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SNP microarray of T-REx-293-based clones overexpressing the DNA cytosine deaminase APOBEC3B

ABSTRACT: A conditional and isogenic system for APOBEC3B (A3B) induction in T-REx-293 cells. Cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs and subjected to 10 rounds of A3B-eGFP exposure causing 80-90% cell death. Control pools (eGFP) were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. DNA was extracted and subjected to SNP analyses using the Human OmniExpress-24v1-0 BeadChip (Illumina, San Diego, CA). Genotyping was performed using the humanomniexpress_24v1-0_a cluster file. SNP data is avaiable for a total of 16 samples, all derived from single cells mother lines A and C. For each series, each consisting of 8 samples, A3B-eGFP and eGFP daugther clones were processed on the SNP microarray, as well as 3 A3B-eGFP and 3 eGFP grand-daugther clones.

ORGANISM(S): Homo sapiens

SUBMITTER: Anita Grigoriadis

PROVIDER: E-GEOD-78710 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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SNP microarray of T-REx-293-based clones overexpressing the DNA cytosine deaminase APOBEC3B

Project description:A conditional and isogenic system for APOBEC3B (A3B) induction in T-REx-293 cells. Cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs and subjected to 10 rounds of A3B-eGFP exposure causing 80-90% cell death. Control pools (eGFP) were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. DNA was extracted and subjected to SNP analyses using the Human OmniExpress-24v1-0 BeadChip (Illumina, San Diego, CA). Genotyping was performed using the humanomniexpress_24v1-0_a cluster file.

2016-06-30 | GSE78710 | GEO

Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

Project description:Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

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Project description:48 ǂKhomani San living in the South African Kalahari were genotyped using the Illumina OmniExpress, OmniExpress Plus, and HumanHap550 arrays

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Homo sapiens strain:T-REx-293

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Upregulation of FOXM1 Induces Genomic Instability in Human Epidermal Keratinocytes

Project description:Genome-wide 10k SNP profiling of FOXM1B-transduced N/TERT and primary normal human epidermal keratinocytes (NHEK). The aim of this study was to study the cancer initiation role of UVB and FOXM1B upregulation in NHEK. Upregulation of FOXM1B alone (without UVB) was found to directly induce genomic instability in the form of copy number aberration (CNA) and low levels of loss of heterozygosity (LOH) in primary NHEK. The FOXM1B-induced CNA was found to be retained and accumulated in subsequent cell culture passages. UVB-exposure resulted in significant chromosomal LOH and CNA in N/TERT cells expressing FOXM1B but not in EGFP-expressing cells. This indicates that UVB corroborated with FOXM1B to recruit LOH and CNA which may predispose cell to malignant transformation. Collectively, these results indicate that aberrant upregulation of FOXM1B in skin keratinocytes following UVB exposure may be an early mechanism whereby cells acquire genomic changes required for oncogenesis. Keywords: Genome-wide SNP profiling for loss of heterozygosity (LOH) and copy number aberration (CNA), FOXM1, UVB, Keratinocytes, Basal cell carcinoma, genomic instability, carcinogenesis, squamous cell carcinoma. 1) Three individual normal primary NHEK cultures were retrovirally transduced (with either EGFP or EGFP-FOXM1B) and left to grow for 4 days prior to SNP array analysis. 2) EGFP or EGFP-FOXM1B-tansduced NHEK cells were harvested at passage 1, 2 and 3 for SNP array analysis to investigate if genomic instability is maintained and accumulated in subsequent passages. 3) N/TERT cells transduced with either EGFP or EFOX were UVB-irradiated and left to grow for 50 days in culture prior to SNP array analysis.

2010-07-29 | E-GEOD-16937 | biostudies-arrayexpress

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Clonal differences underlie variable responses to sequential and prolonged treatment

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2024-01-23 | GSE253739 | GEO

hsa-miR-92a knock down in Flp-in T-REx 293-PTH-AGO1 cells

Project description:transcriptome profiling of miR-92a inhibitor treated and control cells with the aim of measuring miR-92a influence on its mRNA targets Abstract: MicroRNAs (miRNAs) play key roles in gene regulation, but reliable bioinformatic or experimental identification of their targets remains difficult. To provide an unbiased view of human miRNA targets we developed a technique for ligation and sequencing of miRNA-target RNA duplexes associated with human Ago1. Here we report datasets of more than 18,000 high-confidence miRNA-mRNA interactions. The binding of most miRNAs includes the 5' seed region, but around 60% of seed interactions are noncanonical, containing bulged or mismatched nucleotides. Moreover, seed interactions are generally accompanied by specific, non-seed basepairing. 18% of miRNA-mRNA interactions involve the miRNA 3' end, with little evidence for 5' contacts, and some of these were functionally validated. Analyses of miRNA:mRNA basepairing showed that miRNA species systematically differ in their target RNA interactions, and strongly overrepresented motifs were found in the interaction sites of several miRNAs. We speculate that these affect the response of RISC to miRNA-target binding.

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