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Biology

山中因子の過剰発現によって多能性の成体体細胞のin vivoでの再プログラミング中

Published: December 17, 2013 doi: 10.3791/50837
Automatic Translation
1, 1, 1, 2
1Nanomedicine Lab, UCL School of Pharmacy, University College London, 2Nanomedicine Lab, Faculty of Medical & Human Sciences, University of Manchester

Abstract

定義された要因の強制発現による多能性状態への体細胞の初期化から生じる人工多能性幹（iPS）細胞は、再生医療のための新たな機会を提供しています。 iPS細胞のような臨床用途は、主に既存の再プログラミング方法論の効率が悪く、移植の際に腫瘍を形成する生成されたiPS細胞の危険性のために、これまで限定されていた。

我々は、多能性に向かって体細胞の再プログラミングは、再プログラミング因子の遺伝子導入によりインビボで達成することができるという仮説を立てた。効率的にin vivoで細胞を再プログラミングするためには、山中（OKSM）転写因子の高レベルは、標的組織で発現される必要がある。これは、標的組織に応じて、異なるウ​​イルスまたは非ウイルス性遺伝子ベクターを使用することによって達成することができる。この特定の研究では、プラスミドDNAの流体力学的尾静脈（HTV）の注射を配信するために使用したマウス肝細胞へのOKSM要因。これは実証的証拠大人のin vivoでの再プログラミング、高効率、高速な反応速度を持つ多能性状態への体細胞の提供。さらに、まだ腫瘍または奇形腫形成は、in situで観察されなかった。

これは、 インビボで体細胞を再プログラミングするプロトコルを実行し、将来の異なる組織タイプに適用することができるインビトロと比較して安全に効率よく、迅速に強化された多能性を誘導するために潜在的なアプローチを提供し得ると結論することができる。

Materials

Name Company Catalog Number Comments
pCX-OKS-2A pDNA Addgene 19771 Obtained from Addgene as bacterial stab, plasmid production performed in Plasmid Factory (Germany)
pCX-cMyc Addgene 19772 Obtained from Addgene as bacterial stab, plasmid production performed in Plasmid Factory (Germany)
0.22 μm filter Milliopore SLGP033RS
Isoflurane Abbott B505
HBSS buffer Sigma-Aldrich H6648 Ca2+ and Mg2+ free, with bicarbonate
Liver Digest Medium Gibco 17703-034
Hepatocyte Wash Medium Gibco 17704-024
100 μm cell strainer BD Biosciences 352360
Nucleospin RNA II kit Macherey-Nagel 740955.25 Kit for RNA isolation from cells and tissues
iScript cDNA synthesis kit Bio-Rad 170-8890
iO SYBR Green Supermix Bio-Rad 170-8880
Oct3/4 forward primer Sigma sequence given in comments TGAGAACCTTCAGGAGATATGCAA
Oct3/4 reverse primer Sigma sequence given in comments CTCAATGCTAGTTCGCTTTCTCTTC
Sox2 forward primer Sigma sequence given in comments GGTTACCTCTTCCTCCCACTCCAG
Sox2 reverse primer Sigma sequence given in comments TCACATGTGCGACAGGGGCAG
C-myc forward primer Sigma sequence given in comments CAGAGGAGGAACGAGCTGAAGCGC
C-myc reverse primer Sigma sequence given in comments TTATGCACCAGAGTTTCGAAGCTGTTCG
Nanog forward primer Sigma sequence given in comments CAGAAAAACCAGTGGTTGAAGACTAG
Nanog reverse primer Sigma sequence given in comments GCAATGGATGCTGGGATACTC
Ecat1 forward primer Sigma sequence given in comments TGTGGGGCCCTGAAAGGCGAGCTGAGAT
Ecat1 reverse primer Sigma sequence given in comments ATGGGCCGCCATACGACGACGCTCAACT
Rex1 forward primer Sigma sequence given in comments ACGAGTGGCAGTTTCTTCTTGGGA
Rex1 reverse primer Sigma sequence given in comments TATGACTCACTTCCAGGGGGCACT
Alb forward primer Sigma sequence given in comments GTTCGCTACACCCAGAAAGC
Alb reverse primer Sigma sequence given in comments CCACACAAGGCAGTCTCTGA
Aat forward primer Sigma sequence given in comments CAGAGGAGGCCAAGAAAGTG
Aat reverse primer Sigma sequence given in comments ATGGACAGTCTGGGGAAGTG
Trf forward primer Sigma sequence given in comments ACCATGTTGTGGTCTCACGA
Trf reverse primer Sigma sequence given in comments ACAGAAGGTCCTTGGTGGTG
B actin forward primer Sigma sequence given in comments GACCTCTATGCCAACACAGT
B actin reverse primer Sigma sequence given in comments AGTACTTGCGCTCAGGAGGA
BD Cytofix fixation buffer BD Biosciences 560585
1x BD Perm/Wash buffer BD Biosciences 560585
anti-mouse OCT4-PerCP-Cy5.5 BD Biosciences 560585
anti-mouse Nanog-PE BD Biosciences 560585
2-Methylbutane Sigma-Aldrich M32631
X100-Triton Sigma-Aldrich X100-500ML
Goat serum Sigma-Aldrich G9023
BSA Sigma A2153
rabbit pAb anti-OCT4 Abcam ab19857 Use at a concentration of 3 μg/ml
rabbit pAb anti-SOX2 Abcam ab97959 Use at a concentration of 1 μg/ml
rabbit pAb anti-Nanog Abcam ab80892 Use at a concentration of 1 μg/ml
mouse mAb anti-SSEA1 Abcam ab16285 Use at a concentration of 20 μg/ml
goat pAb anti-rabbit IgG labeled with Cy3 Jackson ImmunoResearch Laboratories Inc. 111-165-003-JIR Use at a concentration of 1/250
goat pAb anti-mouse IgG labeled with Cy3 Jackson ImmunoResearch Laboratories Inc. 115-165-003-JIR Use at a concentration of 1/250
VECTASHIELD mounting medium with DAPI Vector Laboratories H-1200
BCIP/NBT liquid substrate system Sigma B1911
Aqueous mounting medium Sigma
16% Paraformaldehyde Fisher AA433689M Stock solution is 16%, use it at 4%
Hematoxylin and eosin Sigma GH53/HT110216
PAS staining Sigma 395B

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山中因子の過剰発現によって多能性の成体体細胞の<em>in vivoでの</em>再プログラミング<em>中</em>
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Cite this Article

Yilmazer, A., de Lázaro, I.,… More

Yilmazer, A., de Lázaro, I., Bussy, C., Kostarelos, K. In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors. J. Vis. Exp. (82), e50837, doi:10.3791/50837 (2013).

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