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Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination.

ABSTRACT: Cre-mediated recombination is a widely used technique for the re-arrangement of DNA sequences that are bracketed by loxP recognition sites. This bacteriophage P1 enzyme is commonly used to excise the bacterial artificial chromosome (BAC) sequence, a vector sequence used for large herpesvirus genomes for the purposes of propagation and manipulation in Escherichia coli. Most methods utilize cell lines that can be induced for the expression of Cre enzyme to facilitate this excision. In addition, methods have been developed that express Cre from the virus genome and enable auto-excision of the BAC plasmid. We report a versatile and rapid in vitro method based on purified Cre enzyme to carry out the same process in a test tube and does not require cell line generation or cloning into the virus genome. This method greatly increases the repertoire of methods available to modify the genome prior to reconstitution of virus infectivity in a mammalian host.

SUBMITTER: Grzesik P

PROVIDER: S-EPMC6400485 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination.

Grzesik Peter P Ko Nathan N Oldfield Lauren M LM Vashee Sanjay S Desai Prashant J PJ

Journal of virological methods 20180806

Cre-mediated recombination is a widely used technique for the re-arrangement of DNA sequences that are bracketed by loxP recognition sites. This bacteriophage P1 enzyme is commonly used to excise the bacterial artificial chromosome (BAC) sequence, a vector sequence used for large herpesvirus genomes for the purposes of propagation and manipulation in Escherichia coli. Most methods utilize cell lines that can be induced for the expression of Cre enzyme to facilitate this excision. In addition, me ...[more]

PMID: 30092252

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Project description:Emergence of induced pluripotent stem cells (iPSC) technology has paved novel routes for regenerative medicine. iPSCs offer the possibilities of disease modeling, drug toxicity studies as well as cell replacement therapies by autologous transplantation. Classical protocols of iPSC generation harness infection by retro- or lenti-viruses. Although such integrating viruses represent very robust tools for reprogramming, the presence of viral transgenes in iPSCs is deleterious as it holds the risk of insertional mutagenesis leading to malignant transformation. Moreover, remaining reprogramming transgenes have been shown to affect the differentiation potential of iPSCs. More recently, alternative protocols have been explored to derive transgene-free iPSC, including use of transposons, mRNA transfection, episomal plasmid transfection, and infection with non-integrating viruses such as Sendai virus. However, the utility of such protocols remains limited due to low efficiency and narrow range of cell specificity. In this study we aim at combining the robustness of lentiviral reprogramming with the high efficacy of Cre recombinase protein transduction to readily delete reprogramming transgenes from iPSCs. We demonstrate rapid generation of transgene-free human iPSCs by excising the loxP-flanked reprogramming cassette employing direct delivery of biologically active Cre protein. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs do resemble more to human ESCs and has better differentiation potential than iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of superior transgene-free iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies. mRNA extracted from human Fibroblasts (AR1034ZIMA), human Embryonic Stem Cell line I3 (hES I3), three human induced Pluripotent Stem Cell clones 1, 1.2 and 1.4 (fl-ARiPS cl1, del-ARiPS cl 1.2, del-ARiPS cl1.4) has been hybridized on Illumina Human HT-12 (version 4 revision 2) arrays for genome wide expression analysis. Samples were run at least as duplicate technical replicates. Differential gene expression analysis has been performed on the grouped expression data with the human embryonic stem cells (hES I3) group as the reference.

Dataset Information

Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination.

Publications

Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination.

Similar Datasets

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