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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 01, 2023 |
| Title |
SHIELD: A platform for high-throughput screening of barrier DNA elements in human cells |
| Organisms |
Homo sapiens; synthetic construct |
| Experiment type |
Other
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| Summary |
Chromatin boundary elements contribute to the partitioning of mammalian genomes into topological domains to regulate gene expression. Certain boundary elements are adopted as DNA insulators for safe and stable transgene expression in mammalian cells. These elements, however, are ill-defined and less characterized in the non-coding genome, partially due to the lack of a platform to readily evaluate boundary-associated activities of putative DNA sequences. Here we report SHIELD (Site-specific Heterochromatin Insertion of Elements at Lamina-associated Domains), a novel platform tailored for the high-throughput screening of barrier-type DNA elements in human cells. SHIELD takes advantage of the high specificity of serine integrase at heterochromatin, and exploits the natural heterochromatin spreading inside LADs for the discovery of potent barrier elements. We adopted SHIELD to evaluate the barrier activity of 1000 DNA elements in a high-throughput manner and identified 8 novel elements with barrier activities comparable to the core region of cHS4 element. SHIELD should greatly facilitate the discovery of novel barrier DNA elements from the non-coding genome in human cells.
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| Overall design |
To screen the potential barrier activity of DNA elments in a high-throughout manner, we adopted the landing-pad strategy. We first generated a chassis cell line (HCT116) by inserting the landing pad at epigenetically highly repressive LAD loci. Then we integrated pooled reporter plasmids at the selected loci via integrase-mediated recombination. The reporter plasmid carried the EGFP reporter gene flanked by candidate DNA elements. Each DNA element was barcoded with its own sequence. Strong barrier activity would lead to higher EGFP expression at the repressive loci. Following reporter integration, we sorted polyclonal cells based on EGFP expression levels using FACS, extracted the genomic DNA from each population, amplifed genomic DNA with primers targeting the candidate DNA, and performed NGS analysis of the amplicon to determine the relative abundance of each element in the corresponding population. The abundance of a given DNA element in the sorted population determined by NGS would reflect its relative barrier activity.
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| Contributor(s) |
Zhang M, Ehmann ME, Matukumalli S, Boob AG, Gilbert DM, Zhao H |
| Citation(s) |
37699958 |
| Submission date |
Jun 29, 2023 |
| Last update date |
Sep 21, 2023 |
| Contact name |
Meng Zhang |
| Organization name |
Stanford
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| Street address |
443 Via Ortega
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| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platforms (2) |
| GPL15520 |
Illumina MiSeq (Homo sapiens) |
| GPL17769 |
Illumina MiSeq (synthetic construct) |
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| Samples (17)
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| GSM7519624 |
GFP Neg, day 3, replicate 1 |
| GSM7519625 |
GFP High, day 3, replicate 2 |
| GSM7519626 |
GFP Low, day 3, replicate 2 |
| GSM7519627 |
GFP Med, day 3, replicate 2 |
| GSM7519628 |
GFP High, day 15, replicate 1 |
| GSM7519629 |
GFP Low, day 15, replicate 1 |
| GSM7519630 |
GFP Med, day 15, replicate 1 |
| GSM7519631 |
GFP High, day 15, replicate 2 |
| GSM7519632 |
GFP Low, day 15, replicate 2 |
| GSM7519633 |
GFP Med, day 15, replicate 2 |
| GSM7519634 |
GFP Neg, day 15, replicate 2, R1 |
| GSM7519635 |
GFP Neg, day 15, replicate 2, R2 |
| GSM7519636 |
Reporter Plasmid Library Maxiprep from E. coli, R1 |
| GSM7519637 |
Reporter Plasmid Library Maxiprep from E. coli, R2 |
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| Relations |
| BioProject |
PRJNA989234 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE236198_RAW.tar |
158.2 Mb |
(http)(custom) |
TAR (of XLSX) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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