PhiC31 integrase‐mediated genomic integration and stable gene expression in the mouse mammary gland after gene electrotransfer

Luo, Yan; Li, Jun; Wang, Yongsheng; Su, Jianmin; Wu, Yongyan; Hu, Guang‐Wan; Gao, Ming-Qing; Quan, Fusheng; Zhang, Yong

doi:10.1002/jgm.2723

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References 47 publications

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“…Even non‐integrated adeno‐associated recombinant viruses (rAAVs), which currently are the most attractive viral vectors, along with retro‐ and lentiviral vectors, have several serious disadvantages for use in gene therapy and even in gene functional studies, such as low cloning capacity and lack of long‐term transgene expression. Another method is based on integration of a gene into a “hot spot” of a mammalian genome using a bacteriophage P1‐derived Cre recombinase or ΦC31 integrase (Luo et al., 2013). However, efficiency of gene integration with such a system is very low.…”

Section: Commentarymentioning

confidence: 99%

Assembly of Multiple Full‐Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System

et al. 2021

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Human artificial chromosomes (HACs) are gene delivery vectors that have been used for decades for gene functional studies. HACs have several advantages over viral‐based gene transfer systems, including stable episomal maintenance in a single copy in the cell and the ability to carry up to megabase‐sized genomic DNA segments. We have previously developed the alphoidtetO‐HAC, which has a single gene acceptor loxP site that allows insertion of an individual gene of interest using Chinese hamster ovary (CHO) hybrid cells. The HAC, along with a DNA segment of interest, can then be transferred from donor CHO cells to various recipient cells of interest via microcell‐mediated chromosome transfer (MMCT). Here, we detail a protocol for loading multiple genomic DNA segments or genes into the alphoidtetO‐HAC vector using an iterative integration system (IIS) that utilizes recombinases Cre, ΦC31, and ΦBT. This IIS‐alphoidtetO‐HAC can be used for either serially assembling genomic loci or fragments of a large gene, or for inserting multiple genes into the same artificial chromosome. The insertions are executed iteratively, whereby each round results in the insertion of a new DNA segment of interest. This is accompanied by changes of expression of marker fluorescent proteins, which simplifies screening of correct clones, and changes of selection and counterselection markers, which constitutes an error‐proofing mechanism that removes mis‐incorporated DNA segments. In addition, the IIS‐alphoidtetO‐HAC carrying the genes can be eliminated from the cells, offering the possibility to compare the phenotypes of human cells with and without functional copies of the genes of interest. The resulting HAC molecules may be used to investigate biomedically relevant pathways or the regulation of multiple genes, and to potentially engineer synthetic chromosomes with a specific set of genes of interest. The IIS‐alphoidtetO‐HAC system is expected to be beneficial in creating multiple‐gene humanized models with the purpose of understanding complex multi‐gene genetic disorders. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Integration of the first DNA segment of interest into the IIS‐alphoidteto‐HAC Basic Protocol 2: Integration of a second DNA segment of interest into the IIS‐alphoidteto‐HAC Basic Protocol 3: Integration of a third DNA segment of interest into the IIS‐alphoidteto‐HAC Support Protocol: Fluorescence in situ hybridization analysis for the circular IIS‐alphoidtetO‐HAC

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Section: Commentarymentioning

confidence: 99%

Assembly of Multiple Full‐Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System

et al. 2021

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PhiC31 integrase‐mediated genomic integration and stable gene expression in the mouse mammary gland after gene electrotransfer

Abstract: The results obtained in the present study show that the use of phiC31 integrase is a feasible and efficient method for high and stable transgene expression in the mouse mammary gland.

Cited by 1 publication

References 47 publications

Assembly of Multiple Full‐Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System

Assembly of Multiple Full‐Size Genes or Genomic DNA Fragments on Human Artificial Chromosomes Using the Iterative Integration System

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