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A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

ABSTRACT: DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease system targeting HIV long terminal repeats (LTR). Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs) targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

SUBMITTER: Ebina H

PROVIDER: S-EPMC4363575 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

Ebina Hirotaka H Kanemura Yuka Y Misawa Naoko N Sakuma Tetsushi T Kobayashi Tomoko T Yamamoto Takashi T Koyanagi Yoshio Y

PloS one 20150317 3

DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuclea ...[more]

PMID: 25781496

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Project description:AimTo investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.MethodsIn this study, human adipose tissue derived stem cells (hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis. Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1 (Non-integrated LV-PDX1) were constructed using specific plasmids (pLV-HELP, pMD2G, LV-105-PDX1-1). Then, hADSCs were transduced with non-integrated LV-PDX1. After transduction, ADSCs(PDX1+) were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 and insulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCs(PDX1+) in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCs(PDX1+) were implanted into hyperglycemic rats.ResultsHuman ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture. Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs(+PDX1) became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis. Significant expressions of PDX1, Ngn3, glucagon, Glut2 and somatostatin were detected by quantitative RT-PCR. hADSCs(PDX1+) revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCs(PDX1+) in the high glucose medium was 2.32 μU/mL. hADSCs(PDX1+) implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.ConclusionHuman ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.

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A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

Publications

A high excision potential of TALENs for integrated DNA of HIV-based lentiviral vector.

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