Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs. The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions / Romano, Oriana; Cifola, Ingrid; Poletti, Valentina; Severgnini, Marco; Peano, Clelia; De Bellis, Gianluca; Mavilio, Fulvio; Miccio, Annarita. - In: JOURNAL OF VISUALIZED EXPERIMENTS. - ISSN 1940-087X. - 2017:123(2017), pp. e55919-e55919. [10.3791/55919]

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

ROMANO, ORIANA;MAVILIO, Fulvio;
2017

Abstract

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.
2017
2017
123
e55919
e55919
Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions / Romano, Oriana; Cifola, Ingrid; Poletti, Valentina; Severgnini, Marco; Peano, Clelia; De Bellis, Gianluca; Mavilio, Fulvio; Miccio, Annarita. - In: JOURNAL OF VISUALIZED EXPERIMENTS. - ISSN 1940-087X. - 2017:123(2017), pp. e55919-e55919. [10.3791/55919]
Romano, Oriana; Cifola, Ingrid; Poletti, Valentina; Severgnini, Marco; Peano, Clelia; De Bellis, Gianluca; Mavilio, Fulvio; Miccio, Annarita
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1138071
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