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Title: Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

Abstract

The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls didmore » not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.« less

Authors:
 [1];  [2];  [2];  [3]
  1. Department of Radiology, University of Minnesota Medical School, MMC 292, 420 Delaware Street SE, Minneapolis, MN 55455 (United States)
  2. Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States)
  3. (United States)
Publication Date:
OSTI Identifier:
22606162
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 477; Journal Issue: 3; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANIMALS; DNA; GENES; GLOBINS; HOST; METHYLATION; MUTAGENESIS; SLEEP; TRANSPOSONS

Citation Formats

Aravalli, Rajagopal N., E-mail: aravalli@umn.edu, Park, Chang W., Steer, Clifford J., E-mail: steer001@umn.edu, and Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.06.094.
Aravalli, Rajagopal N., E-mail: aravalli@umn.edu, Park, Chang W., Steer, Clifford J., E-mail: steer001@umn.edu, & Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis. United States. doi:10.1016/J.BBRC.2016.06.094.
Aravalli, Rajagopal N., E-mail: aravalli@umn.edu, Park, Chang W., Steer, Clifford J., E-mail: steer001@umn.edu, and Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455. Fri . "Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis". United States. doi:10.1016/J.BBRC.2016.06.094.
@article{osti_22606162,
title = {Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis},
author = {Aravalli, Rajagopal N., E-mail: aravalli@umn.edu and Park, Chang W. and Steer, Clifford J., E-mail: steer001@umn.edu and Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455},
abstractNote = {The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.},
doi = {10.1016/J.BBRC.2016.06.094},
journal = {Biochemical and Biophysical Research Communications},
number = 3,
volume = 477,
place = {United States},
year = {Fri Aug 26 00:00:00 EDT 2016},
month = {Fri Aug 26 00:00:00 EDT 2016}
}