底までピペット。徐々にベクトル上清の下でのショ糖溶液を追加します。別のチューブから上清について、この手順を繰り返します。 </li><li> °Cで4時間ベックマンSW28スイングローターで11000回転と4で上清を遠心分離します。 </li><li>上清を除去します。各遠心チューブに150μlの4％の乳糖を（PBS中で調製）を追加します。ペレットを再懸濁します。 </li><li>すべての遠心チューブから1.5 mlチューブに濃縮されたベクトルを転送します。 15分間チューブを氷上にしておきます。 </li><li>ピペッティングによりベクトル懸濁液を混ぜる。 1分間、フルスピード（16000約g）で遠心してスピン。 </li><li>新しい1.5 mlチューブに上清を移します。 20μlのアリコートに最終的なサンプルを分割し、-80℃の冷凍庫でそれらを格納します。 </li></ol> 3。ベクターの滴定<ol><li>シード5×10 4 /ウェルの10％FBSを添加し1ミリリットルDMEM培地で12ウェルプレートでHT1080細胞の。 </li><li> overnig後HT文化は、1つのウェルから細胞をカウントし、細胞数を獲得。 </li><li>培養液で濃縮されたベクトルの：5倍希釈（および1:625; 1:125 25 1:5、1）を行います。別々のウェルに各希釈ベクトルの私は、液を追加します。サンプルは、精度を向上させるために複製される場合があります。 </li><li>各ウェルを含むベクトルやベクトルせずにウェルに1μlの4 mg / mlのポリブレン（Hexadimethrine臭）を追加します。静かにプレートを振とうして混合します。 48時間インキュベーターに戻す。 </li><li>細胞培養ウェルから培地を除去します。 PBSで各ウェルを洗浄します。細胞に250μlの1X trypsine-EDTA溶液を追加します。細胞は（3-5分）デタッチされたときに、1 mlの培地を追加します。ピペッティングにより細胞を再懸濁します。 1.5 mlの遠心チューブに細胞懸濁液を移します。 </li><li> 6分間900gで遠心します。蛍光レポーター遺伝子（例えばGFP）とのベクトルの場合、FACS分析のために3.7に進みます。レポーターせずにベクトルの場合、リアルタイム定量PCRは3.8に進みます。 </li><li>蛍光を含むベクトルのrescentレポーター遺伝子、上清を除去し、PBS中3.7％ホルムアルデヒド300μlでペレットを再懸濁します。 FACS分析によりレポーター陽性細胞の割合を決定します。力価はミリリットルの濃ベクトル当たり形質導入ユニット（TU / ml）として表されます。 </li></ol><img alt="式（1）" src="/files/ftp_upload/4031/4031eq1.jpg" /> 1月25日μL（0.04μl）をベクトルと30％の細胞はレポーター陽性であると、例えば1×10 5細胞が形質導入された場合、力価は次のようになります。 <img alt="式（2）" src="/files/ftp_upload/4031/4031eq2.jpg" />唯一の希釈液を使用して力価を計算するために追加された陽性細胞の割合とベクトル量との間に直線関係に落ちる。最後の力価は、ベクトルの少なくとも2つの異なる量の伝達経路から得られた抗体価の平均値である必要があります。 <ol start="8"><li>せずにベクトルの蛍光レポーター遺伝子は、製造業者のプロトコルに従って別途準備DNA Mini Kit（キアゲン社）を用いてHT1080細胞からゲノムDNAを抽出します。プライマー（HIV-1のPBS / PSI領域17）の5&#39;-CCGTTGTCAGGCAACGTG-3 &#39;および5&#39;-AGCTGACAGGTGGTGGCAAT-3&#39;とABI PRISM 7000配列検出システム（Applied Biosystems）を用いてゲノムDNAのベクター配列を増幅、およびTaqManプローブ5 &#39;-FAM-AGCTCTCTCGACGCAGGACTCGGC-TAMRA-3&#39;。ゲノムにおけるシングルコピー遺伝子であるアルブミン遺伝子（2コピー/細胞）もプライマー5&#39;-TGAAACATACGTTCCCAAAGAGTTT-3 &#39;および5&#39;-CTCTCCTTCTCAGAAAGTGTGCATAT-3&#39;、およびプローブ5&#39;-FAM-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3で増幅された&quot;内部統制として。次のプログラムで製造元の指示に従って、96ウェルプレートにコピーベクターの数とPCR法によるアルブミンを決定します15秒50℃2分間、95℃で10分間、および95の35サイクルのC°Cと60°Cで2分間。既知濃度のプラスミドの10倍希釈系列（コピーのように表さ数）の鋳型配列を含有するにも未知サンプルの定量化のための標準曲線を作成するために増幅されるべきである。力価はミリリットルの濃ベクトル当たり統合ユニット（IU / ml）として表されます。 </li></ol><img alt="式（3）" src="/files/ftp_upload/4031/4031eq3.jpg" /> 4。大脳新皮質文化の伝達ニューロンとグリアの両方を含む新皮質の培養物は、前述の18として二段階めっきプロシージャを使用して、マウスの大脳皮質から調製される。 14から16日間の妊娠で胎児マウスから得られたneocortexの複数形は、10％FBS、24ウェル組織培養プレートに20mMグルコースおよび2mMグルタミンを添加したMEMで以前に確立されたグリア細胞単層上に播種されています。 <ol><li> in vitroで 5日後、neocで10μMシトシンアラビノシド（ARA-C）を追加する非神経細胞分裂を阻害するortical文化。文化の2日間細胞を続行します。 </li><li> 5〜10分間37の暖かい培養培地は、°Cの水浴。 ARA-Cは新鮮な培養液（500μL/ウェル）とを含む培地に置き換えます。 </li><li>文化への理想のMOI（標的細胞の数にベクター粒子数の比率感染多重度）を持つベクトルを追加します。 24時間培養を継続します。我々は、プライマリ皮質培養で（通常は5）1-10 MOIを使用しています。 </li><li>新鮮な培地で培養液を交換してください。培養を継続します。ベクターコンストラクトにレポーター遺伝子がある場合は、2日間導入後、蛍光顕微鏡で細胞を確認してください。レポーター遺伝子の発現は、ベクターの設計と使用量に応じて、導入後のニューロン2-7に表示されます。 </li></ol> 5。代表的な結果このプロトコルの範囲で生産レンチウイルスベクターの力価は10 8 -10 10 IU / mlで、whichは、in vitroおよび in vivo の両方で CNSから種々の細胞型の伝達に適しています。 表1および図2は、このプロトコルによって生成されたベクターを用いた代表的な結果を示しています。我々は、形質導入 シナプシン（SYN）プロモーターまたはグリア線維性酸性タンパク質（GFAP）のプロモーターによって制御される緑色蛍光タンパク質（GFP）を発現するレンチウイルスベクターによるマウスの新皮質の文化。七日間の導入後、私たちはそれぞれ、抗NeuNおよび抗GFAP抗体を用いて神経細胞やアストロサイトを標​​識するために免疫染色を行っ。 表1及び図に示すように。図2Aは 、ニューロンの90％（NeuN +細胞）を介して、シナプシンプロモーターを有するベクターで形質導入した後にGFPを発現していないアストロサイト（GFAP +細胞）は、このレポーター遺伝子を発現する。 GFAPプロモーターは、ベクターコンストラクト（ 図2B）、アストロサイトの約80％（GFAP +細胞）発現に使用される場合のGFP、GFAPとNeuNで標識した細胞でのGFPの発現の有無と局在によって確認としてすべてのGFP +細胞はアストロサイトがあります。これらの結果は、レンチウイルスベクターは、中枢神経系および細胞特異的遺伝子発現から細胞に導入遺伝子を提供する非常に効率的であることを示す適切なプロモーターが使用されているときに達成することができます。 <img alt="図1" src="/files/ftp_upload/4031/4031fig1.jpg" /> 図1。 HIVベースのレンチウイルスベクターとパッケージングプラスミドの概略図。HIV-1プロウイルスは上部に表示されます。ベクトル生産のための要素は4つの異なるプラスミドに分けられています。プラスミドレンチ転送は、U3領域はパッケージングシグナル（ψ）、RRE配列を、中央polypurine路（cPPT）、目的の遺伝子（例えば、サイトメガロウイルス（CMV）プロモーターに置換されているハイブリッドの5 &#39;LTRを含む蛍光選択肢のプロモーターと一緒にレポーター）、および3 &#39;LTRのシス調節配列は完全にU3領域から削除されます。 pMDLg / pRREは、CMVプロモーターの制御下でHIV-1からgagおよびpol遺伝子とRRE配列を含んでいる。 PRSV-RevはRSVプロモーターにより駆動されるリビジョンのコーディング配列を含んでいます。のpCMV-Gは、CMVプロモーターの制御下でVSV-G蛋白質遺伝子を含んでいます。 PAは、ヒトβ-グロビン遺伝子からのポリアデニル化シグナルを示しています。 <img alt="図2" src="/files/ftp_upload/4031/4031fig2.jpg" /> 図2。マウス新皮質の混合培養におけるレポーター遺伝子の発現は、細胞型特異的プロモーターを担持するレンチウイルスベクターで導入した。培養はLV-SYN-GFP（A）または（B）MOI 5でLV-GFAP-GFPベクターを形質導入した。七日間の導入後、細胞は、抗NeuN又は抗GFAP抗体で免疫染色した。上部のパネルには、GFPの蛍光を示し、中央のパネルには、免疫染色を示し、下側のパネルは、（GFP：緑色、NeuN又はGFAP：赤）の画像をマージされます。 </P&gt; <table border="1"><tbody><tr><td> ベクトル </td><td> 神経細胞におけるGFP +細胞 </td><td> GFP +アストロサイトにおける </td></tr><tr><td> LV-SYN-GFP </td><td> 92.2±7.3 </td><td> 0 </td></tr><tr><td> LV-GFAP-GFP </td><td> 0 </td><td> 78.3±11.5 </td></tr></tbody></table> 表1。マウスの新皮質の培養物中のGFP発現の比較は、異なるプロモーター運ぶレンチウイルスベクターで導入した。 マウス新皮質の培養物は、（5×10 5 /ウェルで24ウェルプレート）MOI 5でLV-SYN-GFPまたはLV-GFAP-GFPで形質導入した。導入7日後には、文化は固定されており、NeuN又はGFAPのために免疫染色した。 GFPとNeuN / GFAPを発現する細胞の数は、実験条件ごとに10分野からの画像で計数した。値は、ニューロンの割合（NeuN +細胞）を表すか、また、GFPレポーター遺伝子を発現するアストロサイト（GFAP +細胞）。示された値は、平均±SD、3つの独立した実験からである。

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Gene Ther. 12, 981-997 (2001).</li><li>Broussau, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+packaging+cells+for+large-scale+production+of+lentiviral+vectors+in+serum-free+suspension+culture.">Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.</a> Mol. Ther. 16, 500-507 (2008).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J. Vis. Exp. (32), e1499 (2009).</li><li>Sastry, L., Johnson, T., Hobson, M. J., Smucker, B., Cornetta, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Titering+lentiviral+vectors:+comparison+of+DNA,+RNA+and+marker+expression+methods.">Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods.</a> Gene Ther. 9, 1155-1162 (2002).</li><li>Snider, B. J., Lobner, D., Yamada, K. A., Choi, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioning+heat+stress+reduces+excitotoxic+and+apoptotic+components+of+oxygen-glucose+deprivation-induced+neuronal+death+in+vitro.">Conditioning heat stress reduces excitotoxic and apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro.</a> J. Neurochem. 70, 120-129 (1998).</li><li>Mazarakis, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies+virus+glycoprotein+pseudotyping+of+lentiviral+vectors+enables+retrograde+axonal+transport+and+access+to+the+nervous+system+after+peripheral+delivery.">Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.</a> Hum. Mol. Genet. 10, 2109-2121 (2001).</li><li>Kato, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-specific+gene+transfer+through+retrograde+transport+of+lentiviral+vector+pseudotyped+with+a+novel+type+of+fusion+envelope+glycoprotein.">Neuron-specific gene transfer through retrograde transport of lentiviral vector pseudotyped with a novel type of fusion envelope glycoprotein.</a> Hum. Gene Ther. 22, 1511-1523 (2011).</li><li>Segura, M. M., Garnier, A., Durocher, Y., Ansorge, S., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+protocol+for+lentiviral+vector+mass+production.">New protocol for lentiviral vector mass production.</a> Methods Mol. Biol. 614, 39-52 (2010).</li><li>Kutner, R. H., Puthli, S., Marino, M. P., Reiser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+production+and+concentration+of+HIV-1-based+lentiviral+vectors+using+HYPERFlask+vessels+and+anion+exchange+membrane+chromatography.">Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography.</a> BMC Biotechnol. 9, 10 (2009).</li><li>Laughlin, M. A., Chang, G. Y., Oakes, J. W., Gonzalez-Scarano, F., Pomerantz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+butyrate+stimulation+of+HIV-1+gene+expression:+a+novel+mechanism+of+induction+independent+of+NF-kappa+B.">Sodium butyrate stimulation of HIV-1 gene expression: a novel mechanism of induction independent of NF-kappa B.</a> J Acquir Immune Defic Syndr Hum Retrovirol. 9, 332-339 (1995).</li><li>Gasmi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirements+for+efficient+production+and+transduction+of+human+immunodeficiency+virus+type+1-based+vectors.">Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors.</a> J. Virol. 73, 1828-1834 (1999).</li><li>Palsson, B., Andreadis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physico-chemical+factors+that+govern+retrovirus-mediated+gene+transfer.">The physico-chemical factors that govern retrovirus-mediated gene transfer.</a> Exp. Hematol. 25, 94-102 (1997).</li><li>Lizee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum. Gene Ther. 14, 497-507 (2003).</li><li>Lee, J. K., Chung, J., McAlpine, F. E., Tansey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulator+of+G-Protein+Signaling-10+Negatively+Regulates+NF-{kappa}B+in+Microglia+and+Neuroprotects+Dopaminergic+Neurons+in+Hemiparkinsonian+Rats.">Regulator of G-Protein Signaling-10 Negatively Regulates NF-{kappa}B in Microglia and Neuroprotects Dopaminergic Neurons in Hemiparkinsonian Rats.</a> J. Neurosci. 31, 11879-11888 (2011).</li><li>Shevtsova, Z., Malik, J. M., Michel, U., Bahr, M., Kugler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promoters+and+serotypes:+targeting+of+adeno-associated+virus+vectors+for+gene+transfer+in+the+rat+central+nervous+system+in+vitro+and+in+vivo.">Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo.</a> Exp. Physiol. 90, 53-59 (2005).</li></ol>

利害の衝突が宣言されません。

このプロトコルでは、新皮質の培養物中のこれらのベクトルのレンチウイルスベクターと、アプリケーションの生産を示している。我々は、これらの方法で製造されたベクターを用いた効率的な細胞型固有の伝達を示した。シナプシンプロモーターが使用されている場合、GFPの発現は厳密には特定のニューロンである。 GFAPプロモーターが使用されている場合、GFPの発現はアストロサイトで?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich社</td><td> D5796 </td></tr><tr><td> MEM </td><td>インビトロジェン</td><td> 11090-081 </td></tr><tr><td>ウシ胎仔血清</td><td>ハイクローン</td><td> SV3001403 </td></tr><tr><td> PBS </td><td>メディアテック</td><td> 21から040-CM </td></tr><tr><td>トリプシン-EDTA </td><td> Sigma-Aldrich社</td><td> T3924 </td></tr><tr><td>酪酸ナトリウム</td><td> Sigma-Aldrich社</td><td> B5887 </td></tr><tr><td> Hexadimethrineブロマイド（ポリブレン） </td><td> Sigma-Aldrich社</td><td> H9268 </td></tr><tr><td> 293T細胞</td><td> ATCC </td><td> CRL-11268 </td></tr><tr><td> HT1080細胞</td><td> ATCC </td><td> CCL-121 </td></tr><tr><td>ファルコン100×20ミリメートル組織cultureの皿</td><td> BDバイオサイエンス</td><td> 353003 </td></tr><tr><td> 1×3½polyallomoerの遠心チューブに</td><td> Beckman-Coulter社</td><td> 326823 </td></tr><tr><td> 0.2ミクロンのシリンジフィルタ</td><td>コー​​ニング</td><td> 431219 </td></tr><tr><td>別途準備DNA Miniキット</td><td>キアゲン</td><td> 51304 </td></tr></tbody></table>

production of lentiviral vectors for transducing cells from the central nervous system

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

Watch this Scientific Journal Video about Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System at JoVE.com

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

このプロトコルでは、生産、精製、およびレンチウイルスベクターの滴定について説明します。我々は初代培養神経細胞やアストロサイトにおけるレンチウイルスベクターによる遺伝子送達の例を示します。私たちの方法は、他の細胞型にも適用される場合があります<em&gt; in vitroで</em&gt;と<emin vivoでの&gt;</em&gt;。

中枢神経系から細胞を変換するためのレンチウイルスベクターの生産

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

production-lentiviral-vectors-for-transducing-cells-from-central

Research

JoVE Journal

Neuroscience

35.8K ビュー.  Washington University School of Medicine. このプロトコルでは、生産、精製、およびレンチウイルスベクターの滴定について説明します。我々は初代培養神経細胞やアストロサイトにおけるレンチウイルスベクターによる遺伝子送達の例を示します。私たちの方法は、他の細胞型にも適用される場合があります in vitroでと。

ビデオ: Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

齧歯類モデルで中枢神経系の疾患を治療するための全身および局所薬物送達

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

神経科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

マウスの中枢神経系における幹細胞注入のマルチモーダルイメージング

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

の中枢神経系における単一細胞のラベリング<em&gt;キイロショウジョウバエ</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

齧歯中枢神経系への薬物の直接脳室内デリバリー

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

ラット中枢神経系および末梢リンパ節の組織切片での免疫組織化学的解析

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

ポリリボソーム画によって中枢神経系Translatomeの生体尋問中

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

齧歯類の中枢神経系の生体光遺伝学的刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

パッシブCLARITYを使用したマウス中枢神経系の光学クリア

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三令キイロショウジョウバエの中枢神経活動の電気生理学的記録

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Here, we describe a method for delivering drugs to the rat central nervous system by implanting a catheter into the lumbar intrathecal space of the spine. We focus on the delivery of antisense oligonucleotides, though this method is suitable for delivery of other therapeutic modalities as well.

ラット中枢神経系におけるアンチセンスオリゴヌクレオチドの子宮内送達

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central ...