JP5697123B2 - 酸性化ポリエチレンイミンを用いる細胞への核酸導入方法 - Google Patents
酸性化ポリエチレンイミンを用いる細胞への核酸導入方法 Download PDFInfo
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- JP5697123B2 JP5697123B2 JP2009174234A JP2009174234A JP5697123B2 JP 5697123 B2 JP5697123 B2 JP 5697123B2 JP 2009174234 A JP2009174234 A JP 2009174234A JP 2009174234 A JP2009174234 A JP 2009174234A JP 5697123 B2 JP5697123 B2 JP 5697123B2
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- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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Description
1. 下記工程を含む、細胞への核酸導入方法:
(1)ポリエチレンイミン(PEI)と核酸とを、pH 3.5からpH 4.5の酸性条件下で混合する工程、および
(2)得られた混合物で細胞を処理する工程、
2. 前記工程(2)についで下記工程を更に含む前記1.の細胞への核酸導入方法:
(3)細胞の培養培地を新鮮培養培地と交換する工程、
3. PEIが、直鎖PEIである前記1.または2.の細胞への核酸導入方法、
4. PEIが、分子量約25,000の直鎖PEIである前記1.または2.の細胞への核酸導入方法、
5. PEIと核酸との重量比が、4:1から8:1である前記1.から4.のいずれかの細胞への核酸導入方法、
6. 前記1.から5.のいずれかに記載のPEIが、pH 0.5からpH 2.0の溶液(PEI溶液)である前記1.から5.のいずれかの細胞への核酸導入方法、
7. 前記1.から5.のいずれかに記載のPEIが、PEIの塩酸溶液である前記1.から5.のいずれかの細胞への核酸導入方法、
8. 前記1.から5.のいずれかに記載のPEIが、PEIを0.2N 塩酸溶液に溶解することにより調製された溶液である前記1.から5.のいずれかの細胞への核酸導入方法、
9. 下記工程を含む、細胞への核酸導入方法:
(1)ポリエチレンイミン(PEI)と核酸とを、pH 3.5からpH 4.5の酸性条件下で混合する工程、
(2)得られた混合物で細胞を処理する工程、および
(3)細胞の培養培地を新鮮培養培地と交換する工程、
ここで、PEIは分子量約25,000の直鎖PEIを0.2N 塩酸溶液に溶解することにより調製された溶液であり、PEIと核酸との重量比が4:1から8:1である、
10. ポリエチレンイミン(PEI)溶液を含む細胞への試薬キットであって、PEI溶液のpHが0.5から2.0であることを特徴とする細胞への核酸導入用試薬キット、
11. 前記PEI溶液がPEIの塩酸溶液である前記10.の細胞への核酸導入用試薬キット、
12. 前記PEI溶液がPEIを0.2N 塩酸溶液に溶解することにより調製された溶液である前記10.の細胞への核酸導入用試薬キット、
13. 前記PEI溶液に含まれるPEIが直鎖PEIである前記10.から12.のいずれかの細胞への核酸導入用試薬キット、
14. 前記PEI溶液に含まれるPEIが分子量約25,000の直鎖PEIである前記10.から12.のいずれかの細胞への核酸導入用試薬キット、
15. ポリエチレンイミン(PEI)溶液を含む試薬キットであって、PEI溶液が分子量約25,000の直鎖PEIを0.2N 塩酸溶液に溶解することにより調製された溶液であることを特徴とする細胞への核酸導入用試薬キット、
16. ポリエチレンイミン(PEI)からなる細胞への核酸導入試薬であって、PEI溶液のpHが0.5から2.0であることを特徴とする該導入効率が保持された細胞への核酸導入試薬。
(1)PEIと核酸とを、重量比4:1から8:1の範囲内で、pH 3.5からpH 4.5の範囲内の酸性条件下で混合する工程、
(2)得られた混合物で細胞を処理する工程、および
(3)細胞の培養培地を新鮮培養培地と交換する工程、
直鎖PEI(分子量25,000)は、ポリサイエンシズ社(Polysciences, Inc.)より購入した。PEI粉末は0.2N HClに5mg/mlの濃度で溶解し、そして一部を4℃または-80℃で保管した。HEPES緩衝生理食塩水(以下、HBSと略称する:20 mM HEPES-KOH、pH 7.4、および150 mM NaCl)および乳酸緩衝生理食塩水(以下、LBSと略称する:20 mM 乳酸ナトリウム, pH 3.5、4.0、または4.5、および150 mM NaCl)を調製した。TransIT-LT1トランスフェクション試薬は、マイラスバイオLLC社(Mirus Bio LLC)より購入した。強化緑色蛍光タンパク質(enhanced green flulorescent protein;以下、EFGPと略称する)をコードするベクターpcDNA4-TO-EGFPは、pcDNA4/TOベクター(Invitrogen社製)を用いて従前報告された方法で構築した(Nakayama, Y. et al., (2006) Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus. Exp. Cell Res. 312, 2252-2263.)。CD25をコードするpCAGベクター(pCAG/TetR-IRES-CD25)は従前報告された方法で構築した(Miyazaki, J. et al., (1989) Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5. Gene 79, 269-277.)。
ヒト上皮HeLaおよびHeLa S3細胞(Japanese Collection of Research Bioresources、大阪)は、5%牛胎仔血清(FBS)および5%牛血清(BS)をそれぞれ含むイスコフ改変ダルベッコ培地(Iscove's modified Dulbecco's medium;IMDM)中で培養した。ヒト巨核球系のDami細胞は、従前報告されたように、10%ウマ血清を含むIMDM中で浮遊培養により維持し(Greenberg, S.M. et al., (1988) Characterization of a new megakaryocytic cell line: The Dami cell. Blood 72, 1968-1977.;Hirao, A. et al., (1998) Overexpression of C-terminal Src kinase homologous kinase suppresses activation of Lyn tyrosine kinase required for VLA5-mediated Dami cell spreading. J. Biol. Chem. 273, 10004-10010.;Sato, I. et al., (2009) Differential trafficking of Src, Lyn, Yes and Fyn is specified by the state of palmitoylation in the SH4 domain. J. Cell Sci. 122, 965-975.)、2.5% FBSおよび2.5% ウマ血清を含むIMDM中で2-3日間培養することにより培養ディッシュに付着させた。
HeLa細胞を35mm培養ディッシュにディッシュ当たり1×105細胞となるように播種し、1-2日間培養した。トランスフェクション前に培養培地を新鮮な血清含有培地と交換した。PEI/DNAポリプレックスを形成させるため、1μgのpcDNA4-TO-EGFPを、100μlのLBSまたはHBS中で、後述するPEI/DNA比でPEIと混合し、そして当該混合物を室温で20分間放置した。初期の実験では1μgのpcDNA4-TO-EGFPと5μgのPEIを100μlのLBS中で直接的に混合したが、以降の実験では1μgのpcDNA4-TO-EGFPと5μgのPEIをそれぞれ50μlのLBSで別々に希釈してから混合した。得られたPEI/DNAポリプレックスは500μlの予め温めた血清不含IMDMで希釈し、そして各培養ディッシュに滴下して添加した。トランスフェクション後24時間目に、0.05% EDTAを含むリン酸緩衝生理食塩水(PBS)中で静かにピペッティングすることにより細胞を剥離させた。遠心処理後、3% FBSおよび1μg/ml ヨウ化プロピジウム(PI)を含むPBSに細胞を再度浮遊させ、そして、蛋白質発現を検証するためにEGFP蛍光を、死細胞の検証のためにPI染色を、MoFloセルソーター(Beckman Coulter社製)を用いてフローサイトメトリーにより分析した。培養ディッシュで増殖したトランスフェクション細胞は、トランスフェクション後24時間目にツァイス LSM 5 パスカル デコンボリューション顕微鏡(Zeiss LSM 5 Pascal deconvolution microscope、Carl Zeiss社製)下で観察した。
HCT116細胞はトランスフェクション試薬の毒性に敏感である。PEIの毒性を低減させると共にトランスフェクション効率を増加させるために、逐次トランスフェクション方法を開発した。具体的には、DNA/PEIポリプレックスを、7.5μgのPEIと6μgのpCAG/TetR-IRES-CD25を用いて調製し、35mmの培養ディッシュそれぞれに滴下により添加し、そして8時間インキュベーション後に培養培地を新鮮な血清含有培地と交換した。更なる次のトランスフェクションのために、新たに調製したDNA/PEIポリプレックスを1回目のトランスフェクションにおいて示したように細胞培養に添加し、さらに8時間インキュベーション後に培地を新鮮な血清含有培地と再び交換した。細胞を更に16時間培養して、抗CD25抗体で染色し、そして細胞表面のCD25発現をMoFloセルソーターを用いてフローサイトメトリーにより従前報告されたように分析した(Nakayama, Y. et al., (2005) Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase. Exp. Cell Res. 304, 570-581.)。
HeLa S3細胞を60mm培養ディッシュにディッシュ当たり3×104細胞となるように播種し、1日間培養した。PEI/DNAポリプレックスを形成させるため、97.5μgのPEIと15μgのpcDNA4-TO-EGFPをそれぞれ50μlのLBS(pH 3.5)中に別々に希釈し、そして希釈物を混合して上記のようにトランスフェクションに用いた。トランスフェクション後24時間目に培養ディッシュ中で増殖した細胞を4つのディッシュに等分に分け、そして、安定トランスフェクション細胞の選択を、250μg/ml ゼオシン(Invitrogen社製)中でトランスフェクション後48時間目から実施した。
Claims (6)
- 下記工程を含む、細胞への核酸導入方法:
(1)ポリエチレンイミン(PEI)と核酸とを、pH 3.5からpH 4.5の酸性条件下で混合する工程、および
(2)得られた混合物で細胞を処理する工程、
ここで、該PEIは分子量25,000の直鎖PEIである。 - 前記工程(2)についで下記工程を更に含む請求項1に記載の細胞への核酸導入方法:
(3)細胞の培養培地を新鮮培養培地と交換する工程。 - ポリエチレンイミン(PEI)と核酸との重量比が、4:1から8:1である請求項1または請求項2に記載の細胞への核酸導入方法。
- 前記ポリエチレンイミン(PEI)が、pH 0.5からpH 2.0の溶液(PEI溶液)であることを特徴とする請求項1から3のいずれか1項に記載の細胞への核酸導入方法。
- 前記ポリエチレンイミン(PEI)が、PEIを0.2N 塩酸溶液に溶解することにより調製された溶液であることを特徴とする請求項1から3のいずれか1項に記載の細胞への核酸導入方法。
- 下記工程を含む、細胞への核酸導入方法:
(1)ポリエチレンイミン(PEI)と核酸とを、pH 3.5からpH 4.5の酸性条件下で混合する工程、
(2)得られた混合物で細胞を処理する工程、および
(3)細胞の培養培地を新鮮培養培地と交換する工程、
ここで、該PEIは分子量25,000の直鎖PEIを0.2N 塩酸溶液に溶解することにより調製された溶液であり、PEIと核酸との重量比が4:1から8:1である。
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